key: cord- -zn tm ww authors: sokoloski, kevin j.; nease, lauren m.; may, nicholas a.; gebhart, natasha n.; jones, claire e.; morrison, thomas e.; hardy, richard w. title: identification of interactions between sindbis virus capsid protein and cytoplasmic vrna as novel virulence determinants date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zn tm ww alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. while the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. a clip-seq approach was used to screen for candidate sites of interaction between the viral capsid protein and genomic rna of sindbis virus (sinv), a model alphavirus. the data presented in this report indicates that the sinv capsid protein binds to specific viral rna sequences in the cytoplasm of infected cells, but its interaction with genomic rna in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. mutational analyses of the cytoplasmic viral rna-capsid interaction sites revealed a functional role for capsid binding early in infection. interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. rather mutation of the cytoplasmic capsid-rna interaction sites negatively affected the functional capacity of the incoming viral genomic rnas leading to decreased infectivity. furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic rna, while not essential for particle formation, are necessary for genomic rna function early during infection. this previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant. a a a a a alphaviruses are positive-sense rna viruses that exhibit a broad host range; and as evidenced by the emergence of chikungunya virus (chikv), represent a significant burden on the public health systems of developed and underdeveloped communities [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . alphaviral disease is broadly classified based on the symptomology associated with clinical infection. arthritogenic alphavirus infections, such as those involving chikv, ross river virus (rrv), and sindbis virus (sinv), induce debilitating arthritis in infected individuals [ ] . in contrast to the arthritogenic alphaviruses, infection with encephalitic alphaviruses may result in severe neurologic disease and significant mortality, primarily in young children [ , ] . regardless of disease symptomology the members of the genus alphavirus exhibit highly similar single-cell replication cycles [ , ] . mature infectious alphavirus particles are approximately nm in diameter, and consist of two concentric protein shells divided by a host derived lipid envelope [ , ] . the arrangement of the outer protein shell, consisting of the viral glycoproteins e and e , and the inner protein shell, comprised of capsid protein, are arranged with icosahedral symmetry [ , ] . the innermost icosahedral structure is the nucleocapsid core, which consists of the viral capsid protein and the rna cargo. however, the assembly of mature alphavirus particles is a highly selective process as, to date, many characterizations of alphaviral particles have agreed that the viral genomic rna is the predominant rna molecule within the nucleocapsid core [ ] [ ] [ ] [ ] [ ] . several studies have identified a region of the genomic rna associated with the selective packaging of the viral genome during the assembly of infectious particles. this element, termed the packaging signal, consists of a highly-structured region found within the open reading frame of the nonstructural polyprotein [ ] [ ] [ ] [ ] [ ] [ ] . while these studies provided an excellent definition of the cis-acting elements involved in the selection of the cargo rna, they did not identify nor describe the interaction between the capsid protein and the viral rna cargo [ ] . the interactions between the alphaviral capsid protein and the viral genomic rna is an under characterized, yet vitally important, rna:protein interaction. alphaviral in vitro assembly systems have largely indicated that the assembly of capsid:nucleic acid complexes is a nonspecific process [ ] [ ] [ ] [ ] [ ] . however it should be noted that the direct interaction between the viral capsid protein and viral rna has not been exhaustively characterized in the cytoplasm of infected cells, or in mature viral particles leaving our understanding of the molecular interactions between these essential components of the virus incomplete. this report details our efforts to identify and characterize the sites of interaction between the viral capsid protein and the genomic rna using the model alphavirus sindbis virus (sinv). a clip-seq approach was used to screen for potential sites of capsid:rna (c:r) interaction within sindbis virus (sinv) particles and cytoplasmic nucleocapsid complexes. interestingly, while the c:r interactions of purified viral particles are relatively evenly distributed across the genome, the c:r interactions in cytoplasmic rna:protein complexes exhibit a discrete binding profile. further characterization of these candidate c:r interaction sites showed a decrease in capsid interaction when the sites were mutated, and indicated that the sites are essential for efficient viral growth as mutation of individual c:r interaction sites significantly reduces the infectivity of the mature viral particles. further analysis indicated that the c:r interactions are necessary at an early stage of virus infection and stabilize incoming viral genomic rna. moreover, the cytoplasmic c:r interaction sites represent a series of novel pathogenicity determinants as c:r interaction site mutants are significantly attenuated in a neurotropic mouse model of infection. collectively, the results presented indicate a new role for capsid protein during the viral replication cycle and identify a novel determinant of viral pathogenesis expanding our understanding of how capsid:rna interactions regulate viral infection beyond particle assembly. to identify candidate site(s) of interaction between the viral genomic rna and the capsid protein we utilized a clip-seq method to develop cdna libraries of c:r interactions [ ] [ ] [ ] . briefly, either purified viral particles or infected tissue culture cells were irradiated with shortwavelength uv radiation to form covalently cross-linked rna:protein complexes. the rna components of cross-linked complexes were then fragmented via rnase digestion. the fragmented c:r complexes were then selectively immunoprecipitated from the lysates using anticapsid polyclonal antibodies (supporting information, s fig) . from the immunoprecipitated materials, cdna libraries were generated using an adaptor ligation process and deep sequenced. in addition to anti-capsid clip-seq libraries, derived from purified virions and infected cells, nonspecific control libraries were also developed. for all cdna libraries the sequencing data were aligned and clustered to a reference genome ( fig a, and supporting information s data), consisting of the input sinv toto parental sequence. the c:r interactions in mature virions are dispersed and nonspecific. clip-seq analysis of purified sinv particles indicates that the contacts between the viral capsid proteins and the genomic rnas are extensive, and likely nonspecific in nature. as shown in fig b, the interactions between the encapsidated genomic rna cargo and the viral capsid protein in purified viral particles are widely dispersed across the genome. nonetheless, two regions of the genome exhibited decreased c:r interaction relative to the entirety of the genomic rna. interestingly, one of these sites corresponds to the previously identified packaging signal [ ] . the second region of low coverage corresponds to the subgenomic promoter and the nucleotides corresponding to the 'utr of the subgenomic rna [ , ] . the c:r interactions of cytoplasmic nucleocapsid complexes exhibit specificity. in contrast to the sequence coverage observed with purified virions, c:r complexes derived from infected cell lysates exhibit remarkable specificity. co-precipitation of viral rna and capsid protein was dependent on crosslinking and the use of antibodies specific for capsid (s fig) . subtractive analysis of nonspecific control and anti-capsid cdna library sequencing data revealed several discrete regions of significant enrichment in the anti-capsid library (fig c) . the most highly enriched regions correspond to the coding sequence of the structural orf, in particular the e and e genes. nonetheless, several minor peaks are found in other regions of the genomic rna, including the ' and ' utrs and the nonstructural and structural coding regions. interestingly, these peaks do not correlate with the previously described packaging signal or other known cis-acting features of the viral rna [ , ] . to prioritize which peaks would be further characterized we utilized a statistical method to examine the relative enrichment of individual sequences against the entire subtractive data set. as depicted in fig d, statistical analysis of these data indicated that several of the regions with substantial depth-ofcoverage were statistically enriched (as determined by z-score) relative to other sequence clusters. collectively, these data indicate that the intracellular c:r interactions occur at discrete interaction sites on the viral genome and that the c:r interaction sites found within the structural orf are the most significantly enriched. for reference the graphs in panels b through d are aligned with this schematic. b) coverage of anti-capsid clip-seq libraries derived from purified, mature infectious viral particles. depth of coverage is represented by the y-axis, with the xaxis representing nucleotide position. c) depth of coverage for anti-capsid libraries derived from cytoplasmic fractions. plotted on the y-axis is the depth of coverage of the anti-capsid libraries following subtractive analysis, with the x-axis representing nucleotide position. d) statistical analysis of the data presented in panel c, with the y-axis indicating the zscore of all represented sequences. the x-axis represents the nucleotide position, with all panels above being aligned with the indicated nucleotide numbering intervals. as described earlier, several of the intracellular c:r interaction sites were statistically enriched relative to others. these sites, henceforth referred to as nt , nt , and nt based on their approximate nucleotide locations in the viral genome, were prioritized for further characterization. initially, we sought to determine if the intracellular c:r interaction sites exhibited similar rna secondary structures, or primary sequence motifs. bioinformatic analysis of the nt , nt , and the nt regions (including up to flanking nucleotides) using mfold failed to identify any rna secondary structures that were energetically favorable [ ] . similarly, analysis of the nt , nt , and nt c:r interaction sites did not reveal primary sequence motifs, and did not overlap with known subgenomic promoter elements [ ] . nonetheless, bioinformatic analysis of the individual intracellular c:r interaction sites indicates that the regions identified as highly enriched c:r interaction sites are well conserved across sinv strains. as shown in fig a, the prevalence of single nucleotide polymorphisms, as determined by the sequence alignment of independent sinv strains (supporting information s table) , indicated that the regions of interest exhibited few snps. numerous residues within the c:r interaction sites were absolutely conserved; furthermore, the overall incidence of snps was on average comparable to or below neighboring sequences that were not identified as c:r interaction sites. mutation of the candidate interaction sites reduced capsid binding confirming the relevance of the rna sequence for the interaction with capsid. analysis of the individual c:r interaction site mutants by quantitative immunoprecipitation following cross-linking indicated that mutation of each of the candidate c:r interaction sites reduced capsid binding. as shown in fig c, the retention of the individual c:r interaction sites following capsid immunoprecipitation was significantly reduced relative to parental virus for each of the c:r interaction sites. these data support the identification of the c:r interaction sites as bona fide capsid: rna interaction sites and indicate that the mutational approach is capable of reducing capsid interaction at these specific sites. following the identification of the cytoplasmic sinv c:r interaction sites we next sought to determine their biological importance to viral infection. to this end we developed a series of c:r interaction site mutants. due to the c:r interaction sites being present in open reading frames it was necessary to maintain amino acid identity during mutation. therefore, mutations were limited to substitutions of the wobble-base position of each codon ( fig b) . importantly, the mutants were designed to maintain codon usage rarity to prevent changes in translational efficiency. in some instances no alternative codons were available (such as for methionine), and as a result the corresponding codons were not altered. the one-step growth kinetics of the c:r interaction site mutants were characterized in mammalian tissue culture cells. as shown in fig a, mutation of each of the individual sinv c:r interaction sites significantly reduced the number of infectious particles produced during infection. on average, a reduction of~ -fold in the yields of infectious virus at hpi was detected for the c:r nt , nt , and nt , compared with wildtype sinv. since a potential consequence of disrupting the interactions between the viral capsid protein and the genomic rna could be a reduced production of viral particles, we next sought to determine if particle production was negatively affected in c:r interaction mutant viruses. interestingly, as shown in fig b, the total number of viral particles produced (as measured by genome equivalents per unit volume) by both wildtype sinv and c:r interaction site mutants were numerically, and statistically, equivalent. therefore, the production of viral particles was not perturbed by mutation of the c:r interaction site mutations. however, the capacity of the fig . the right y-axis indicates the prevalence of single nucleotide polymorphisms (snps) across a curated set of sinv genomic rnas; higher values indicate increased base identity variation at a particular nucleotide but are not informative as to relative base conservation. nucleotide position is reported on the x-axis. b) the individual nucleotide sequences of the c:r interaction sites are described in regards to parental sequence (top) and mutant sequence (bottom). the mutated nucleotides are highlighted in red. c) quantitative analysis of the individual c:r interaction sites following mutation indicates that capsid:rna binding is significantly reduced relative to parental virus. data shown is the mean of three independent biological replicates, with the error bar representing the standard deviation of the mean. to extend our understanding of the molecular impact of c:r interaction site mutation we assayed viral rna synthesis and viral gene expression. as demonstrated in fig a mutation of the c:r interaction sites did not significantly alter either the accumulation, or relative ratios of the individual sinv rna species. for each of the c:r interaction site mutants, the accumulation of the minus-strand rna was slightly increased relative to wildtype levels. while this was consistent over multiple biological replicates, the observed differences in minus-strand accumulation are relatively minor. analysis of viral gene expression by metabolic labeling of hek cells at and hours post infection indicates that viral gene expression is similarly unperturbed by c:r interaction site mutation ( fig b) . additionally, host cell shutoff, as indicated by the relative intensity of the actin band, is equivalent at and hpi for the c:r interaction site mutants and wildtype sinv at equal moi in terms of infectious units per cell. the data reported above indicates that the c:r interaction sites identified by clip-seq analysis of cytoplasmic complexes are not directly involved with nucleocapsid assembly and particle release. moreover, the lack of an apparent defect in viral rna synthesis or structural gene expression indicates that the sinv gene products are functioning normally, and that the c:r interaction site mutants are, late during infection, equivalent to wildtype virus. however the infectivity of sinv c:r interaction site mutants is significantly diminished relative to parental virus. hence, these data suggest that mutation of the c:r interaction sites negatively affects an early event of the viral lifecycle prior to viral rna synthesis. the data acquired indicted that while the c:r mutants did not inhibit particle production the infectivity of the particles was decreased. however, once infection was established the synthesis of viral rnas and viral gene expression was unaffected. these data implied that the c:r mutations disrupted particle infectivity at an early stage of infection and that the cytoplasmic c:r interactions were less essential at later stages of infection. previously, we demonstrated that alphaviral infectivity is largely determined by the functional capacity and stability of the genomic rna immediately following viral entry [ ] . on this basis we determined the genomic rna half-lives for each of the individual c:r interaction site mutants at very early times postinfection during a synchronized infection of hek cells. to this end, hek cells cultured in the presence of the uridine analogue -thiouridine ( su) were infected at an moi of infectious units per cell at ˚c to allow for viral adsorption but not entry. after the initial adsorption period, the cell monolayers were extensively washed to remove unbound particles and pre-warmed media containing su was added to release the block to viral entry. at the indicated times post-infection the total cellular rna was harvested and the incoming genomic rnas purified from the nascent transcribed rnas. the viral genomic rnas were then quantified using qrt-pcr as described in the materials and methods. as shown in fig a, the stability of the c:r interaction site mutant viral rnas was decreased in hek cells. b) quantitative analysis of the sinv c:r interaction site mutants and parental wild type virus in regards to the number of infectious units (left y-axis), and the total number of viral particles produced as measured by qrt-pcr (right y-axis). c) the infectivity of the individual sinv c:r interaction site mutants and parental wild type virus as reported as the ratio of total particles per infectious unit as determined using bhk- cells. all quantitative data in this figure represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean. statistical significance, as indicated on the individual panels above, are the p-values obtained from student's t-test. compared with the stability of wildtype sinv rna. importantly, the rna half-life observed during these studies is very similar to that previously reported [ ] . however, and interestingly, while wildtype sinv exhibits a steady monophasic decay profile, the individual c:r interaction site mutants exhibit multi-phasic decay, with an initial period of notable instability followed by a prolonged period of stability. calculation of the individual half-lives for each of the viruses used in this study indicates that mutation of c:r interaction sites destabilizes the incoming viral genomic rnas significantly, decreasing the mean half-life by -fold on average ( fig b) . collectively, these data indicate that mutation of the individual c:r interaction sites results in destabilization of the incoming genomic rnas early during infection. since efficient function of the incoming viral genomic rna is essential to the establishment of viral infection, it is, a priori, understandable that failure of the genomic rna early in infection, such as rna instability, would result in reduced viral infectivity. aside from persisting in the host cytoplasm, another important molecular function of the incoming genomic rna is to act as an mrna for the synthesis of the viral replication machinery. moreover, since the translational capacity of a transcript often correlates with the relative stability of an rna it is likely that the earliest translation events of c:r interaction site mutants are also perturbed [ ] . as such, we next sought to determine if the translational activity of the incoming viral genomic rnas differed amongst wild type and c:r interaction site mutant viruses. to this end we utilized a reporter sinv strain that expresses nanoluciferase internal to the nsp protein. this construct enables highly sensitive detection of gene expression early during infection, similar to previously described. to assess the translational capacity of the c:r interaction site mutants we developed a series of individual c:r interaction site mutants in the nsp nanoluciferase reporter backbone. unfortunately, despite many attempts we were unable to recover sinv nsp .nanoluc nt mutant virus as the resulting mutant was so severely attenuated. the translational activity of the parental wild type sinv nsp nanoluciferase construct and the nt and nt c:r interaction site mutants was assessed in hek cells. briefly, hek cells were infected at an moi of infectious units per cell. after the removal of unbound viral particles the cells were harvested at min, hr, and hrs post infection and assayed for nanoluciferase activity. as shown in fig c , wild type parental virus exhibited steady expression during early infection. however, both nt and nt exhibited significantly reduced translational activity early during infection. indeed, at -minutes post infection the normalized nanoluciferase activity was reduced . -fold and . -fold for the nt and nt mutants, respectively. at -hour post-infection the nanoluciferase activity of the nt mutant was further reduced, approximately -fold relative to wild type levels. however, at the same time point, the nt mutant exhibited an increase in nanoluciferase activity to more or less wild type levels, indicating that the defect imparted by the nt mutant is short lived. moreover, the nanoluciferase expression levels at two-hours post-infection for the nt mutant are~ -fold less than that of wild type sinv; however, the nt mutant has surpassed that observed for wild type sinv by approximately . -fold. to ensure that the mutations had not compromised the inherent translatability of the genomic rnas we checked translation using an in vitro system (supporting information, s fig). no differences in translation from the mutant and wt genomic rnas were observed in vitro. interaction site mutants and parental wild type virus were determined by qrt-pcr analysis as described in the materials and methods. plotted is the relative abundance of the incoming viral genomic rnas (y-axis) with regards to time (x-axis). regression analysis was utilized to determine the rna decay profile (as shown with the solid line) and the dashed lines represent the % confidence intervals of the aforementioned regression. b) the half-lives of the individual genomic rnas as determined using the calculations reported in dolken et al., as determined by the first point at which the relative abundance has reached . . c) the levels of nanoluciferase activity for wild type parental virus and the nt and nt c:r interaction site mutants were determined as reported in the materials and methods at the indicated times post infection. all quantitative data in this figure represents the mean of at least three independent biological replicates. comparative analysis was performed using variable bootstrapping, as described in the materials and methods, with the error bar representing the standard deviation of the mean. statistical significance, as indicated on the individual panels above, are the p-values obtained from student's t-test. https://doi.org/ . /journal.ppat. .g together, these data support the conclusion that the c:r interaction sites are important to early genomic rna function during viral infection. indeed, mutation of the individual c:r interaction sites reduces the rna stability of the incoming genomic rna; and, at least for nt and nt , reduces or delays the translational activity early during infection. however, the deficit created by the mutation of the c:r interaction sites appears to be specific to the early stages of the viral molecular lifecycle. this notion is supported by the observation that, at late stages of infection, the steady state levels of the three viral rna species are unperturbed, and the viral gene expression profiles are more or less equivalent. therefore, we posit that the c:r interactions are critical for molecular events at the earliest stages of viral infection, and that the c:r interaction sites represent a means by which the incoming viral genomic rna is stabilized, and perhaps licensed for translation. once this initial critical event is surpassed, the biological role of the c:r interaction sites become less essential for reasons currently unclear. regardless, diminished viral genomic rna function early during the viral lifecycle appears to significantly impact the progression of infection [ , ] . previously, we demonstrated that the rapid rna decay of alphaviral genomic rnas correlated with an increased elicitation of a type-i ifn response [ ] . to determine if the sinv c:r interaction site mutants induced a more robust ifn response we quantified the soluble type-i ifn produced during infection using a tissue culture model [ , , ] . as reported in fig a , the individual c:r interaction site mutants produced on average -fold more soluble type-i ifn relative to wildtype sinv infection. the induction of a robust ifn response is undoubtedly a significant consequence of the mutation of the cytoplasmic c:r interaction sites. indeed, the instability of the viral rna and apparent reduction of translational capacity early during infection likely contributes to the inability of the virus to successfully limit the induction of a soluble type-i ifn response. together, these findings indicate that the c:r interaction site mutants are liable to be highly restricted in ifn competent systems [ , [ ] [ ] [ ] [ ] [ ] [ ] . interactions between sindbis virus capsid protein and cytoplasmic vrna neurotropic sindbis virus is significantly attenuated in a mouse model the ifn findings described above suggested that c:r interaction site mutants would be attenuated, at least in regards to replication, in ifn competent models of infection, including immunocompetent wt mice. to test this hypothesis, we characterized the sinv c:r interaction site mutant viruses in a mouse model of infection. as shown in fig , wt c bl/ mice infected interactions between sindbis virus capsid protein and cytoplasmic vrna with parental wild type ar sinv, which uniquely among sinv strains remains neurovirulent in adult wt mice [ ] , displayed significant mortality and weight loss, with a median time to death of~ . days post infection (fig a and b ). in contrast, the nt c:r interaction site mutant virus was significantly attenuated relative to wild type infected mice, with only a fraction of the animals infected with nt mutant virus succumbing to disease. analysis of sinv.nt infected mice indicated that the disease associated with infection was mild compared with wild type ar -infected mice as indicated by the timing and severity of weight loss. similarly, but to a much greater extent, the sinv.nt mutant virus was also attenuated in wild type mice as none of the sinv.nt infected animals succumbed to infection, or demonstrated overt signs of disease as indicated by the absence of weight loss. it is important to note that these animals were productively infected with sinv, as infectious units were recovered from central nervous system tissue. as shown in fig c, the viral load detected in the brains of sinv.nt infected mice was decreased greater than -fold relative to wild type ar infected mice. in contrast to sinv.nt and sinv.nt , mice experimentally infected with sinv. nt exhibited mortality comparable to wild type ar -infected animals; however, disease progression was delayed with a median time to death of~ days post infection ( fig a) . indeed, as indicated by animal weight loss and health monitoring, the disease progression of sinv.nt was distinct from that observed in mice infected with wild type ar as sinv. nt infected animals exhibited delayed weight loss and experienced severe rapid onset paralysis at day , requiring the animals to be humanely euthanized (fig b) . collectively, these data suggest that the sinv c:r interaction sites identified via clip-seq are biologically important to infection in tissue culture models of infection and in vivo. given the behavior of the sinv c:r interaction site mutants in tissue culture cells, diminished viral replication in an in vivo model was anticipated. the obvious difference in disease phenotype for the and c:r mutants versus the c:r mutant indicates there is an unappreciated degree of complexity in the regulation of viral processes associated with the c:r interaction. the alphaviral capsid protein is a~ kda protein which surrounds the viral genomic rna in viral particles and in the cytoplasm in the form of nucleocytoplasmic cores. architecturally, the alphaviral capsid protein is globular in nature with an n-terminal domain that is implicated in rna binding and dimerization [ , ] . the alphavirus capsid protein is expressed as part of the structural polyprotein, which due to the serine-like protease activity of the c-terminal domain of the capsid protein, is autoproteolytically processed into free capsid protein [ ] . in addition to its well-known roles in particle assembly, the alphavirus capsid of new world alphaviruses, in particular venezuelan equine encephalitis virus (vee), is also directly involved in the shutoff of host macromolecular synthesis via the interruption of nuclear export [ , ] . nonetheless, a larger role for the alphavirus capsid protein in the regulation of alphaviral infection has not been previously described; and, hence, the observations described in this report represent a novel contribution of the c:r interaction during alphaviral infection and pathogenesis. positive-sense rna virus capsid proteins: more than just packaging in many positive-sense rna viruses the viral capsid proteins serve additional roles for the viral capsid protein / rna interactions outside of the context of particle assembly. these functions include the regulation of viral translation and rna synthesis. for instance, the ms coat protein dimer binds to the viral genomic rna of the ms bacteriophage to regulate the expression of the viral rna-dependent rna polymerase [ ] [ ] [ ] . similarly, the core protein of hepatitis c virus (hcv) binds to the ' ires during infection where it modulates the level of translation in a seemingly concentration dependent context [ ] [ ] [ ] [ ] [ ] . furthermore, a similar mechanism of translational regulation has been reported for other members of the alphaviruslike superfamily, in particular members of the bromoviridae, including brome mosaic virus (bmv) [ , ] . however, unlike these previous reports where the capsid protein expressed during the course of infection serves to regulate viral gene expression, the observations reported here indicate that the incoming capsid protein's association with the sinv genomic rna is necessary for viral rna translation early during infection. therefore, this phenomenon appears to be more similar to that previously described for alfalfa mosaic virus (amv), where the association of the amv capsid protein with distinct elements of the 'utr is required for rna function [ ] [ ] [ ] . nonetheless, in contrast to amv, the regulatory binding sites for sinv are found within the structural coding region of the viral rnas. it is worth mentioning that this orf, in the context of the genomic rna, is indeed part of a large non-translated region. examples of capsid protein / rna interactions that regulate rna synthesis can readily be found in bmv, amv, and with members of the coronaviridae. binding of the capsid proteins of amv and bmv to rna regulatory elements have been implicated in the regulation of viral rna synthesis and promoter recognition [ , ] . additionally, members of the coronaviridae require functional n protein, likely via the association of the highly charged n-terminal domain with the transcription-regulating sequences, to achieve efficient viral replication [ ] [ ] [ ] [ ] . nonetheless, from the data presented here we are unable to assign a role for an alphaviral c:r interaction in regards to viral rna synthesis. for, as reported above, the accumulations of the viral rna species is similar between the individual c:r interaction mutants and wild type parental virus. this implies that rna synthesis / promoter utilization is not negatively affected. however, it remains possible that degeneracy between the individual c:r interaction sites is able to compensate for this activity. unfortunately, the development of combined c:r mutants has, so far, been unsuccessful. several studies have described interactions between the alphaviral capsid protein and host ribosomal rnas. indeed, co-sedimentation studies indicated that the host s rrnas interact with viral capsid proteins in vertebrate cells during infection [ , ] . this interaction has been proposed to be a leading mechanism in particle disassembly [ ] . nonetheless, these interactions have not been exhaustively characterized and further examination is needed to complete the mechanistic understanding of alphaviral nucleocapsid disassembly. collectively, the observations reported here are indicative of a novel role for the c:r interactions in regard to the function of the sinv genomic rna early during viral infection. as demonstrated by the data in this report, mutation of the individual candidate c:r interaction sites identified via clip-seq screen significantly reduced the function of the genomic rna early during infection. a key novel observation of these studies is that the mutation of the c:r interaction sites reduced the rna stability of the incoming viral genomic rna. a role for a viral capsid protein in the stabilization of viral rnas, to our knowledge, has not yet been described for positive sense rna viruses. the data above leads to the conclusion that the interaction sites of the sinv capsid protein with the viral genomic rna identified in this study by clip-seq, while not required for rna function late during infection, are vital to early genomic rna stability, and function. moreover, mutation of the c:r interaction site diminished translation of the viral genomic rna early during infection, likely resulting in increased type-i ifn production. currently the precise mechanism of c:r-mediated regulation is unclear; however, the c:r interaction sites are clearly involved in the stabilization of the incoming viral genomic rna and the regulation of early viral translation. we suggest a model in which capsid protein of incoming nucleocapsid complexes remains associated or re-associates with specific sites in the genome following nucleocapsid disassembly enhancing rna stability and facilitating translation (fig ) . this is a working model, whether the effects on rna function are due only to capsid binding, or could be affected by the binding of other rna binding proteins at proximal or overlapping sites is unknown. ongoing studies in the sokoloski and hardy laboratories have indicated that the c: r interaction sites may be common sites of virus and host protein binding. the precise role(s) and contributions of these interactions are currently being characterized. further potential mechanisms include direct or indirect roles in the recruitment of the translational machinery, or beneficial host rna-binding proteins to the viral rna; or perhaps the evasion of toll-like receptors and rna-helicase sensors such as rig-i and mda , or the eluding of the host cellular rna surveillance machinery such as the host nonsense-mediated rna decay pathway. while our data cannot completely rule out this possibility, we do not believe that the decreased rna stability observed in the c:r interaction site mutants is due to nonspecific recruitment of cellular endonucleases (for instance rnase l), as mutation of off-target sites does not result in an altered phenotype ( supplementary information, s fig) . whereas, the mutation of the c:r interaction sites reduced viral growth kinetics, likely as a result of decreased infectivity due to impaired genomic rna function early during infection. in addition to their molecular role(s), the c:r interaction sites represent a set of previously unidentified pathogenicity determinants in that mutation of the c:r interaction sites significantly attenuates viral infection in vitro and in vivo. as shown in this study mutation of the sinv c:r nt and nt interaction sites reduced the viral load, pathology, and morbidity of a neurotropic sinv infection. these findings have great potential as a means by which viruses may be attenuated for the purpose of rational vaccine development. given that c:r interaction site mutation limits viral rna function early during infection, but not late during infection, implies that viral dissemination but not immunogenicity would be limited. however, further exploration is necessary, as not all c: r interaction sites resulted in complete attenuation; as the sinv.nt mutant exhibited significant mortality and morbidity in experimentally infected mice. currently, the precise mechanism behind this phenotype is unclear. one possibility being investigated is that this site is also bound by a host factor and this contributes to the phenotype observed with this particular mutant. it should be noted that while the c:r interaction at obviously is important in maintaining genome stability following infection the site may also play a role in the function of the viral subgenomic mrna that this may be affected by the binding of a host factor. several studies have indicated that the association of the alphaviral capsid protein with viral rna is nonspecific in regards to primary nucleotide sequence and secondary structure, and is likely driven by electrostatic interactions between the n-terminus of capsid and the phosphodiester rna backbone [ ] [ ] [ ] [ ] [ ] . while these studies primarily focused on the interactions of nucleic acids and the viral capsid proteins in vitro, it is highly likely that these observations are valid during bona fide infections as evidenced by the distributive nature of the capsid:rna interactions observed in particles. however, collectively the data presented in this report indicate that the interactions, or at least the strength thereof, between the sinv capsid protein and the genomic rna may be context dependent. the disseminated pattern of binding of the viral genomic rna to capsid observed in purified viral particles supports a model where the c:r interactions are largely nonspecific, and perhaps based on charge-charge interactions [ , , ] . however, from this data set it is impossible to identify if the interactions between the capsid protein and cargo rna are mutually saturating (where each capsid monomer interacts with~ nt of genomic rna), or unsaturated (where not every nt is associated with capsid monomer) but nonspecifically distributed along the length of the genome. further examination of the pattern of binding observed in purified viral particles reveals an interesting phenomenon-there are two regions of the sinv genome where sequence coverage is greatly decreased. these regions roughly correspond to the previously described packaging signal, and the subgenomic promoter region [ , , ] . importantly, the decreased coverage of these regions cannot be simply explained by bias in the library generation (as is the case for the extreme ' and ' termini of the genome). in these instances, the decrease in coverage was due to genomic rna having a ' cap and ' poly(a) tail, which effectively prevented the linkage of the ' and ' adaptors as these rna features were not modified prior to library construction. nonetheless, decreased sequence coverage at the packaging signal could be potentially explained due to the presence of a high degree of secondary structure within the element. the nuclease fragmentation approach utilized in these studies is selective for single-stranded rna, therefore regions with a high degree of dsrna may be excluded from nuclease digestion. the net result would be the formation of rna fragments that lay outside the region of interest / selection during the preparation of the cdna libraries. alternatively, if the capsid interaction is exceptionally avid at this location the fragmentation of the viral rna may be similarly diminished, resulting in decreased library coverage at the packaging signal. further analysis of the decreased coverage at the cis-acting sites is separate and ongoing research focus. examination of the interactions between the capsid protein and the viral genomic rna in a cytoplasmic context indicated that several discrete interaction sites were readily observable. however, from our data we cannot conclude as to the molecular context of these interactions, as the milieu of capsid interactions likely expands from monomers (or as some models suggest dimers) to complete intact nucleocytoplasmic cores. from our current data it is impossible to determine the molecular nature of the interaction in regards to protein:rna stoichiometry and molecular context. unfortunately, given the promiscuous interaction of alphaviral capsid proteins with nucleic acids (including dna) deciphering the supramolecular nature of the interaction through a reductionist in vitro approach presents significant challenges [ , , ] . moreover, it is unclear if these interactions are exclusive of other c:r binding events, or simply the cytoplasmic c:r interactions with high relative affinities. it should be noted that the method of cross-linking used in these studies is inefficient, which increases the specificity but reduces overall signal intensity during the clip-seq analyses; it is therefore likely that the observed cytoplasmic c:r interaction sites are exceptionally stable relative to other c:r interactions. tissue culture cells bhk- (gift from charles rice, rockefeller university), hek , and l cells (both cell lines were gifts from pranav danthi, indiana university) were cultured in minimal essential media (mem, cellgro) supplemented with % fetal bovine serum (fbs, atlanta biologicals), x antibiotic / antimycotic solution (cellgro), x nonessential amino acids (cellgro) and l-glutamine (cellgro). all cell lines utilized in this study were cultured at ˚in the presence of % co . wild type sinv toto , sinv p (a toto derived strain containing gfp in frame with nsp ), sinv ptoto -naluc (a toto derived strain containing nanoluciferase in frame with nsp ), wild type sinv ar , and all derivatives thereof (described below), were prepared by electroporation as previously described [ ] . briefly, ug of in vitro transcribed rna were electroporated into bhk- cells via a single pulse from a gene pulser xcell system (bio-rad) at . kv, ma and ohms. after the development of cytopathic effect (typically to -hours post-electroporation) the tissue culture supernatants were collected and clarified via centrifugation at , xg for minutes at ˚c. the resulting stocks were aliquoted and either used immediately or stored at - ˚c for later use. sinv particles were purified via a two-step process [ ] . first, supernatants from x hek cells infected with sinv toto at an moi of pfu/cell were harvested at -hours post infection. the viral particles were concentrated via pelleting through a % (m/ v) sucrose cushion prepared in hne buffer ( mm hepes [ph . ] / mm nacl / mm edta) by centrifugation at , xg for . hours in a ti rotor. second, the pelleted viral particles were then resuspended in hne buffer and applied to a % / % (m/v) sucrose step gradient prepared in hne. the sinv particles were then banded via centrifugation at , xg for . hours in an sw rotor at ˚c. the purified sinv particles were collected via needle aspiration, aliquoted and stored at - ˚c for later use. per clip-seq library, either purified sinv particles or infected tissue culture cells were crosslinked via exposure to shortwave ( nm) uv irradiation. briefly, approximately sinv toto particles in a volume of μl, or x hek cells infected with sinv toto at an moi of pfu/cell at hpi, were irradiated with x μjoules per square centimeter in a stratalinker. after cross-linking the rna-protein complexes were solubilized in ripa buffer ( mm tris [ph . ] / mm nacl / . % np- / . % sodium deoxycholate / . % sds) and frozen at - ˚c for later use. prior to immunoprecipitation the lysates were thawed on ice, vortexed and clarified via centrifugation at , xg for minutes at ˚c. the clarified supernatants (~ μl) were transferred to a fresh tube. the cross-linked lysates were then incubated with ul packed volume of protein g sepharose beads for minutes at ˚c prior to being clarified via centrifugation at , xg for five minutes. the resulting pre-blocked lysates were then immunoprecipitated using a polyclonal anti-capsid antibody, or control rabbit igg sera. the cross-linked rna:protein complexes were incubated in the presence of antibody at ˚c for a period of hours under constant agitation. after antibody binding, ul packed volume of protein g sepharose was added to each sample and further incubated for another hours. after resin binding the rnas were fragmented via the addition of rnase t (thermoscientific) to each immunoprecipitation. the fragmentation reactions were allowed to incubate for minutes at room temperature. after fragmentation, the immunoprecipitated complexes were purified via centrifugation at , xg for minutes and washed three times with ripa buffer and twice more with sterile xpbs. the resulting purified, fragmented rna: protein complexes were treated with proteinase k for minutes at ˚c to release the rna fragments from the immunoprecipitated complexes. the purified rna fragments were then extracted using trizol and resuspended in a minimal volume. the rna fragments were then used as the input materials for cdna library generation via the nebnext sequencing kit, as according to the manufacturer's instructions. the resulting cdna libraries were sequenced using the miseq platform. the specificity of the immunoprecipitation protocol described above was confirmed independently using small scale analytical replicates and metabolic labeling. hek cells were either mock treated, or infected with sinv at an moi of pfu/cell, and allowed to incubate for hours under normal conditions. thirty minutes prior to the labeling period the infected monolayers were washed twice with xpbs and the media was replaced with methionine / cysteine free dmem supplemented with % dialyzed fbs. after the depletion period the media was removed and replaced with methionine/cysteine free dmem supplemented with l-azido-homoalanine (l-aha) at a concentration of um. the cells were allowed to incubate under normal conditions for a period of two hours prior to removal of the culture media. prior to harvesting the monolayers were washed three times with xpbs. whole cell lysates were generated via the addition of ripa buffer. protein concentration was quantified via bradford assay and equivalent amounts of protein were precipitated via methanol / chloroform treatment. the precipitated proteins were resuspended in xpbs containing um dibo-alexafluor and incubated for hour at room temperature while shielded from light. after the labeling period, the samples were diluted in x volumes of ripa buffer and immunoprecipitated identically to that described for the clip-seq experiments. these treatments only differed on the basis of scale-as the total materials used were reduced -fold relative to those used for library generation. after immunoprecipitation x laemmli buffer was added to a final concentration of x and the samples analyzed via sds-page. protein species were detected via a pharos molecular imager using the appropriate excitation and emission detection settings. as shown in supporting information (s fig) , the anti-capsid sera specifically immunoprecipitated the sinv capsid protein from infected extracts. in addition, no other host proteins were enriched during immunoprecipitation with anti-capsid sera in either infected or mock infected lysates. furthermore, the nonspecific antibody control did not enrich for any host, or viral, protein. collectively these data are indicative of the specificity and purity of the immunoprecipitations used for cdna library generation. the cdna libraries were trimmed to remove indices and adaptors prior to alignment to a reference genome consisting of the parental toto strain of sinv. alignments were performed using lastz with standard parameters. only reads corresponding to the positive-sense genomic rna were mapped to the reference genome sequence. sequence coverage was clustered at the nucleotide level of resolution from the aligned sequence reads. analysis of the clustered sequence data relied on a subtractive method whereby the sequence coverage of capsidspecific libraries was compared to nonspecific control libraries. for these analyses the coverage of both the capsid and nonspecific control libraries were normalized internally by dividing each base by the total number of represented nucleotides to gain a percentage which was then multiplied by an arbitrary amount to generate a standardized measure of each nucleotides relative representation amongst independent library sets. the resulting values from the control libraries were averaged and subtracted from the capsid-specific libraries to generate a difference map (as shown in fig c) . z-scores for each individual nucleotide were calculated from the statistical mean and standard deviation from the subtractive analysis data sets. for these studies statistical significance was rigorously defined as nucleotide clusters with differential coverage values greater than standard deviations from the mean, which correlates to a zscore of~ − . the sequencing data directly relevant to the studies described herein are available accompanying this document as s data. this supplemental information also includes a statistical summary of the entire rna sequencing dataset and analysis of read size for the regions of interest and a control region. the entire rna sequencing dataset has been deposited in the national center for biotechnology information (ncbi) gene expression omnibus, and can be accessed using the following url: https://urldefense.proofpoint.com/v /url?u= http- a__www.ncbi.nlm.nih.gov_geo_query_acc.cgi- facc- dgse &d=awieag&c= sgmrq dbjbgx e zsshgezx a iaf so aj bnrhlk&r=oeul-bhg_ jju s ftlcvxycn jpesq pjohuawmnty&m=s zyvrm qbr me xeyir dqzkoyopa rz m xm q&s=m w fvnsvbtk bssvmljiua pxourfnbzukiy xy jg&e= following the identification and prioritization of the nt , nt , and nt c:r interaction sites mutant sinv strains were developed using the q site directed mutagenesis kit (neb). briefly, pcr amplification of the parental toto , or ar , infectious cdna clones were performed using the sequences indicated in fig . q reaction products were confirmed visually using standard agarose electrophoresis and diagnostically digested to confirm product size. the individual c:r interaction site mutant sinvs were completely sequenced to confirm the presence of the mutation and sequence relative to wild type virus prior to being utilized as templates for the production of infectious virus as described above. the quantitative assessment of the capsid:rna interactions of the individual c:r interaction sites for parental and c:r interaction site mutants utilized small scale extracts generated identically to that described above for the clip-seq process. briefly, hek cells were infected at an moi of prior to cross-linking via uv irradiation. after the preparation of whole cell lysates, the capsid:rna complexes were immunoprecipitated and the bound rnas fragmented with rnase t . the immunoprecipitates were washed extensively and the retained rna fragments were release via proteinase k digestion. the rna fragments were extracted via trizol according to the manufacturer's instructions. the precipitated rnas and paired input controls, were used as the input materials for reverse transcription using random hexamer. the resulting cdnas were assessed quantitatively via qrt-pcr as described below using the following primer pairs: sinv.nt f '-gcaccgccatcaagcaatgtgtggc- '; sinv.nt r '-caatttc ccttgggccgtgtggtcg- '; sinv.nt f '-tgttccaaatgtgccacagataccg- '; sinv.nt r '-aatgt actcttggttggtggaaggc- '; sinv.nt f '-cagcagattgcgcgtctgaccatgc- '; sinv.nt r '-gactcc gttcacgtacacatctagg- '. the viral growth kinetic assays performed in these studies were essentially as previously described [ ] . briefly, hek cells were infected with either wildtype or c:r interaction site mutant viruses at a multiplicity of infection of pfu/cell. the infected monolayers were washed with xpbs to remove unbound virus and fresh whole media was added. at regular times post infection the tissue culture supernatant was harvested and replaced with fresh growth media. all samples were frozen at - ˚c until completion of the time course. viral titers were determined using the standard plaque assay protocols involving bhk- cells and a % agarose overlay. plaque assays were allowed to incubate until plaques were readily visible prior to being fixed with . % formaldehyde and stained with crystal violet. at the indicated times post infection, infected hek cells were washed twice with xpbs and harvested into trizol. total rna was extracted according to the manufacturer's directions, with carrier glycogen being added to the precipitations. equal amounts of total rna ( . μg) were used as template for the synthesis of cdna and assessed quantitatively via qrt-pcr using the method and oligonucleotide primers previously described [ , , ] . briefly, the positive and negative sense viral rnas were selectively reverse transcribed using specific rt primers. the absolute quantities of the genomic and total positive sense viral rnas (consisting of the genomic and subgenomic rnas) were determined using paired standard curves; and the absolute quantity of the subgenomic rna itself was determined via subtraction of the number of genomes from the total positive sense rnas. the minus strand was detected in isolation from the positive sense rnas. all qrt-pcr reactions were normalized to the level of s rrna present as previously described [ , , ] . to determine the relative infectivity of each sinv c:r interaction site mutant the number of total viral particles per unit volume, as measured by the genome equivalents per ml, was determined using qrt-pcr, as previously described [ ] . briefly, the viral genomic rnas from bhk- tissue culture cell supernatants were utilized as the source template for cdna synthesis. the resulting cdnas were then assessed using qrt-pcr and the absolute quantity of viral particles per unit volume determined using standard curve analysis. the number of infectious units was determined using standard plaque assay on bhk- cells, and the infectivity was determined by comparing the total number of particles per infectious unit. the quantitative analyses of viral gene expression described in these studies was performed via one of two methods-gross viral and cellular gene expression was determined via metabolic labeling of infected cells [ , ] . briefly, hek cells were infected with sinv at an moi of pfu/cell and allowed to incubate for the indicated times under normal conditions. thirty minutes prior to the labeling period the infected monolayers were washed twice with xpbs and the media was replaced with methionine / cysteine free dmem supplemented with % dialyzed fbs. after the depletion period the media was removed and replaced with methionine/cysteine free dmem supplemented with s-labeled methionine and cysteine at a specific activity of μci/ml. the cells were further incubated under the labeling conditions for the indicated time periods. at the end of the labeling period the cells were washed three times with xpbs prior to lysis in ripa buffer. equal volumes of cell lysates were analyzed by sds-page and radiolabeled proteins were detected using standard phosphorimaging practices. quantitative analysis of genomic rna gene expression early during infection was performed using a process previously described, with one notable exception [ , ] . specifically, the reporter construct, and hence detection system used, consisted of an in-frame fusion of the nanoluc reporter gene with nsp at a position identical to that of ptoto -fluc [ , ] . nanoluciferase signal was assayed according to the manufacturer's instructions. all analysis methods were identical to that previously described [ ] . viral rna half-lives were assessed as previously described [ ] . hek cells, cultured in media supplemented with um -thio uridine (sigma), were infected with either parental sinv toto or the individual sinv c:r interaction site mutant sinvs at an moi of pfu per cell. at the indicated times post infection the tissue culture supernatant was removed and the cell monolayers were washed three times with pbs prior to trizol (invitrogen) extraction. a total of μg of total rna was biotinylated using hpdp-biotin (pierce). the biotinylated rnas were bound to ultralink streptavidin resin (pierce) to remove newly transcribed viral rnas. the unbound rnas were then phenol extracted and ethanol precipitated prior to use as a template for cdna synthesis via reverse transcription using random hexamer. the resulting cdnas were assayed via qrt-pcr to determine the relative abundances of the incoming sinv genomic rnas normalized to the cellular s rrna. rna half-lives were calculated as reported in dolken et al from data pertaining to the initial phase of decay [ ] . the production of soluble type-i ifn was assayed as previously described [ ] . briefly, the ifn competent l cell line was infected with the c:r interaction site mutant derivatives or parental wildtype sinv ar at an moi of pfu/cell for hour at room temperature. prior to further incubation the infected tissue culture cells were washed three times with xpbs and fresh media was added. the tissue culture supernatants were harvested hpi and clarified via centrifugation. infectious viral particles in the supernatant were inactivated via acidification and uv irradiation. the inactivation of samples was confirmed via standard plaque assays. the inactivated supernatants were then serially diluted and tittered onto fresh l cells in a -well format. after a period of hours of treatment the media was removed and the cells were challenged with a fluorescent chikungunya mcherry reporter strain at an moi of pfu/cell. viral gene expression was detected at hpi via a typhoon phosphorimager and quantified via densitometry. after hpi the cells were fixed and assayed for cell death via crystal violet staining; the formation of cpe was highly consistent with viral mcherry expression. relative ifn production was calculated as a function of the dilution needed to attain a % reduction in viral gene expression. four-week old wt c bl/ j mice were obtained from the jackson laboratory. mice were inoculated in the left rear footpad with virus in diluent (phosphate-buffered saline [pbs] supplemented with % fbs) in a volume of μl. on the termination day of each experiment, mice were sedated with isoflurane and euthanized by thoracotomy, blood was collected, and mice were perfused extensively by intracardiac injection of pbs. serum was obtained by collecting blood in serum separator tubes (bd). pbs-perfused tissues were removed by dissection, placed into pbs- % fbs and homogenized using a magna lyser (roche). the amounts of infectious virus in tissues were quantified by standard plaque assays using bhk- cells. this study was conducted in accordance with the recommendations in the guide for the care and use of laboratory animals and the avma guidelines for the euthanasia of animals. all animal experiments were performed with the approval of the institutional animal care and use committee at the university of colorado school of medicine (assurance number: a - ) under protocol b- ( ) e. experimental animals were humanely euthanized at defined endpoints by exposure to isoflurane vapors followed by thoracotomy. unless otherwise noted, the quantitative data reported in this study are the means of a minimum of three independent biological replicates. where appropriate, the statistical analysis of comparative samples was performed using variable bootstrapping, as previously described [ ] . the error bars indicate the standard deviation from the mean. when indicated, the p-values associated with individual data sets are the result of the student's t-test for the corresponding data. supporting information s table. sinv genomes used for single nucleotide polymorphism analysis. (pdf) s dataset. sequence data used for clip-seq analyses. statistical summary of sequencing data. sinv-specific sequence reads. read size analysis across the identified capsid interaction sites and a control region in the nsp coding sequence. qc was performed using trimmomatic. quality scores were averaged over a sliding window of nucleotides and nucleotides with values less than were removed. (xlsx) s fig. the immunoprecipitation of sinv capsid protein is specific. a) metabolically labeled mock control and infected hek cells cell lysates were immunoprecipitated with either non-specific control sera or anti-capsid sera using conditions identical to those used for the preparation of the cdna libraries used in clip-seq. the input lane represents / th of the starting material for the ip. data shown is representative of several biological replicates. b) sinv infected hek cells were either mock-or uv-crosslinked via shortwave uv irradiation at hpi. the cell monolayers were harvested via gentle scraping and solubilized in ripa buffer to form whole cell lysates. the cell lysates were then precipitated with antibodies specific for either sinv capsid, or control rabbit igg, as indicated on the figure. all purification conditions were identical to those described for the development of the clip-seq cdna libraries utilized in this study, with the only exception being that rna fragmentation was omitted. after purification, cdna was generated from the immunoprecipitated materials, and the presence of the nsp coding region was detected via rt-pcr and agarose gel electrophoresis. data shown is representative of three biological replicates. in vitro transcribed genomic rnas for parental sinv, and each of the individual c:r interaction site mutants were assessed for translation using rabbit reticulocyte extracts according to the manufacturer's directions. the amount of translation was detected using nanoluciferase detection. data shown is the mean of three independent biological replicates, with the error bar representing the standard deviation of the mean. (tif) the one-step growth kinetics of parental and a non c:r interaction site mutant as observed in hek cells. briefly, a region of the sinv genomic rna detected, but identified as statistically insignificant, by clip-seq analysis was mutated using identical parameters to the bona fide c:r interaction site mutants. this region corresponds to nt - of the viral genomic rna, which includes the genuine start site of nsp . the quantitative data in this figure represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean. nowcasting the spread of chikungunya virus in the americas centers for disease c, prevention. notes from the field: transmission of chikungunya virus in the continental united states-florida, . mmwr morbidity and mortality weekly report reemergence of an unusual disease: the chikungunya epidemic. seminars in pediatric infectious diseases morbidity and impaired quality of life months after chikungunya infection: comparative cohort of infected and uninfected french military policemen in reunion island epidemiology of chikungunya infection on reunion island, mayotte, and neighboring countries a major epidemic of chikungunya virus infection on reunion island post-epidemic chikungunya disease on reunion island: course of rheumatic manifestations and associated factors over a -month period clinical infectious diseases: an official publication of the infectious diseases society of america arrival of chikungunya virus in the new world: prospects for spread and impact on public health the alphaviruses: gene expression, replication, and evolution. microbiological reviews equine encephalitis in massachusetts medically important arboviruses of the united states and canada alphavirus rna synthesis and non-structural protein functions nucleocapsid and glycoprotein organization in an enveloped virus . a cryo-em structure of an enveloped alphavirus venezuelan equine encephalitis virus packaging signals in alphaviruses venezuelan equine encephalitis virus nsp protein regulates packaging of the viral genome into infectious virions conservation of a packaging signal and the viral genome rna packaging mechanism in alphavirus evolution encapsidation of hostderived factors correlates with enhanced infectivity of sindbis virus nucleotide-resolution profiling of rna recombination in the encapsidated genome of a eukaryotic rna virus by next-generation sequencing venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype the efficient packaging of venezuelan equine encephalitis virus-specific rnas into viral particles is determined by nsp - 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of the hepatitis c virus (hcv) core protein specifically inhibit hcv ires-dependent translation in hepg cells, and inhibit both hcv ires-and capdependent translation in huh and cv- cells. the journal of general virology down-regulation of the internal ribosome entry site (ires)-mediated translation of the hepatitis c virus: critical role of binding of the stem-loop iiid domain of ires and the viral core protein hepatitis c virus internal ribosome entry sitemediated translation is stimulated by cis-acting rna elements and trans-acting viral factors brome mosaic virus capsid protein regulates accumulation of viral replication proteins by binding to the replicase assembly rna element rna binding by the brome mosaic virus capsid protein and the regulation of viral rna accumulation specific rna binding by amino-terminal peptides of alfalfa mosaic virus coat protein. the embo journal coat protein binding sites on rna of alfalfa mosaic virus removal of the n-terminal part of alfalfa mosaic virus coat protein interferes with the specific binding to rna and genome activation rna-binding proteins that inhibit rna virus infection the nucleoprotein is required for efficient coronavirus genome replication selective replication of coronavirus genomes that express nucleocapsid protein coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes functional transcriptional regulatory sequence (trs) rna binding and helix destabilizing determinants of murine hepatitis virus (mhv) nucleocapsid (n) protein. the journal of biological chemistry role of ribosomes in semliki forest virus nucleocapsid uncoating semliki forest virus capsid protein associates with the s ribosomal subunit in infected cells promoter for sindbis virus rna-dependent subgenomic rna transcription sequence requirements for sindbis virus subgenomic mrna promoter function in cultured cells the ' untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and mammalian cells sindbis virus usurps the cellular hur protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells heterogeneous nuclear ribonuclear protein k interacts with sindbis virus nonstructural proteins and viral subgenomic mrna role for subgenomic mrna in host translation inhibition during sindbis virus infection of mammalian cells expression of the zinc-finger antiviral protein inhibits alphavirus replication fluorophore-nanoluc bret reporters enable sensitive in vivo optical imaging and flow cytometry for monitoring tumorigenesis high-resolution gene expression profiling for simultaneous kinetic parameter analysis of rna synthesis and decay sindbis virus infectivity improves during the course of infection in both mammalian and mosquito cells we would like to thank the members of the sokoloski, hardy, mukhopadhyay, and danthi labs for their input regarding the development of this project and preparation of this manuscript. key: cord- - rog s authors: hemida, maged gomaa; ye, xin; thair, simone; yang, decheng title: exploiting the therapeutic potential of micrornas in viral diseases: expectations and limitations date: - - journal: mol diagn ther doi: . /bf sha: doc_id: cord_uid: rog s new therapeutic approaches are urgently needed for serious diseases, including cancer, cardiovascular diseases, viral infections, and others. a recent direction in drug development is the utilization of nucleic acidbased therapeutic molecules, such as antisense oligonucleotides, ribozymes, short interfering rna (sirna), and microrna (mirna). mirnas are endogenous, short, non-coding rna molecules. some viruses encode their own mirnas, which play pivotal roles in viral replication and immune evasion strategies. conversely, viruses that do not encode mirnas may manipulate host cell mirnas for the benefits of their replication. mirnas have therefore become attractive tools for the study of viral pathogenesis. lately, novel therapeutic strategies based on mirna technology for the treatment of viral diseases have been progressing rapidly. although this new generation of molecular therapy is promising, there are still several challenges to face, such as targeting delivery to specific tissues, avoiding off-target effects of mirnas, reducing the toxicity of the drugs, and overcoming mutations and drug resistance. in this article, we review the current knowledge of the role and therapeutic potential of mirnas in viral diseases, and discuss the limitations of these therapies, as well as strategies to overcome them to provide safe and effective clinical applications of these new therapeutics. therapeutic strategies based on mirna technology for the treatment of viral diseases have been progressing rapidly. although this new generation of molecular therapy is promising, there are still several challenges to face, such as targeting delivery to specific tissues, avoiding off-target effects of mirnas, reducing the toxicity of the drugs, and overcoming mutations and drug resistance. in this article, we review the current knowledge of the role and therapeutic potential of mirnas in viral diseases, and discuss the limitations of these therapies, as well as strategies to overcome them to provide safe and effective clinical applications of these new therapeutics. rna interference (rnai) is a system in living cells that regulates the activation and silencing of gene expression. rnai governs the regulation of host cell genes, mainly through two types of rna molecules: small interfering rna (sirna) and microrna (mirna). [ ] sirnas are a class of double-stranded rna molecules, - nucleotides in length, which play different roles in cellular biology. experimental application or targeting of sirnas in various cell types and animal models has shown promise for the potential treatment of diseases induced by viruses such as hepatitis c, influenza, and hiv. [ ] [ ] [ ] the suppression of viral gene expression by sirnas makes them very attractive for antiviral therapy, and some sirnas are already being used in clinical trials. [ ] however, sirnas still have a long way to go before being brought to market, because of their potential side effects. one of the major concerns in using sirnas as molecular therapeutics is their induction of a strong immune response. mirnas, which were discovered in , [ ] are a group of non-coding, single-stranded rna molecules, ranging in size from to nucleotides. [ ] it is now believed that mirnas compose one percent of the total human genome. mirnas are widely expressed in various species, including viruses. the first virally encoded mirna was discovered in the epstein barr virus (ebv) genome. [ ] now, there are more than identified mirnas encoded by viruses, with more expected to be identified in the near future as a result of the improvement in online prediction and validation tools. [ ] cellular mirnas in animals seem to be conserved, while virally encoded mirnas are highly variable even within the same group of viruses. [ ] this may be due to the high frequency of viral mutations relative to eukaryotes. mirnas play an important role in regulation of almost one-third of all known human mrnas. [ ] most mirnas have a specific tissue expression profile. their unique expression pattern may explain their roles in different biologic activities, such as cellular differentiation, environment adaptation, oncogenesis, and host-pathogen interaction. [ ] the therapeutic potential of mirnas was first realized with the discovery that downregulation of mir- and mir- is associated with development of b-cell leukemia. [ ] shortly after that, the potential for treatment of several other cancers was realized. [ ] scientists have aimed to control the expression level of key genes via manipulation of cellular or viral mirnas to treat disease. these approaches include anti-mirna oligonucleotides (amos), peptide nucleic acids, and mirna sponges. [ ] initial experiments have obtained promising results in controlling various viral infections. [ ] [ ] [ ] in addition, it has been found that restoring or over-expressing certain mirnas may also be beneficial for reverse pathologic conditions, especially in cancer treatments. [ ] the goal of this article is to review the recent progress in the understanding of the roles of mirnas in viral diseases, and to discuss the potential of these molecules in serving as a therapeutic target or as a useful therapeutic tool. we also specifically highlight the major obstacles faced by mirna technology in both therapeutics and vaccine strategies. viruses are obligate intracellular parasites. they lack the essential machinery required for their replication. thus, viruses adopt several clever strategies to ensure the success of their replication in a suitable host, one of which is manipulation of the host mirnas to modify the cellular environment for their own benefit. [ ] some dna viruses are capable of encoding their own mirnas to modulate both the viral and cellular protein expression in order to provide a favorable environment for viral replication. [ ] answering back, certain host mirnas alter the cell gene expression to defend the cells against the viral infection by interfering with viral proteins or other cellular factors as a type of immune response against these particular viruses. [ ] therefore, the relationship between viruses and mirnas is complicated, to say the least. since mirnas play essential roles in viral infections, they are considered to be promising therapeutic targets in infectious diseases. their endogenous nature, small size, and flexible function make mirnas very good candidates, as they may trigger lower immunogenic responses and have fewer side effects than sirnas. [ , ] in view of the current data regarding the roles of different viral and cellular mirnas in various viral replication cycles, we believe that manipulation of these mirnas will have a promising therapeutic role in infectious diseases. [ , , [ ] [ ] [ ] [ ] currently, there are more than mirnas encoded by the human genome alone. [ ] we will discuss the roles and therapeutic potential of cellular as well as viral mirnas (if any) in the pathogenesis and treatment of different viral diseases. human polyomaviruses (hpyvs) are a group of oncogenic, circular, non-enveloped, double-stranded dna (dsdna) viruses. [ ] five polyomaviruses have been found to infect humans. of particular interest are two strains of these viruses, named (according to the initials of the first affected patients) bk virus (bkv) and james canyon virus (jcv). [ ] the reservoir species for human infection is the rhesus macaque. humans have also acquired simian virus (sv ) infection from contaminated poliovirus vaccines, and recent studies have reported horizontal transmission between people. [ ] hpyvs induce a wide range of tumors affecting almost all body organs (including the brain, bones, colon, pancreas, stomach, and urogenital tract), as well as lymphomas and leukemia. [ , ] the viral genome of sv encodes five proteins; two large t antigen (lt), one small t antigen (st-ag), and three that encode the capsid proteins (vp , vp and vp ). hpyvs are able to encode viral mirnas for their own benefit. both bkv and jcv encode the same mirna, named mir-j . it is upregulated in the brain in progressive multifocal leukoencephalopathy syndrome, suggesting a major role in this particular disease. [ ] sv encodes mirnas called v-mirnas during the late stages of infection. [ ] they are complementary to the viral mrnas produced at the early stage of viral infection. [ ] these v-mirnas slow down the expression of the viral t-antigen genes and lower the level of interferon (ifn)-g produced by cytotoxic t lymphocytes, thus reducing the influx of inflammatory cells and facilitating evasion of the immune response. [ ] human papillomaviruses (hpvs) are also oncogenic viruses. [ ] they are usually associated with different forms of both benign and malignant tumors, especially those affecting the skin and the genital tract. [ ] these viruses are usually classified, on the basis of their virulence, into either low-or high-pathogenic variants. [ ] only a few hpv strains can produce mirnas during their replication. [ ] for example, hpv- encodes viral mirnas at a very early stage of the infection, but these mirnas are usually degraded once latent infection takes place. [ ] in another independent study, both hpv- and hpv- were found to encode viral mirnas, but they are not involved in cell transformation or cancer development. [ ] it is well known that host mir- a is involved in inhibition of abnormal cell growth in tumors. [ ] mir- a inhibits cell cycle progression at the g phase and subsequently induces apoptosis. [ ] studies have reported success using the tumor suppressor complex (mrna-cellular mirna- a), which targets downstream genes of tumor protein p (tp ). mir- a is usually downregulated during hpv infection in primary keratinocytes. [ ] thus, restoration of the normal expression level of this mirna is a potential strategy for therapeutic intervention. [ ] adenoviruses are a group of non-enveloped dsdna viruses. over serotypes have been identified in different clinical diseases, such as respiratory, gastrointestinal, urogenital, and eye diseases. [ ] adenovirus usually encodes several small noncoding rna molecules called virus-associated rnas, such as va and va . [ ] they facilitate immune evasion by inhibiting dsrna-induced protein kinase r (pkr), which blocks ifn-a activity. [ ] one study reported that adenovirus va -rna interferes with the biogenesis of host mirnas and the function of sirna shrna (short hairpin rna), through inhibition of the nuclear transport of the pre-mirnas and the shrnas and direct inhibitory action of dicer. [ ] usually, a small part of the va rna is subjected to processing by dicer, and this results in generation of mirna. use of anti-mirna antisense inhibitors ( -o-methyl amo) to downregulate this mirna resulted in inhibition of virus production. [ ] herpesviruses are a group of enveloped dsdna viruses, classified into three subfamilies (a, b, and g). they are characterized by induction of latent infections in their target hosts. [ ] these virus-encoded mirnas play important roles in the establishment of latent infection, as well as the pathogenesis of virally induced diseases. according to the most recent studies, herpesviruses utilize their encoded mirnas in a wide range of biologic functions, such as inhibition of apoptosis, immune evasion, control of cellular proliferation, and regulation of viral replication. [ ] [ ] [ ] in the following section, we will discuss herpesvirus-encoded mirnas. one of the most important genes encoded by herpes simplex virus (hsv) is called the latency-associated transcript (lat). [ ] this gene does not encode proteins but may be involved in the production of mirnas or in cell survival after viral infection. [ ] there has been debate around the origin of mir-lat as to whether it is a virus-encoded or cell-encoded mirna. [ ] this mirna is believed to act by downregulating transforming growth factor (tgf)-b and smad . tgf-b plays an important role in cell proliferation and induction of apoptosis. smad is a signaling pathway mediator, which is triggered by the action of tgf-b. [ ] hsv- mir-lat-icp . has been recently discovered during hsv- infection. [ ] according to bioinformatic analysis, the hsv- genome encodes mirnas, eight of which were found to be conserved between both hsv and hsv- , and thus are believed to be functional. [ ] six of these mirnas are upregulated in the trigeminal ganglia of mice infected with hsv- . these mirnas are encoded by lat (table i) . [ ] in addition, quantitative reverse transcription pcr showed that both mir- and mir- were highly expressed in vero cells infected with hsv- -as high as and copies, respectively -whereas the other four mirnas showed downregulation, with only copies per infected cell. [ ] the mirna expression profile during hsv- infection revealed that several mirnas among the eight candidates mentioned earlier were upregulated. those mirnas were believed to play major roles in induction of the latent phase of viral infection. this assumption was based on comparison between the mirna expression profiles of the latent and active infections. for example, mir-h was found to be upregulated in latent infection to a level of copies cell, compared with less than copies cell during active infection. [ ] furthermore, mirna- inhibits the production of the infected cell polypeptide (icp)- protein, which is responsible for triggering the active phase of hsv infection. another upregulated mirna, mir- , is responsible for downregulation of icp , which is responsible for the increased expression level of many hsv genes during the active phase of hsv- infection. [ , ] human cytomegalovirus (hcmv) is a herpesviruses affecting humans, and can result in acute or latent infections. [ ] the form of infection largely depends on the immune status of the affected host. [ ] it can be fatal in immune-compromised patients, such as those with aids or recent organ transplants. [ ] it may also be responsible for birth defects and congenital abnormalities in pregnant women. [ ] as with other herpesviruses, there is evidence supporting the presence of mirnas that modulate viral pathogenesis in different tissues. [ ] specifically, hcmv has been recently reported to encode mirnas. [ ] the most commonly expressed three mirnas during the active phase of hcmv infection are mir-ul - p, mir-ul - p, and mir-us , [ ] which target different cellular proteins, such as transcription factors (hfn and tgif ), receptors (cd receptors for interleukin- ), and other proteins involved in signal transduction pathways, such as rab l. [ ] hcmv is able to induce the latent phase of infection by a cunning immune-evasion strategy through the action of mir-ul , which targets several cellular proteins such as major histocompatibility complex (mhc) class i associated proteins, especially the mhc class i-related chain b (micb). [ , ] mir-ul - also regulates early viral protein expression, such as immediate early protein (ie)- . ie is a key mediator in the shift from the latent to the active phase of viral infection, because it suppresses ie . [ , ] thus, if the hcmv infection is synchronized with overexpression of the mir-ul , the ie protein expression level is greatly reduced, which mediates the latent phase of infection. [ ] it is also thought that mir-ul - targets another viral protein called ul . [ ] downregulation of ul protein, using mir-ul - , results in inhibition of viral dna replication and subsequently triggers the latent phase of infection, making the virus able to evade the host immune system. [ ] exploitation of this mechanism is being considered as a potential therapeutic strategy. [ ] ebv is another oncogenic virus affecting humans. it is usually associated with induction of latent infection in more than % of affected patients. [ ] in most cases, benign tumors develop; in some cases, however, malignant tumors may also develop, such as hodgkin's lymphoma, t-cell lymphoma, nasopharyngeal carcinoma, and gastric tumor. [ ] during the active phase of ebv infection, more than genes are usually expressed, whereas only of them are expressed during latent infection. [ ] it has been recently reported that ebv encodes more than mirnas. [ ] these mirnas are divided into two groups: one group is encoded from the intronic regions of a gene called bart, which is expressed at high levels in epithelial cells, but at lower levels in b cells, in the latent phase of infection; the other group is encoded from the untranslated region of a gene called bhrf , a viral bcl homolog that prevents apoptosis. [ ] although the functions of most ebv-encoded mirnas have not been completely identified, it has been found that mirna-bart targets balf mrna, a viral dna polymerase, and mirna-bart induces cleavage in the untranslated region (utr) of balf , resulting in inhibition of the lytic viral infection ( figure and table i) . [ ] according to bioinformatic prediction data, ebv mirna-bart is believed to target puma, a modulator of apoptosis in the bcl protein group regulated by tp . [ ] in some cases, puma can also induce apoptosis through the tp -independent pathway. therefore, targeting puma with ebv mir-bart results in suppression of its action in apoptosis. [ ] other ebv-encoded mirnas target the ifng-inducible chemokine cxcl . [ ] without cxcl , ebv is able to evade the host immune response and subsequently enhance ebv replication. [ ] the same group performed bioinformatic analysis and showed that cxcl contains a target sequence for mir-bhrf - at its utr sequence. targeting cxcl using this viral encoded mirna will have an immunomodulatory effect on the viral-induced tumor. [ ] therefore, it is believed that targeting bhrf - could be a good therapeutic approach for viral ebv-induced tumors. [ ] kshv is one of the gamma-herpes viruses groups and is usually associated with kaposi's sarcoma infection, from which it acquired its name. [ ] like other herpesviruses, kshv usually induces latent infections. [ ] kshv encodes several mi-rna candidates from the genomic region spanning kilobases between orf and orf . [ ] kshv-encoded mirnas target important genes involved in cell proliferation, modulation of the host immune system, apoptosis, and angiogenesis. for example, mir-k targets the mrna of the bcl (prosurvival gene) interacting protein called bclaf . [ ] this leads to reactivation of the kshv lytic infection. [ ] thus kshvencoded mirnas play an important role in the virus host interactions, and silencing of those mirnas using different approaches, particularly the amo, is therefore a promising therapeutic strategy against such virus infection (figure ). [ ] marek's disease virus (mdv) is one of the alpha herpesviruses, characterized by rapid production of t-cell lymphomas in chickens. [ ] the viral genome encodes several mirnas, which are mainly encoded from the oncogenes, especially meq and lat. [ ] the mirnas of this virus are highly expressed in mdv-transformed cells, suggesting an essential role in mdv oncogenesis. however, their target genes have not been well identified. [ , ] it is known that several host cell mirnassuch as mir- , mir- , mir- , and let- i -are involved in the cancer development induced by mdv in chickens. [ ] they are most often upregulated in an msb- (mdv-transformed cd + t-cell line derived from a spleen lymphoma induced by the bc- strain of mdv- ) library after mdv infection. [ ] this makes for an ideal model for the study of mirnas. comparative analysis of mirna expression profiles during mdv infection, using microarray analysis, may enable identification of specific mirnas involved in neoplastic transformation when compared with non-infected cells. this may become a useful diagnostic approach through examination of mirna signatures profiles. [ ] this is supported by a recent report showing an association between downregulation of the host cellular mir- expression and upregulation of mdvencoded mirnas (m - p, m - p, m - p, m - p, m - p, m - p, m - p, m - p, and m - p), a potential marker for mdv-induced tumor formation. [ ] subsequent identification of the putative target of these mirnas will lead to a better understanding of the molecular mechanism of mdv-induced tumorigeneses. [ ] the human immunodeficiency virus (hiv) genome encodes several important genes, such as nef, vef, tat, and vpu. [ ] bioinformatic analysis suggests that the hiv genome encodes five pre-mirnas, which are processed into ten mature mi-rnas, but their definite functions are still not well identified. [ ] the hiv nef gene is located at the end of the hiv genome and is highly expressed in early viral infection. [ ] it has been shown to downregulate the expression levels of cd , cd , and mhc class i molecules. [ ] hiv nef encodes mir-n , which has a unique function at both the transcriptional and translational levels, rather than at the post-transcriptional level. [ , ] the mechanism of action of mir-n is believed to be suppression of hiv promoter activity; however, further studies are required to explain the exact mechanism of such an action. [ ] several studies reported that downregulation of the nef gene results in inhibition of hiv replication. [ , ] this may lead to production of low-pathogenic strains of hiv or may favor latent hiv infection. [ ] targeting of these viral mirnas using different approaches, especially amo treatment, could potentially have a therapeutic effect on hiv. [ hybridizing with corresponding targets of host genes (blue sequences). the viral mirna sequence and the untranslated region (utr) sequence of the targeted host and viral genes were obtained from the referenced articles or from the national center for biotechnology information (ncbi) website and then submitted to the rnahybrid software program (http://bibiserv.techfak. uni-bielefeld.de/rnahybrid/). the predicted secondary structure for the hybridization between viral mirnas and their targets are generated by the rnahybrid program. (a) mir-k encoded by kaposi's sarcoma-associated herpesvirus (kshv), targeting host gene bclaf (bcl -associated transcription factor ). [ ] (b) mir-bart encoded by ebv, targeting the viral dna polymerase gene balf . [ ] (c) mir-ul encoded by herpesviruses, targeting host gene micb (mhc class i-related chain b). [ ] mfe = minimum free energy. currently, there are more than million people affected by hepatitis c virus (hcv) infection worldwide. [ ] drug resistance is one of the major hindrances in treating such viral infection. [ ] hcv induces different forms of tumors in humans and is one of the major causes of liver diseases all over the world, resulting in hepatocellular carcinoma and, finally, complete liver failure. [ ] it has been recently reported that host cellular mirna- has two recognition sites in the utr of the hcv genome, resulting in upregulation of hcv infection. [ ] further investigation showed that interaction between mirna- and the viral genome causes accumulation of viral rna in the liver tissues (table i) . furthermore, the level of viral rna in the liver tissues is usually controlled by mir- binding sites. [ ] interestingly, hcv infection also modulates cellular mirna expression profiles. following hcv infection, three mirnas (mir- , mir- , and mir- a) are upregulated, while other two mirnas (mir- and mir- ) are downregulated. [ ] ura et al. [ ] found that cyclin g acts as a putative target for mir- . use of a primate model targeting mir- with specific antagonists resulted in a reduction in the level of hcv replication in the affected livers, demonstrating the promise of this strategy. [ ] in addition, a recent study demonstrated the therapeutic potential of silencing mir- in chronic hcv viral infection in primate models, whereby chimpanzees that were positive for hcv infection were treated with a specific lna-modified oligonucleotide (spc ). [ ] these lna-oligomers targeted the complement sequence of mir- and resulted in a decrease in the duration of the viremia following acute hcv infection. there were no reports of any side effects or any viral resistance observed after the treatment. [ ] this approach provided long-lasting effects in the hcv-infected animals, as well as great improvement in the liver pathology. [ ] more clinical trials are needed to further confirm the promising results of this new molecular therapeutic approach. [ ] hepatitis b virus (hbv) belongs to the genus orthohepadnavirus in the family of hepadnaviridae. hbv infection progresses into cirrhosis and hepatocellular carcinoma in most cases. [ ] it is believed that hbv encodes mirnas that regulate their own gene expression. [ ] according to bioinformatic predictions, hbv encodes only one mirna. however, several studies have failed to identify any cellular genes regulated by this virus-encoded mirna, implying alternative gene expression mechanisms. [ , ] according to the mirna expression profiles of several patients suffering from cirrhosis due to hbv infection, the host hsa-mirna- - p is usually upregulated. in recent clinical studies, ura et al. [ ] studied the role of different mirnas in the pathogenesis of both hbv and hcv in the context of development of hepatocellular carcinoma in infected liver tissues. [ ] in this study, the differential expression levels of mirnas from hbv and hcv patients were tested using qrt-pcr. according to this study, mirna candidates were highly expressed in both hbv and hcv infections, and mirnas served as markers for the severity of liver damage. it is known that hbv infection triggers pathways associated with dna damage, recombination, and signal transduction pathways -whereas hcv infection usually triggers an immune response, antigen presentation, cell cycle progression, proteosome activation, and lipid metabolism. therefore, the overall conclusion was that certain mirnas may act as important mediators in the pathogenesis of both hbv and hcv infections. [ ] these studies have paved the way to a new era in molecular antiviral therapy through modulation of the expression levels of those key mirnas. there is now a new antiviral therapy for controlling hbv infection, using artificial mi-rnas. this approach has revealed a dramatic reduction in hbv protein expression levels and a remarkable reduction in viral dna replication in vitro. [ ] [ ] [ ] severe acute respiratory syndrome coronavirus (sars-cov) is a single-stranded rna virus, which belongs to the family coronaviridae. although several trials have been performed to treat this pathogen using conventional drugs such as ribavirin, antibiotics, anti-inflammatory steroids, and different kinds of immune stimulators, these approaches still lack viral specificity. sars-cov infection in bronchioalveolar stem cells (bascs) is a prime example of how mirna modulates the virus-host interaction. sars-cov is unable to replicate in well differentiated cells, so it has to control basc cellular differentiation in order to establish a successful viral infection. [ ] this virus usually hijacks cellular mirnas such as mir- * , mir- - p, and mir- for the benefits of its replication and immune evasion. the nucleocapsid and spike glycoproteins downregulate the expression levels of mir- and mir- , respectively. this action enables the virus to hinder basc cellular differentiation and the production of inflammatory chemokines, creating an environment that is optimal for virus replication. restoration of the levels of mir- and mir- poses a potential novel approach in treating sars-cov infection. [ ] the influenza a outbreak of provided a warning about the urgent need for new alternative molecular therapeutic approaches for both the treatment and prophylaxis of such viral infections. molecular therapy using mirna technology may offer a new therapeutic approach to cope with the continuous changes in virus strains every year. recent bioinformatics tools have paved the way for the discovery of new mirnas and their target sequences for the design of nucleic acid-based therapeutics. for example, there are two human-encoded mirnas that have potential binding sites within both the viral polymerase (pb ) and hemagglutinin (ha) genes (mir- and mir- , respectively) [table i]. [ ] the target sequences of these two mirnas are highly conserved among different influenza virus strains. the ha protein is involved in the attachment of the virus to its receptors, and the pb protein is an essential component in the ribonucleoprotein complex, needed for rna transcription and replication. the presence of human mirnas that target conserved regions of influenza rna suggests that the human genome has evolved to use this as a defense mechanism against infection. this supports the argument that targeting viral genes with mirnas may be an effective strategy. this may also suggest that it is a futile attempt, since we have the mirnas and yet still succumb to influenza infections. a recent study has been conducted to determine mi-rna expression profiles after avian influenza virus (aiv) infection in chickens. this study showed changes in the cellular mirna profile in response to the aiv infection, suggesting that the mi-rnas play a role in the host-pathogen interaction during aiv infection. [ ] specifically, there were alterations in the mirna profiles of mir- , which had been previously reported to play a role in immune-related signal pathways in mammals. [ ] one recent study utilized mirna technology in the development of influenza virus vaccines, whereby a new influenza a virus vaccine was developed using mirna-based gene silencing. the method involves introducing an mirna sequence of non-avian origin, known as a mirna-responsive element (mre), into the viral nucleoprotein gene, resulting in construction of new reassortant h n and h n viruses. with this strategy, the degree of the viral attenuation is controlled by the expression level of mir- , which targets the introduced mre sequence. this novel strategy offered a very good vaccine that was species specific, offering a high level of protection. [ ] the nascent viruses were attenuated for mice, while they still propagated well in embryonated chicken eggs and were able to generate high levels of neutralizing antibodies in animals. this novel approach for influenza vaccine development may be used in combination with the currently available vaccine in order to increase both the safety and the efficacy of influenza virus vaccines in the near future. [ ] coxsackievirus, especially coxsackievirus b (cvb ), is the most common pathogen of human myocarditis. anti-cvb drug development has been recently focused on a nucleic acidbased strategy. our laboratory first reported the successful inhibition (> %) of cvb replication in hela cells by transfection of sirnas targeting viral protease a rna. we also found that the antiviral effect was disrupted by mutations in the central strand region, and mismatch was tolerated near the end but not near the end of the sirna; [ ] furthermore, the sirna effect was mediated by the antisense strand to the viral genome, rather than the sense strand complimentary to the viral negative-strand of the replicating intermediate. this finding was further conformed by another report. [ ] when applied systemically to mice, sirna targeting a had a significant protective effect if applied and hours after infection, including reduced viral replication and tissue injury, as well as an increased survival rate. [ ] recently, we tested a packaging rna (prna) vector (a component of the bacterial phage nano-motor) for targeted delivery of sirnas. through conjugation of a folate ligand to the prna vector, we specifically delivered the sirnas targeting cvb a to hela cells and hl- cardiomyocytes that expressed folate receptors. [ ] in addition to the transfection of mature sirnas, overexpression of shrnas was also effective against cvb d rna polymerase and structural protein vp , both in cells and in mice, where viral pancreatitis was significantly reduced. [ ] schubert et al. [ ] used the sidex double expression vector to simultaneously transfect two sirna sequences targeting the cvb d rna polymerase sequence in a green fluorescent protein (gfp) reporter construct. this double expression of both sirnas successfully suppressed reporter expression despite the intentional introduction of an artificial point mutation (simulating an escape mutation or a mirna target) that caused a mismatch with one of the two sirnas. as we have discussed above, there have been some promising results supporting the development of mirnas for the treatment of several viral infections, and some of these mirna-based drugs have reached the clinical trial stage. despite this great progress, their clinical applications are still hampered by several challenges. in the following section, we briefly discuss the current obstacles or limitations facing mirnas-based antiviral therapy. one of the major limitations for the use of mirna-based antiviral therapy is the production of transgene-specific immunity. [ ] delivery of mirnas using viral vectors usually results in the development of immune response against the viral vector. basically, the delivery vector will stimulate an innate immune response in the forms of cytotoxic t-lymphocytes, humoral neutralizing antibody against their viral capsid proteins, and cytokine-mediated inflammatory responses in vivo. [ , ] direct correlation between the immune response to the adenovirus capsid protein and the concentration of the viral vector has been reported; this interaction is usually associated with undesirable side effects in the host, especially if the construct moves from the target tissue into the blood circulation. [ ] the targeted delivery of sirna, mirna, and other nucleic acid-based therapies is another major concern of using these molecular therapeutic approaches. in contrast to the great progress in local administration of both sirna-and mirna-based therapies in the eyes, lung, and vagina, systemic delivery to target organs such as the liver, heart, and intestine is still undergoing optimization. [ , ] it is interesting to note that some studies have shown success in administering sirnas via the intracerebral route; however, the risk is that foreign nucleic acid may be delivered to the central nervous system. [ ] several laboratory techniques -such as real-time pcr, microarray analysis, luminex bead arrays, northern blotting, in situ hybridization, formalin fixation, and paraffin embedding -are currently in use in mirna detection and quantification. however, all of these techniques still require further optimization. [ ] [ ] [ ] once they are optimized, a clear choice for sensitivity and specificity will emerge, and this approach will allow early and sensitive detection of mirna expression in different disease syndromes. this will have a great impact on the early tracking of serious viral diseases. [ ] the off-target effects of sirnas were one of the major concerns in earlier studies using both sirna and shrna technologies in gene therapy. as a new generation of molecular gene therapy, mirnas would be expected to have a high degree of specificity for their targets. however, since mirna action is based on imperfect base pairing with the target sequence in most circumstances, the specificity will be lower than that of sirna. this prediction has been confirmed by recent clinical trials, followed up by microarray analysis, which revealed possible off-target effects of mirnas. [ ] another follow-up study by birmingham et al., [ ] using the combination of bioinformatics and microarray analysis, found that using either the sirna or the mirna could result in off-target silencing. in addition, in vivo studies have revealed that one mirna may target several genes at the same time, and the targets are not clearly identified. this suggests diverse modes of action of a given single mirna. on the other hand, one gene may be regulated by several mirnas, [ ] indicating that the mode of action is more complicated than expected. since drug therapies must precisely target the virus in question and nothing else, a large undertaking is needed to gather all possible information regarding all targets of each mirna that is being considered for drug development. [ , ] although the currently used viral vectors in mirna delivery are non-pathogenic, there is always the possibility of mutations within those viral vectors. these mutations may not only result in abnormal gene expression of the viral mirna construct but may also cause possible insertion of vectors into the human genome, increasing the risk of cancer. [ ] moreover, the targeted viruses (especially the rna viruses) are prone to mutation, which may drive drug resistance. there are currently two possible approaches to conquer these issues: one is the targeting of cellular factors that are essential for virus replication or use of more than one mirna for the same target gene; the other possible solution is the targeting of several conserved regions of the viral genome by different sirnas or mirnas. viruses are among the most common causes of human diseases. because of the unique biologic properties of viruses, there is no effective and specific antiviral therapy available so far. several vaccines and antiviral drugs have shown a limited degree of efficacy for prophylaxis and treatment of some viral infections. however, high mutation rates enable viral diseases to emerge and re-emerge frequently. thus, new strategies for drug and vaccine development must be devised to fight the threat of viral diseases to human health. recent advances in the understanding of mirna structure, function, and particularly their association with the molecular pathogenesis of a variety of complex diseases, have served as a theoretical basis for drug development. on the one hand, as key factors for viral replication and latency, mirnas are ideal targets for inhibition. in this regard, construction of mrnas that contain multiple tandem binding sites of a given mirna may be useful to produce decoys or 'mirna sponges' to inhibit the function of a specific mirna. in addition, chemically synthesized antisense rna oligomers ('antagomirs') targeting a mirna of interest could be also be a promising approach to inhibit mirna activity. on the other hand, mirna expression vectors can be used to overexpress specific mirnas to achieve a long-term effect of reversing the imbalance of mirna expression caused by infection. further, introduction of pre-mirna mimetics for transient replacement is another option for investigation. in summary, although there are 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auto-regulate viral gene expression regulation of human immunodeficiency virus transcription by nef microrna differential expression between hepatitis b and hepatitis c leading disease progression to hepatocellular carcinoma a cellular microrna mediates antiviral defense in human cells human papillomaviruses modulate expression of microrna upon epithelial differentiation to control levels of p proteins micrornas expressed by herpes simplex virus during latent infection regulate viral mrnas herpes simplex virus type icp promotes transcription preinitiation complex formation by enhancing the binding of tfiid to dna severe cytomegalovirus infection in apparently immunocompetent patients: a systematic review identification and function of human cytomegalovirus micrornas suppression of immediate early viral gene expression by herpesvirus encoded micrornas: implications for latency latent epstein-barr virus (ebv) infection and cytomegalovirus (cmv) infection in synovial tissue of autoimmune chronic arthritis determined by rna-and dna-in situ hybridization epstein-barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions tandem array-based expression screens identify host mrna targets of virus-encoded micrornas epstein barr virus encoded microrna mir-bart down-regulates the viral dna polymerase balf the nuclear function of p is required for pumamediated apoptosis induced by dna damage ebv micrornas in primary lymphomas and targeting of cxcl- by ebv-mir-bhrf - micrornas of kaposi's sarcoma-associated herpes virus marek's disease: a model for herpesvirus oncology analysis of the expression profiles of marek's disease virus-encoded micrornas by real-time quantitative pcr differential expression of micrornas in marek's disease virus-transformed t-lymphoma cell lines micrornas accurately identify cancer tissue origin are viral-encoded micrornas mediating latent hiv- infection? hiv- encoded candidate micro-rnas and their cellular targets hiv- nef suppression by virally encoded microrna challenges in modern drug discovery: a case study of boceprevir, an hcv protease inhibitor for the treatment of hepatitis c virus infection mechanisms of drug resistance to current and future antiviral therapies for hepatitis c virus infection occurrence of hcc in asymptomatic hcv-related chronic hepatitis therapeutic silencing of microrna- in primates with chronic hepatitis c virus infection mir- regulation of lipid metabolism revealed by in vivo antisense targeting association between hepatitis b virus and pancreatic cancer hbv-encoded microrna candidate and its target targeted deletion of dicer in the heart leads to dilated cardiomyopathy and heart failure expressed anti-hbv primary micro-rna shuttles inhibit viral replication efficiently in vitro and in vivo inhibition of hepatitis b virus gene expression and replication by artificial microrna micrornaome of splenic macrophages in hypersplenism due to portal hypertension in hepatitis b virus-related cirrhosis an animal model of sars produced by infection of macaca mulatta with sars coronavirus oncogenic hpv infection interrupts the expression of tumor-suppressive mir- a through viral oncoprotein e inhibition of coxsackievirus b replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand maintaining inhibition: si-rna double expression vectors against coxsackieviral rnas targeting a protease by rna interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice targeted delivery of anti-coxsackievirus sirnas using ligand-conjugated packaging rnas expression of short hairpin rnas against the coxsackievirus b exerts potential antiviral effects in cos- cells and in mice prospects and obstacles to using small interfering rnas as small molecule drugs progress and prospects: immune responses to viral vectors lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons inhibition of respiratory viruses by nasally administered sirna an sirna-based microbicide protects mice from lethal herpes simplex virus infection identification of micrornas and other small regulatory rnas using cdna library sequencing mustering the micromanagers analysis of microrna expression by in situ hybridization with rna oligonucleotide probes transcription and processing of human microrna precursors expression profiling reveals off-target gene regulation by rnai a protocol for designing sirna with high functionality and specificity a pattern-based method for the identification of microrna binding sites and their corresponding heteroduplexes serum response factor regulates a muscle-specific microrna that targets hand during cadiogenesis correspondence: professor decheng yang, - burrard street, the heart and lung institute, st paul's hospital this work was supported by grants from the canadian institutes of health research and the heart and stroke foundation of bc and yukon. dr. maged gomaa hemida is a recipient of the cihr-impact postdoctoral training fellowship. xin ye is supported by a ugf award from the university of british columbia.the authors have no conflicts of interest that are directly relevant to the content of this review. key: cord- -guiy x authors: cojocaru, florina-daniela; botezat, doru; gardikiotis, ioannis; uritu, cristina-mariana; dodi, gianina; trandafir, laura; rezus, ciprian; rezus, elena; tamba, bogdan-ionel; mihai, cosmin-teodor title: nanomaterials designed for antiviral drug delivery transport across biological barriers date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: guiy x viral infections are a major global health problem, representing a significant cause of mortality with an unfavorable continuously amplified socio-economic impact. the increased drug resistance and constant viral replication have been the trigger for important studies regarding the use of nanotechnology in antiviral therapies. nanomaterials offer unique physico-chemical properties that have linked benefits for drug delivery as ideal tools for viral treatment. currently, different types of nanomaterials namely nanoparticles, liposomes, nanospheres, nanogels, nanosuspensions and nanoemulsions were studied either in vitro or in vivo for drug delivery of antiviral agents with prospects to be translated in clinical practice. this review highlights the drug delivery nanosystems incorporating the major antiviral classes and their transport across specific barriers at cellular and intracellular level. important reflections on nanomedicines currently approved or undergoing investigations for the treatment of viral infections are also discussed. finally, the authors present an overview on the requirements for the design of antiviral nanotherapeutics. today, we are living through a so-called fourth great transitional period, after the other three waves of epidemiological transitions, namely early agrarian-based settlements, early eurasian civilizations and european expansionism (more details can be found in mcmichael's description [ ] ). the current configuration and variety of infectious diseases closely followed the combined evolutions in demography, environment, technology, social change and behaviours. medicine itself has created new opportunities for microbes either through blood transfusions, organ transplants, the use of hypodermic syringes or the excessive use of antibiotics thus contributing to the induction of iatrogenic effects in some treatments for infections such as hepatitis c, hiv and others [ ] . in recent decades, old concerns have been reactivated at both the official and the general public levels regarding infectious diseases as a threat to public health. mcmichael [ ] analyzed the reflection of this issue in social media and noticed the "emergence and resurgence" of infectious diseases (determined by environmental, sociological and economic changes) and a so-called "public anxiety" set on this topic. despite their widespread and increasing transmission, there is still a poor understanding of global economic impact of viral diseases, which makes difficult to evaluate the societal costs and the cost-effectiveness of preventive efforts. the issue of estimating a general impact of viral infections involves several aspects that hinder this approach, namely: the variety of viral infections, the incidence of associated co-morbidities, social and psychological issues having economic repercussions (hidden costs), the variety of treatments (only direct-acting antiviral (daa) and vaccinations can be used in the evaluation) and the presence of negative externalities [ ] , meaning that the disease consequences are not limited to their patients infected or potentially but also to the related families who may experience social distress as well. also, human mobility and long-distance trade have increased; ever-larger cities, often girded with slums, have become highways for microbial traffic; poverty perpetuates vulnerability to infectious disease; and sexual practices, drug injecting, intensified food production and much modern medical technology all create new "audience" for microbial opportunism, and new management issues for public health decision makers [ ] . for all these reasons, a global assessment of socio-economic impact seems practically impossible or, if contrary, it could not meet all the criteria to be relevant. rather, the impact can be split by relevant categories of viral infections, were the literature is more precise but even so, there remain some inconsistencies regarding the unification of the methodologies from the various studies that will ensure the comparability of the data. figure presents statistical facts related to the most "burden-generator" viral infection diseases [ ] [ ] [ ] [ ] [ ] . pharmaceutics , , x for peer review of in recent decades, old concerns have been reactivated at both the official and the general public levels regarding infectious diseases as a threat to public health. mcmichael [ ] analyzed the reflection of this issue in social media and noticed the "emergence and resurgence" of infectious diseases (determined by environmental, sociological and economic changes) and a so-called "public anxiety" set on this topic. despite their widespread and increasing transmission, there is still a poor understanding of global economic impact of viral diseases, which makes difficult to evaluate the societal costs and the cost-effectiveness of preventive efforts. the issue of estimating a general impact of viral infections involves several aspects that hinder this approach, namely: the variety of viral infections, the incidence of associated co-morbidities, social and psychological issues having economic repercussions (hidden costs), the variety of treatments (only direct-acting antiviral (daa) and vaccinations can be used in the evaluation) and the presence of negative externalities [ ] , meaning that the disease consequences are not limited to their patients infected or potentially but also to the related families who may experience social distress as well. also, human mobility and long-distance trade have increased; ever-larger cities, often girded with slums, have become highways for microbial traffic; poverty perpetuates vulnerability to infectious disease; and sexual practices, drug injecting, intensified food production and much modern medical technology all create new "audience" for microbial opportunism, and new management issues for public health decision makers [ ] . for all these reasons, a global assessment of socio-economic impact seems practically impossible or, if contrary, it could not meet all the criteria to be relevant. rather, the impact can be split by relevant categories of viral infections, were the literature is more precise but even so, there remain some inconsistencies regarding the unification of the methodologies from the various studies that will ensure the comparability of the data. figure presents statistical facts related to the most "burden-generator" viral infection diseases [ ] [ ] [ ] [ ] [ ] . hiv/aids, considered as one of the major burdens of disease globally, became a chronic disease after the introduction of multiple antiretroviral therapy (art), and therefore it needs to provide long- hiv/aids, considered as one of the major burdens of disease globally, became a chronic disease after the introduction of multiple antiretroviral therapy (art), and therefore it needs to provide long-term care and support for the ill person, demanding a higher level of treatment costs for the hiv-affected households. consequently, hiv/aids causes depletion of savings and productive assets, and increases the indebtedness of the hiv-affected households [ , ] . moreover, the higher health care expenditure of the households reduces investment for nutritional food for the family members, investment for farming or business, and the children education. death during the working age of the patient is a major factor in the economic impact of hiv/aids. the household level impact of hiv/aids includes direct costs, including medical and non-medical costs, and productivity costs such as loss of labour time, as a result of the morbidity of hiv positive household members, as well as time spent by others caring for them [ ] . this evidence suggests that hiv/aids places significant economic pressure on households trying to pay for health care costs, and trying to make up for lost income. the difficulty in accurately quantifying and explaining the morbidity and mortality related to viral hepatitis stems from the fact that hepatitis deaths are caused by five distinct viruses (hepatitis a-e) with different routes of transmission, or from the fact that death occurs decades after infection, and that when people die with hepatitis-related liver cancer and cirrhosis, these deaths are not always linked to the underlying infection. although antiviral therapy appears to be expensive (for example, average cost for a treated hcv case ranges from $ , to $ , [ ] ), is considered to be also cost effective when compared with other well-accepted medical interventions [ ] due to sustained viral response to therapy, the cost savings and quality-of-life improvement and prolongation of life expectancy from the prevention of hcv complications. in the era of new daas, the statement "provide treatment to hcv-patients" generates savings compared to not provide it. low and middle-income countries may consider hcv-treatment as a cost-saving intervention for the health system, not only in a long-term horizon, but in - years [ ] . economic impact of no treatment is higher than treatment costs itself. but still, the enthusiasm for daa therapies, however, has been tempered by two major concerns: the price, still very high, of these medications, and, the challenges patients and clinicians face with respect to drug access in many countries. hsv and herpes zoster (varicella-zosterian virus-vzv reactivation) are some of the most common infections in humans, with no effective treatment available at this time. the impact on the health degree varies from a very small impairment to severe, disabling forms, in most cases limiting methods are applied to local infection and reducing side effects (pain, manifestations dermis etc.). also, specific psychological issues are related with this disease, such as negative feelings correlated to the condition following diagnosis, in particular if they have acquired the genital form of the disease. feelings can include depression, fear of rejection, feelings of isolation, fear of being found out, and self-destructive feelings [ ] . as mentioned above, all the available reports present crude estimates rather than precise measures of the economic costs of the illness, because most of the costs are calculated directly on interventions or on medical budgetary chapters, without taking into account the societal losses. moreover, different approaches are discrepant (can be explained, at least in part, with the influence of compliance to treatment and possible under sampling of subpopulations in the data set) [ ] . also, limitations must be placed on the ability to generalize the results beyond the sample. moreover, not only the cost matters. the costs of an intervention have to be compared with the results of interventions because the effectiveness of treatments and the efficiency can produce societal gains that must be offset by losses [ ] . the usual most accurate methodology to estimate a burden (associated with a disease) is the so-called "cost-of-illness" [ ] , and measures all the costs of a particular disease, including the direct, indirect, and intangible dimensions. it is widely accepted that estimating the total social cost of a disease is useful in establishing policy decisions [ ] . but there are also other methods as the cross-sectional surveys of samples of primary and secondary care physicians, analyzing health care resource utilization or approaches based on the analysis of a large administrative data sets, such as values of spending or drugs consuming lists [ ] . other approaches are used to estimate the number of patients seeking medical treatment, the average medical expenditures (as health inputs employed per unit multiplied by number of units) and estimated national costs. these comprehensive studies can often be advantageous in allocating total national expenditures among the major diagnostic categories [ ] . however, regardless of the method, such analyzes are not possible otherwise than inside countries (where the impact determined by cultural and social aspects can vary substantially). but even in the absence of global data of this nature, we still can extract from the information presented the relevant issue for the topic of this paper: the current arrangements in the management of viral infections (treatments, prevention and limitations of spread) are costly and less effective, unaffordable in some cases and burdensome for medical systems. for example, according to the current analysis of globe newswire reports and data [ ], the global antiviral drugs market was valued at $ . billion in and is expected to reach $ . billion by year . sales of antivirals increased by approximately % each two years. moreover, thanks to better diagnostics, innovative drugs and new therapeutics, the market is likely to witness even further future growth. however, the list of viral diseases for which antiviral therapies are available is still relatively short [ ] . there are several factors that hinder the development of antiviral drugs: • dependence of viruses replication on host cell biosynthetic machinery [ ] , that leads to a limited number of virus-specific metabolic functions can be targeted by antiviral drugs without any damage to the host; • the viruses' functions are specific to each virus, preventing the development of a broad-spectrum antivirals fighting against different viruses that cause similar symptoms. antivirals developed for some viruses (as hsv and hiv) can treat the acute illness, but do not cure the latent infection. this leads to recurrent or chronic diseases that require treatment for longer periods of time [ ] . all these limitations prompted the need for a paradigm shift. the great challenge of antiviral therapies is to move on to developing new drug formulas. this involves changing the physico-chemical and bio-pharmaceutical properties of antiviral molecules using new scientific strategies during the preparation or in dosage configuration. viruses are sub-microscopic intracellular parasitic particles of genetic material contained in a protein coat, totally dependent by host for cell replication, showing both living and non-living characteristics [ ] . living characteristics of the viruses are represented by the high rate of multiplication (only in living host cells) and by the ability to mutate. the non-living characteristics for viruses consist in acellularity (lack of cytoplasm and organelles), the replication only by using host cell's metabolic machinery and the composition with dna or rna [ ] . in humans, viral infections are responsible for different diseases as briefly presented in table . according to international committee on taxonomy of viruses (ictv) there are phylum, subphylia, six classes, orders, seven suborders, families, subfamilies, genera, subgenera and species [ ] . currently, viruses are classified based on their type of nucleic acid (dna, rna, single-stranded, double-stranded) and their way of replication, known as baltimore classification [ ] , divided as seven baltimore classes: • i-dsdna viruses (e.g., adenoviruses, herpesviruses, poxviruses): enter to the host nucleus and are dependent by host cell polymerases to replicate viral genome. the virus may induce the cell to forcefully undergo cell division, which may lead to transformation of the cell and, ultimately, to cancer. • ii-ssdna viruses (+ strand or "sense") dna (e.g., parvoviruses), consists of viruses that have a single-stranded dna genome of the same polarity as the mrna. excepting parvoviruses, most of them have circular genomes and are replicating within nucleus. • iii-dsrna viruses (e.g., reoviruses): not dependent by host replication polymerases and their replication (monocistronic) is realized into capsid (in cytoplasm). • iv-(+)ssrna viruses (+ strand or sense) rna (e.g., picornaviruses, togaviruses): the rna can be directly accessed by ribosomes of the host to form proteins, and use a simple reproduction pathway (viruses with polycistronic mrna) or a more complex transcription pathway (for which subgenomic mrnas, ribosomal frameshifting, and proteolytic processing of polyproteins may be used). [ ] [ ] [ ] [ ] [ ] gastroenteritis adenoviruses, rotaviruses, noroviruses, astroviruses, coronaviruses [ ] [ ] [ ] sexually transmitted diseases herpes simplex type , human papillomavirus hiv [ ] there are distinct stages of viral replication (cell entry, uncoating, transcription of viral genome, translation of viral proteins, post-translational modifications and assembly of virion components) and the classes of antiviral agents that can act at each stage, the correspondence between stage of replication and classes of selective inhibitors being described in detail in reference [ ] and their pharmacological properties as it follows in the next section. according to our knowledge (based on found studies) only the antivirals summarized in figure were applied in nanomedicine. the therapy of the large hsv family of double-stranded dna viruses, widely distributed among humans, includes highly selective and effective antivirals, from which only acyclovir (acv) and ganciclovir (gcv) were incorporated into nanomaterials; a complete classification of hsv antivirals pharmaceutics , , of can be found in described in detail in references [ ] [ ] [ ] [ ] [ ] . briefly, acv pharmacology can be explained as follows: • the acv selectivity is dependent by interaction with viral hsv thymidine kinase and dna polymerase, therefore the cellular uptake and first phosphorilation are facilitated by hsv thymidine kinase that presents a high affinity for acv; • then, intracellular enzymes convert monophospate to triphosphate acv and this form of acv inhibits viral dna polymerase and, to a much lesser extent, cellular dna polymerase; acv triphosphate is also integrated into viral dna and acts as a chain terminator, as it binds to viral dna polymerase and determines its irreversible inactivation by a mechanism called suicide inactivation [ ] [ ] [ ] ; • occurrence of resistance to acv could be acquired by three mechanisms: impaired production of viral thymidine kinase (the most common), altered thymidine kinase substrate specificity (e.g., phosphorylation of thymidine but not acyclovir), or altered viral dna polymerase (rare). alterations in viral enzymes are caused by point mutations and base insertions or deletions in the corresponding genes [ ] . according to our knowledge (based on found studies) only the antivirals summarized in figure were applied in nanomedicine. the therapy of the large hsv family of double-stranded dna viruses, widely distributed among humans, includes highly selective and effective antivirals, from which only acyclovir (acv) and ganciclovir (gcv) were incorporated into nanomaterials; a complete classification of hsv antivirals can be found in described in detail in references [ ] [ ] [ ] [ ] [ ] . briefly, acv pharmacology can be explained as follows:  the acv selectivity is dependent by interaction with viral hsv thymidine kinase and dna polymerase, therefore the cellular uptake and first phosphorilation are facilitated by hsv thymidine kinase that presents a high affinity for acv;  then, intracellular enzymes convert monophospate to triphosphate acv and this form of acv inhibits viral dna polymerase and, to a much lesser extent, cellular dna polymerase; acv triphosphate is also integrated into viral dna and acts as a chain terminator, as it binds to viral dna polymerase and determines its irreversible inactivation by a mechanism called suicide inactivation [ ] [ ] [ ] ;  occurrence of resistance to acv could be acquired by three mechanisms: impaired production of viral thymidine kinase (the most common), altered thymidine kinase substrate specificity (e.g., phosphorylation of thymidine but not acyclovir), or altered viral dna polymerase (rare). alterations in viral enzymes are caused by point mutations and base insertions or deletions in the corresponding genes [ ] . the mechanism of gcv inhibits viral dna synthesis [ ] as briefly explained below:  it is monophosphorylated intracellular by viral thymidine kinase during hsv infection and by a viral phosphotransferase encoded by the ul gene during cmv infection, while diphosphate and gcv triphosphate forms are produced by cellular enzymes; the mechanism of gcv inhibits viral dna synthesis [ ] as briefly explained below: • it is monophosphorylated intracellular by viral thymidine kinase during hsv infection and by a viral phosphotransferase encoded by the ul gene during cmv infection, while diphosphate and gcv triphosphate forms are produced by cellular enzymes; • cmv can become resistant to gcv by mutations in the viral phosphotransferase encoded by the ul gene and by mutations in viral dna polymerase [ ] . the conventional treatment (prophylaxis or therapy) of an influenza virus infection, as a major public health concern worldwide, is designed to target viral proteins and could be used, either alone or in combination [ ] . these include also amantadine and neuraminidase inhibitors (zanamivir and oseltamivir), that have been encapsulated into nanoparticles as specified in table . potent vacuolar atpase (v-atpase) inhibitors, namely diphyllin and bafilomycin, previously shown to have broad-spectrum antiviral activity represent another possibility against influenza virus infection [ ] [ ] [ ] . briefly, the antiviral mechanism of amantadine is based on nterference with the viral protein, m (an ion channel), the protein needed for the viral particle to become uncoated once it is taken inside the cell by endocytosis [ ] . also, oseltamivir carboxylate mechanism implies a selective inhibition of influenza virus neuraminidase enzymes, which are glycoproteins found on the virion surface, very important for viral entry into uninfected cells, for the release of recently formed virus particles from infected cells, and for the further spread of the infectious virus in the body [ , ] . hepatitis viruses have been the subject of intense study in the last years, with a special attention on therapy. as mentioned in the first section, hepatitis treatment depends upon the type of hepatitis, therefore different antivirals are considered and summarized in detail in references [ ] [ ] [ ] [ ] [ ] [ ] . currently, interferons (ifns) α, β, and γ have antiviral activity, the first two being produced by nearly all cells as response to viral infections, while the third is restricted to t-lymphocytes and nk cells. ifn-induced proteins include - -oligoadenylate [ - (a)] synthetase and a protein kinase, either of which can inhibit protein synthesis in the presence of double-stranded rna. the - (a) synthetase produces adenylate oligomers that activate a latent cellular endoribonuclease (rnase l) to cleave both cellular and viral single-stranded rnas. antiretroviral therapy-art-refers to the treatment with hiv medicines. according to the last updated list approved by food and drug administration (fda) these drugs can be classified as can be seen in figure [ , ] . in the following paragraph the authors describe the pharmacological mechanism of the medicines for hiv treatment that were included/encapsulated/incorporated into nanomaterials. the representative nucleoside reverse transcriptase inhibitor (nrti) zidovudine (azt) is phosphorylated intracellular by kinases specific to azt -triphosphate, a metabolite responsible for termination in elongation of proviral dna because it is incorporated by reverse transcriptase into nascent dna but lacks a -hydroxyl group. resistance to azt is associated with mutations at reverse transcriptase codons , , , , , , and [ ] . also, lamivudine (lam), another nrti agent, enters cells by passive diffusion, and then is converted to the monophosphate by deoxycytidine kinase, and undergoes further phosphorylation by deoxycytidine monophosphate kinase and nucleoside diphosphate kinase to yield lamivudine -triphosphate, which is the active anabolite [ ] . tenofovir disoproxil is a derivative of adenosine -monophosphate lacking a complete ribose ring, and it is the only nucleotide analogue currently marketed for the treatment of hiv infection, being active against hiv- , hiv- and hbv. after a rapid hydrolysis, tenofovir is formed, being then phosphorylated by cellular kinases to its active metabolite, tenofovir diphosphate which is a competitive inhibitor of viral reverse transcriptases and is incorporated into hiv dna, causing chain termination [ , ] . non-nucleoside reverse transcriptase inhibitors (nnrtis) include a variety of chemical substrates that bind to a hydrophobic pocket in the p subunit of the hiv- reverse transcriptase and induce a conformational change in the d structure of the enzyme that greatly reduces its activity, and thus they act as non-competitive inhibitors. unlike nucleoside and nucleotide reverse transcriptase inhibitors, these compounds do not require intracellular phosphorylation to attain activity [ ] . also, the binding site for nnrtis is virus-strain-specific and the approved agents are active against hiv- but not hiv- or other retroviruses and should not be used to treat hiv- infection. hiv protease inhibitors (pis) are peptide-like chemicals that competitively inhibit the action of the virus aspartyl protease (a homodimer consisting of two -amino acid monomers). the preferred cleavage site for this enzyme is the n-terminal side of proline residues, especially between phenylalanine and proline. these drugs prevent proteolytic cleavage of hiv gag and pol precursor polypeptides that include essential structural (p , p , p , and p ) and enzymatic (reverse transcriptase, protease, and integrase) components of the virus, preventing the metamorphosis of hiv virus particles into their mature infectious form [ , ] . only sqv, indinavir, atv, rtv, nfv and lopinavir are currently employed in nanotechnology research. there are two drugs available in the entry inhibitors class: enfuvirtide-enf and maraviroc-mcv, with different mechanisms of action, both incorporated into nanoparticles as specified in table . enf inhibits fusion of the viral and cell membranes mediated by gp and cd interactions, while mcv is a chemokine receptor antagonist and binds to the host cell ccr receptor to block binding of viral gp . as such, mcv is the only approved antiretroviral drug that targets a host protein [ ] [ ] [ ] . raltegravir (ral), the first approved hiv integrase inhibitor, has potent activity against both hiv- and hiv- , and also retains activity against viruses that have become resistant to antiretroviral agents of other classes because of its unique mechanism of action [ ] [ ] [ ] [ ] . ral was encapsulated into a polymeric plga nano-formulation and gold nanoparticles (see table ). the first line of defence [ ] that any substance encounters is the biological barriers penetration into the organism. the "security" system includes physiological barriers, such as blood-brain barrier (bbb), epithelium, stratum corneum, air-blood lung barrier [ ] , reproductive system barrier, etc. all of which control the extracellular and intracellular access and trafficking of foreign substances such as bacteria, viruses, fungi, and chemicals [ ] but also provide selective access to "suitable candidates" such as nutrition and/or therapy molecules. it is well established that more than one mechanism may be involved in intracellular drug delivery. the mechanisms involved in nano-based intracellular drug delivery include passive diffusion of free drug, non-specific phagocytosis of the nanocarrier, nanocarrier uptake by pinocytosis, and receptor-mediated endocytosis [ ] . in this section, we will discuss in particular how to overcome biological barriers such as mucus, skin, cell membrane and bbb in antiviral therapy. the gastrointestinal tract, respiratory system, the urogenital cavities, eyes and mouth, are all covered with mucosal membranes. the highly adhesive mucus acts as protective layer as well as for lubrication purposes. although large molecules cannot pass, small ones along with viruses can easily penetrate. these are also the reasons that drug delivery is so challenging. mucus contains % of water along with mucin fibres, lipids, salts, cholesterol and proteins. it is continuously produced but the thickness, ph and amount differ by its position. two strategies have to be followed for passing through mucus, mainly depending how fast the turnover is: fast mucosal penetration or highly adhesive particles (slow turnover) to increase the drug's residence on the targeted mucosa. mucoadhesion can be the resultant of interactions like hydrophobic, hydrogen bonding, ionic bonding or van der waals ones. other possible attractive interactions can be covalent bond formation between catechols, maleimides, thiols, and acrylates with domains of mucin glycoproteins rich in cysteine. chitosan, alginate, pectin or cellulose polymers are mostly used for achieving adhesion on mucosa. furthermore, it is known that thiolation of the mentioned polymers develops high mucoadhesive properties. mucopenetration is the second strategy found to permeate the mucus layers by two potential mechanisms: active strategy characterized by the interaction with the mucus and chemically shifting the features of the mucus or their own structures and passive strategy that uses hydrophilicity enhancers to penetrate the mucus [ ] . the classical hsv therapy includes daily dosing of orally administered acv [ ] that is effective in most cases, and of course problematic in other cases, for example, in long-term use of acv patients report resistance against the drug followed by renal injury [ , ] . another issue produced by standard hsv treatment in this case for the topical form of trifluridine (tft) and ganciclovir (acv analog) gels, is denoted by low retention time on vaginal and corneal mucus followed by multiple doses up to times [ ] . the nanotechnology field offers a great deal of drug delivery modalities in order to overpass the biological barriers, deliver efficiently the incorporated active principle in a controlled and targeted pharmaceutics , , of manner, reduce circulating drug levels and attenuate the renal damage. a recent review points out the nanogels based on the above-mentioned materials capabilities as an adequate example to pass through these types of biological barriers [ ] . for example, the synthesized nano-drug delivery micelles based on chitosan-g-oligo (nipaam) copolymers stabilised by ionotropic crosslinking by raskin et al. [ ] , gave good results for delivering antiretroviral drugs (efv) through mucosa. more studies on how acv penetrates different barriers can be found in table and detailed in section . it is important to mention a "special barrier" namely the ocular mucus that changes completely in to min, making the drug absorption unfavourable. in addition, the eye is protected by blood-anterior chamber and blood-retinal barriers. in case of drug administration, a combination of penetration and adhesion of the substances is necessary. polymers like phosphotyrosine could be the solution. it was demonstrated that if intestinal alkaline phosphatase is present, polymers can manifest a zeta potential change, thus causing their immobilization after penetration. another kind of construct for combining adhesion and penetration is through thiolated systems with mucolytic enzymes, ph dependent. therefore, at acid ph, the absence of disulphide bonds formation with cysteine-rich domains in mucins does not manifest mucosal adhesion unless they are near the epithelium [ ] . skin, as the largest organ of our body, protects us from microorganisms and chemicals, regulates our body temperature and maintains hydroelectrolytic balance. the two layers of the skin are the epidermis and the dermis. the first one is avascular and is composed of stratified, keratinized squamous epithelium, in four layers from bottom up: basale, spinosum, granulosum and corneum. thick skin (palms and soles) has a fifth layer (under the most superficial corneum) called lucidum. the dermis consists of two layers (reticular and the more superficial papillary) of connective tissue of elastin and collagenous fibres and has in its component blood and lymphatic vessels network, nerves, touch receptors (meissner corpuscles), adipocytes, phagocytes, hair follicles and sweat glands [ ] . topical, through skin drug delivery, has a local effect, requiring less drug for the targeted outcome. transdermal therapy results in fewer side effects with no need of regular treatment but systemic distribution of the drug. both methods of treatment have a common blockage, stratum corneum. to pass through this barrier, different approaches were developed based mostly on disrupting this structure chemically with substances like surfactants, alcohols, esters, amines, terpenes, alkanes phospholipids, or mechanically by using ultrasounds, micro needling, magnetophoresis, iontophoresis, electroporation or lasers. excessive use, though, can damage the skin. analysing the literature data, we can suggest that there are different processes and mechanisms that govern the penetration of small/large molecules through skin barrier. according to schneider et al. [ ] review there are two general pathways for skin absorption: through skin appendages or through the stratum corneum and the underlying layers. the lipophilic statum corneum medium determines the first mechanism of skin penetration, namely absorption of lipophilic compounds [ ] . the three transport routes of substances across stratum corneum can be classified in transcellular, intercellular and trans-appendageal pathways as defined by liang et al. [ ] . more examples are available but the conclusion is the same: "the full understanding of the penetration or absorption processes is still under evaluation" due to the challenges associated with delivering complex burdens through the skin barrie [ ] . as specified in section , the current antiviral therapy for hsv infection includes topical formulations of acv that is unable permeate stratum corneum and target the virus site at the basal epidermis due to its polarity and solubility, leading to poor clinical efficacy due to delayed antiviral activity and sub-inhibitory concentrations [ ] . nanotechnology strategies [ ] seem to facilitate the "admission fees" due to the rationally design and innovative functionalities of the synthesized nano-platforms as presented in figure . one more problem in dermal drug passing is related to inflammation skin pathology. the barrier is changed, and the drug resides much less on the targeted site because of fast penetration. the thermoresponsive drug delivery nanogels used in this purpose have encouraging results for overcoming the abovementioned problems. ph sensitive nanogels can also be utilised for controlled medicinal release. epidermis due to its polarity and solubility, leading to poor clinical efficacy due to delayed antiviral activity and sub-inhibitory concentrations [ ] . nanotechnology strategies [ ] seem to facilitate the "admission fees" due to the rationally design and innovative functionalities of the synthesized nanoplatforms as presented in figure . one more problem in dermal drug passing is related to inflammation skin pathology. the barrier is changed, and the drug resides much less on the targeted site because of fast penetration. the thermoresponsive drug delivery nanogels used in this purpose have encouraging results for overcoming the above-mentioned problems. ph sensitive nanogels can also be utilised for controlled medicinal release. thermoresponsive nanogels can be controlled through irradiation with infrared lamp. another way of skin penetration is the hair follicle. the stratum corneum is less intact in the lower infundibulum, so the nanoparticle's (nps) passage is dependent on size and not on composition [ ] . the cell membrane separates the content of a cell from the exterior surroundings. besides the standard protection around the cell, the cell membrane controls what substances go in and out. the composition is based on a bilayer of phospholipids, internally hydrophobic (tails) and externally hydrophilic (heads) with different proteins and cholesterol between them. the membrane is permeable selectively, permitting only some materials to pass through its lipid layer by active (through protein pumps or vesicles) or passive (diffusion) processes of transportation. water passes the membrane through a process called osmosis, which occurs when an imbalance of solutes appears outside and inside the cell [ ] . in most of the cases, hydrophobic and small molecules can pass through diffusion. nanoscale drug delivery systems depend upon an active mechanism (endocytosis). over this process, the cell unit takes in ions, solid particles and molecules. there are studies that prove that positive charged nanogels can bind the membrane of the cell (negative charge) through electrostatic intercommunication. more than that, receptor-mediated endocytosis can provide a mechanism through which selectively attracted cell groups are targeted. in addition, hydrophobic nanoplatforms can grow the adhesion to the membrane and the amount of drug entered in the cell [ ] . thermoresponsive nanogels can be controlled through irradiation with infrared lamp. another way of skin penetration is the hair follicle. the stratum corneum is less intact in the lower infundibulum, so the nanoparticle's (nps) passage is dependent on size and not on composition [ ] . the cell membrane separates the content of a cell from the exterior surroundings. besides the standard protection around the cell, the cell membrane controls what substances go in and out. the composition is based on a bilayer of phospholipids, internally hydrophobic (tails) and externally hydrophilic (heads) with different proteins and cholesterol between them. the membrane is permeable selectively, permitting only some materials to pass through its lipid layer by active (through protein pumps or vesicles) or passive (diffusion) processes of transportation. water passes the membrane through a process called osmosis, which occurs when an imbalance of solutes appears outside and inside the cell [ ] . in most of the cases, hydrophobic and small molecules can pass through diffusion. nanoscale drug delivery systems depend upon an active mechanism (endocytosis). over this process, the cell unit takes in ions, solid particles and molecules. there are studies that prove that positive charged nanogels can bind the membrane of the cell (negative charge) through electrostatic intercommunication. more than that, receptor-mediated endocytosis can provide a mechanism through which selectively attracted cell groups are targeted. in addition, hydrophobic nano-platforms can grow the adhesion to the membrane and the amount of drug entered in the cell [ ] . the bbb is a highly selective semipermeable structure composed of five parts: the basement membrane, the astrocytes, the immune cells, the pericytes and an endothelial cell layer of capillaries. the area between basement membrane and the neurons is called virchow-robin space. in this region there is interstitial fluid in which reside microglia. all the above components are called neurovascular unit. the kinetics of this unit is crucial to the role of bbb and its states of illness. the capillaries of bbb compose a layer of squamous epithelial cells that fold to form a circular vessel. these cells are linked with strong connections for blocking entrance or exit of materials through central nervous system. protein transportation facilitates the selective flow of molecules through vessel lumens, essential biomolecules being in higher concentrations, like glucose and at the same time eliminating toxins. the bbb is a physical and metabolic "obstacle", physiologically important and active, which survey blood-brain traffic and control it, restricting the paracellular diffusion between the endothelial cells (microvessels) and the efflux pumps activity that quickly expel back into the capillary lumen a wide variety of xenobiotics. bbb integrity and function is critically influenced by what is now referred to as "the extended neurovascular unit" that incorporates not only microvascular endothelial cells and adjacent pericytes, astrocytes and neurons, but also neighbouring smooth muscle cells and microglia in the brain, and blood cells in the capillary lumen such as polymorphonuclear cells, lymphocytes and monocytes [ ] . transport at the bbb level is assured by numerous transport mechanisms that provides to the brain the necessary nutrients and also protects from the toxic xenobiotics. the main transport mechanisms are represented by free diffusion of small lipophilic substances or by active transport (carrier mediated, receptor mediated and active efflux transport). active efflux transport is assured by two major types of transporters that extrude metabolic waste, xenobiotics and a large number of drugs from the brain back into the blood. the first superfamily of bbb efflux transporters is the solute carrier proteins (slc) superfamily, being represented at the level of bbb by slc and slco (slc ) efflux transporters. the second is the atp-binding cassette (abc) efflux transporter family represented by permeability glycoprotein (p-gp), breast cancer resistance protein (bcrp) and the multidrug resistance associated proteins (mrps) [ ] [ ] [ ] . based on their localisation, the abc efflux transporters prevent lipophilic and amphiphilic environmental toxic compounds or drugs, including anti-inflammatory, immunosuppressive, anti-infectious, antineoplastic drugs, some antiepileptic, antidepressant and psychotropic agents, and drug conjugates by an energy-dependent, unidirectional direct transport mechanism, from entering specific substrates [ ] . the bbb's efflux machinery does an excellent job of recognizing xenobiotics, but a poor job on distinguishing between toxicants and therapeutic drugs, creating an important obstacle to treatment of brain cancer, epilepsy and neuro aids [ ] . the penetration of the bbb for drug delivery, although challenging, captivates the interest of numerous researchers in antiviral therapy since the mechanisms by which for example hsv- penetrates the cns remain unclear. the most likely routes include retrograde transport via the olfactory or trigeminal nerve fibres, occasionally leading to herpes simplex encephalitis (hse) caused by hsv- [ ] . another studied virus that is involved in encephalitis and bbb disruption is hiv, known to cause severe neurological disorders and leading to hiv-related encephalitis [ ] since bbb is impermeable to % of antiretroviral drugs [ ] . the possible mechanism responsible for bbb disruption in hiv- encephalitis is considered a "trojan horse" mechanism, where hiv infects specific t-lymphocytes and circulating monocytes, then entering to cns through bbb gaps and followed by inflammatory reactions [ ] , but, in the last years nanotechnology has been intensely explored and several experimental attempts have been carried out in order to enhance the bbb permeability toward antiretroviral drugs, briefly described below based on the nano-based formulation composition, since it is well known that the size and surface functionalization influence transport properties within tissues: • polymeric polybutylcyanoacrylate (pbca) nanoparticles with two incorporeated antiretroviral drugs (azt and lamivudine) showed a - and - fold increase in bbb permeation, by three possible mechanisms as presented by the authors: prolonged interaction interval between drug-loaded nanoparticles and brain-microvascular endothelial cells elevated the concentration gradient between blood and the brain, polysorbate covering on the periphery of nanoparticles was able to be absorbed and degraded nanoparticles improved drug absorption [ ] ; • spherical transferrin coated-pegylated albumin nanoparticles encapsulating azt prepared by ultra-emulsification method using chemical cross-linking by glutaraldehyde gained an access across the bbb through the transferrin receptor mediated endocytosis on the membrane [ ] ; • transferrin-conjugated quantum rod nanoparticles conjugated with saquinavir crossed an in vitro bbb model by exploiting a receptor-mediated transport [ ] ; • magnetic liposomal nanoformulations of azidothymidine -triphosphate (the active form of azidothymidine) migrate across bbb in vitro, either directly or by a monocyte-mediated transport, under the influence of an external magnetic field [ ] ; • novel nanodrug consisting of an iron oxide nanoparticle coated with pma amphiphilic polymer and functionalized with the antiretroviral peptide enfuvirtide crossed the bbb by a passive diffusion, probably mediated by the absorption of the amphiphilic coating on the cell membrane [ ] . as briefly presented the preliminary results are more than encouraging. certainly, future investigations on the mechanisms about bbb disruption are needed along with novel, innovative, safe and efficacious therapeutic approaches. up to now we have concluded that current antiviral therapy has not yet achieved the ideal shape and efficiency and also that the complex biological barriers are major obstacles, but can we critically say that nanotechnology could be the identified solution? search engine queries on "nanotechnology" generate more than , items on specialized platforms (science direct, for example) that represents potential and challenges in different fields from biosensors and industry-related applications up to nanomedicine and biomaterials. when the search keywords are "nanotechnology as antiviral therapy" the same engine only returns results starting from . it is a given fact that nanotechnology is defined as the application of materials on the nanometer scale. according to the literature data results, namomaterials designed with different shapes and morphologies display numerous advantages for use in antiviral therapy, namely: nanometric size that permits drug delivery through impermeable barriers [ ] , large surface area to volume ratios for large drug payloads incorporation [ ] and improved efficacy, surface modification and/or backbone functionalization versatility that facilitates cellular membranes passage [ ] or enhancing stability and bioavailability [ ] , virucidal activity against a series of viruses (hiv, hsv, hbv, etc.) due to biomimetic properties [ ] , increased specificity, improved antiviral delivery and controlled drug release to the target [ ] through engineered moieties, decrease the emergence of drug resistance, personalized therapy possibility, protection of the drugs and low adverse drug side effects mainly due to the composition. the mechanisms of nanomaterial-mediated drug delivery are determined by the chemistry, the architecture and the specific properties of each nanosystem (as presented in the schematic representation in figure ). the design of new drug delivery systems for the antiviral therapy is focused on manipulating these features that are relevant in viral diseases where high drug doses are compulsory, implies high costs and the patient is depended on the administration protocol. lembo and cavalli [ ] present the current status up to in the nanoparticulate delivery systems in antiviral therapy area, highlighting their perspective on the challenges that must be tackled before the nanotechnology can be translated into clinical use as safe and effective antiviral formulations. therefore, the nanoparticulate antiviral systems synthesised up to consisted mainly of micelles, polymeric nps, solid lipid nps (slns), nanostructured lipid carriers (nlcs), liposomes, nanocapsules, vesicles, dendrimers, nanogels, cyclodextrin-based systems and emulsions. tackled before the nanotechnology can be translated into clinical use as safe and effective antiviral formulations. therefore, the nanoparticulate antiviral systems synthesised up to consisted mainly of micelles, polymeric nps, solid lipid nps (slns), nanostructured lipid carriers (nlcs), liposomes, nanocapsules, vesicles, dendrimers, nanogels, cyclodextrin-based systems and emulsions. in , liu and chen [ ] summarized in a review paper an interesting perspective of nanotechnology use in hiv/aids vaccine development. their overview underline the potential of various nanomaterials and nano-architectures to be used as hiv vaccine carriers or adjuvants due to proven capabilities to improve delivery, permeability, stability, solubility and pharmacokinetics of traditional hiv vaccine approaches. the authors exhibit also the desired features of nano-carriers and adjuvants with high benefits-cost ratio. in , milovanovic et al. [ ] outlined, beside, the virus replication cycle and mechanism of actions of antiviral agents, an overview of particulate carriers for drug delivery. the review summarized several classes of the mostly considered carriers namely liposomes, micelles, microspheres, dendrimers and nps synthesized as alternative supports for antiviral therapy. table highlights part of their summary and described based on virus classification in section . in , cao and woodrow [ ] reviewed the nanotechnology solutions used to eradicate hiv reservoirs and also the gene delivery and immunotherapy nanocarriers used in cancer with potential in hiv treatment. in section . nanocarriers for eradicating hiv reservoirs" the authors focused mainly on nanocarriers incorporating combination therapeutics developed in order to boost drug effectiveness and minimize toxicity. several examples are presented in table and described in section . in , liu and chen [ ] summarized in a review paper an interesting perspective of nanotechnology use in hiv/aids vaccine development. their overview underline the potential of various nanomaterials and nano-architectures to be used as hiv vaccine carriers or adjuvants due to proven capabilities to improve delivery, permeability, stability, solubility and pharmacokinetics of traditional hiv vaccine approaches. the authors exhibit also the desired features of nano-carriers and adjuvants with high benefits-cost ratio. in , milovanovic et al. [ ] outlined, beside, the virus replication cycle and mechanism of actions of antiviral agents, an overview of particulate carriers for drug delivery. the review summarized several classes of the mostly considered carriers namely liposomes, micelles, microspheres, dendrimers and nps synthesized as alternative supports for antiviral therapy. table highlights part of their summary and described based on virus classification in section . in , cao and woodrow [ ] reviewed the nanotechnology solutions used to eradicate hiv reservoirs and also the gene delivery and immunotherapy nanocarriers used in cancer with potential in hiv treatment. in section . nanocarriers for eradicating hiv reservoirs" the authors focused mainly on nanocarriers incorporating combination therapeutics developed in order to boost drug effectiveness and minimize toxicity. several examples are presented in table and described in section . recently, arca-lafuente et al. [ ] overviewed nanotechnology-based systems as reliable alternative diagnostic tools for hcv infectious disease. even if our review does not cover screening, it is important to mention that new diagnostic methods are required in order to overcome current drawbacks of hcv under-diagnosed infection as highlighted in the above-mentioned review. the nanotechnology-based tools described in the review seem to fulfil the necessary features for hcv elimination. with the aim of developing new personalized diagnostic tools, farzin et al. [ ] summarized current strategies and under-development tools for early diagnosis of hiv. their review combines the use of nanomaterials such as carbon nanostructures, nanoclusters, quantum dots, metallic and metal oxide nps as advanced structures for hiv detection with possible biosensing strategies targeting to offer innovative outlooks for developing intelligent, sensitive and specific nano-objects for in situ and real-time detection of hiv. in this section the authors point out the suitability of nanomaterials (recent data) for antiviral therapy, highlighting the enhanced features pursued to overcome the identified issues as related above. donalisio et al. [ ] have reported the preparation by a modified nano-emulsion method of chitosan nanospheres (ns) loaded with . % acv as a topical formulation against both hsv- and hsv- herpes virus strains. the main component, chitosan, a natural polycationic polysaccharide, was selected as a material for acv release, due to its distinctive properties: hydrophilic character, in situ gelling, mucoadhesion, permeation enhancing, in addition to a low cytotoxicity, biocompatibility and bioresorbability features [ ] . the obtained gel formulation based on acv-loaded ns proved an enhanced ability to penetrate porcine skin to about % (at h) greater than the commercial cream product ( %). ic values against hsv- and hsv- were also determined on vero cell cultures infected with above-mentioned strains, displaying significant reduced values of . µm and . µm, respectively, when using the ns formulation as compare to . µm and . µm for free acyclovir. this nano-technological approach attests the higher efficacy of the described formulation and with promising expectations for further preclinical and clinical experiments. yadavalli et al. [ ] have explored the potential of highly porous activated carbon (hpac) particles as a model for restricting hsv- and hsv- from entering target cells. they have considered this material due to the charcoal surface-active that could provide antiviral effects through virion sequestration approach. furthermore, acv molecules adsorbed or encapsulated inside the hpac pores revealed sustained drug release acting in a synergistic manner to obtain an enhanced therapeutic effect. the hpac compound prove d a to % reduction in hsv- and hsv- entry for concentrations as low as mg/ml. the ic value of hpac corresponding to hsv- and hsv- infection in prophylactic administration was found of . and mg/ml, respectively, significantly lower than clinically accepted tc value (half maximal toxicity concentration) of hpac. following the promising outcomes from in vitro tests, further determinations of antiviral efficacy on in vivo studies using a murine model of ocular (hsv- ) and genital (hsv- ) infection were performed. as a result, the acv loaded hpac acts by capturing the virus and releasing the encapsulated drug, hindering inflammation and immune cell infiltration in targeted tissue. the strong antiviral activity of this product was assigned to the charged surface of its pores which may interact with the cell's surface, stimulating an active exchange of ions (na + , k + , ca + , cl − , and oh − ), when sustained or slow release of acv has been acquired. moreover, these particles exhibited both prophylactic and therapeutic effects against hsv- /hsv- cells, unlike the free drug that did not demonstrate a prophylactically antiviral response. gold and silver nps using seaweed sargassum wightii with anti-herpetic activity biogenic gold (au) and silver (ag) nps were prepared using the seaweed sargassum wightii (sw) and investigated for their antiviral activity against hsv- and hsv- strains [ ] . the nps synthesis resided in an eco-friendly method, previously described in the literature [ ] , replacing the use of different reducing agents. the obtained nps, sw-au and sw-ag, were evaluated concerning both cytotoxic and antiviral effect, using mtt and cpe (cytopathic effect) assays on vero cells. the results showed that cell viability ranged from % to % when the concentration ranged between . and µl per sample in sw-au, and from % to . % for concentrations of . and µl per sample in sw-ag. the antiviral assay have shown a % decrease of cpe on both hsv- and hsv- when vero cells were treated with and µl sw-au, whereas sw-ag exhibit similar reduction of cpe at a concentration of only . µl per sample. higher concentrations of sw-ag are not accepted due to an increased cytotoxic effect. the authors claimed that the obtained results are in agreement with other published research and they inferred that functionalized metallic nps act as antiviral agents by blocking the virus attachments and cell access, depending on particle size. cagno et al. [ ] conducted a research study concerning broad-spectrum antiviral products, which usually mimic heparan sulfate proteoglycans (hspg), as well-preserved target of "viral attachment ligands" (vals). the antiviral effect relies on the binding mechanism of the nanoparticles to the virus surface, thus preventing virus-cell attachment. in most cases, the reversibility of these bonds is reported [ ] , so that by increasing the dilution viral inhibition is lost, causing those compounds not to be considered antiviral drugs. the aforementioned authors have designed antiviral nanoparticles of virucidal effect based on long and flexible linkers simulating hspg, leading to irreversible viral deformation. of the synthesized compounds, the most notable virucidal effect was found in the aunps coated with a : mixture of decanesulfonic acid (mus) and -octanethiol (ot). mus allows a multivalent binding [ ] as a consequence of its structure comprising a long hydrophobic chain, sulfonic acid terminated. the enhanced activity of mus:ot-nps was assigned to the new construct using mus linker that caused local distortions and then a global virus deformation, leading to irreversible loss of infectivity. the mus:ot-nps exhibited efficient virucidal effect against hsv- and hsv- , human papilloma virus (hpv- ), respiratory syncytial virus (rsv), dengue and lenti virus. the in vivo testing on balb/c mice infected with rsv reveals the efficacy of mus:ot-nps treatment that prevented the pulmonary dissemination of the infection. these results are in agreement with previous published data [ ] , which assessed the relationship between the surface structure of nano-objects and their ability to cross cell membranes. both in vivo and in vitro tests on cell cultures have proven the lack of toxicity of mus:ot-nps. coumestrol is an isoflavonoid-like compound having the ability to inhibit the replication of hsv- (both acyclovir sensitive and resistant strains) and also some strains of hsv- [ ] . argenta et al. [ ] have designed a formulation in an effort to obtain a topical product for coumestrol delivery at the level of mucosa. in this approach, the bioactive compound was entrapped by fluid or rigid phospholipid nanoemulsions (dioleylphosphocholine, dopc and distearoylphosphocholine, dspc, respectively) dispersed in a hydroxyethylcellulose gel. the effectiveness of the proposed antiviral agents was tested regarding permeation and retention ability on intact and damaged porcine esophageal mucosae and for antiherpes activity on cell culture assays using vero and gmk ah cell lines. the greatest performance of both coumestrol-loaded nanoemulsions ne-cou/dopc and the same product thickened with hydroxyethylcellulose, hne-cou/dopc, as compared to those based on dspc, relies largely on the physico-chemical properties of the nanoemulsion. the positively charged nanoemulsion showing highest values of ζ-potential may interact with negatively charged surface of mucosa membrane, with beneficial consequences relating to transmucosal delivery of coumestrol [ ] . the length of phospholipids alkyl chain, the number of unsaturations, the lipophilic/hydrophilic balance of the active principle also contributed to the global effect, so that the fluid-state of hydrocarbon chain induced by dopc explained the interaction between the oil-water interface and mucosa, increasing coumestrol permeation and retention. the low ic values proved an enhanced antiviral activity against hsv- and hsv- after coumestrol formulation using nanoemulsions based on dopc, which could be considered for advanced studies in order to be introduced in therapy. the huge socio-economic impact of hiv, as mentioned in section , determined a continuously increased trend of studies related with finding an almost perfect treatment. since the seven classes of antiretroviral drugs defined by fda contain a large number of active principles, plenty of studies regarding their incorporation in different types of nanosystems can be found in the literature. several examples are presented below. rtis classes, nrtis and nnrtis, include some of the drugs often used in hiv treatment plans. recently, grande et al. [ ] published a complex review on rtis nanosystems for controlled drug delivery and our review complements part of the data presented in this article, emphasizing the penetration of biological barriers in vitro or in vivo by nanosystems containing rtis. azt, a high bioavailable drug, has serious side effects, the most common being bone marrow suppression, toxicity for some organs, neutropenia and anaemia. specific target drug delivery using different nanosystems is a promising solution [ ] . atz has been incorporated in hybrid nps based on alginate and stearic acid-poly ethylene glycol. c glioma, neuro brain and hela cells have been used to study the cellular uptake and the cytotoxicity of the nps in vitro. the results proved that these nanosystems are nontoxic and have significant brain cellular uptake, suggesting that they can be used for more complex internalization in brain cells studies [ ] . in another study, sol-oil chemistry has been used to prepare small nps lactoferrin loaded with azt ( - nm in size), stable in biological simulated fluids (gastric and intestinal). antiviral activity of nps has been analysed using supt cells infected with hiv- in virus and the results suggested that the encapsulation of azt in lactoferrin does not influence the drug activity. the nps loaded with azt and the drug alone have been orally administrated to wistar rats of both genders, the performed assays (bone marrow micronucleus, histopathological and biochemical analysis) showing that azt loaded in nps is more efficient and less toxic, compared with the soluble form [ ] . lamivudine-lam, a water-soluble drug with two main drawbacks: its half-life is only h and has a deficient bioavailability, especially in paediatric patients ( %) [ ] . lam has been included in nps based on poly(ε-caprolactone)-pcl [ , ] , poly lactic-co-glycolic acid-plga [ ] , chitosan and sodium alginate [ ] , eudragit e [ ] . the physico-chemical characterization of the obtained nps has shown an adequate size of the nps and a good stability. in vitro drug release tests indicate that nps can support the drug delivery for h, indicating a less frequent administration [ , ] . sneba et al. [ ] have reported a more complex study, where lam-polymeric non-cytotoxic nps have been included in films for drug delivery through the buccal mucosa barrier. four mucoadhesive polymers: polyvinyl alcohol-pva, polyvinyl pyrrolidone-pvp, sodium carboxymethylcellulose-scm, hydroxypropyl methylcellulose-hpmc have been used to prepare the films. moreover, ozturk et al. [ ] , obtained plga nps loaded with lam and proved that are physicochemical stable and slowly released the drug, a great property attributed to ester end-group of plga. because these nps were intended for oral administration, the authors evaluated the gastrointestinal stability of the nps in vitro, using different fluids of biological interest with ph in the range . - . phosphate buffer solution, intestinal fluid phosphate buffer solution, physiological serum and distilled water; the tests have been developed at • c for h. the results indicated that plga nps are promising intestinal targeted drug delivery system for lma, being stable in tested media. in clinical practice, lam is frequently administrated together with azt, therefore this combination of drugs is being studied for target delivery using different types of nps. sankar et al. [ ] used plga, methylmethacrylate-sulfopropylmethacrylate-mma-spm, poly lactic acid-pla, and poly methyl methacrylate-pmma to prepare different types of nps by emulsion polymerization, as drug delivery nanosystems for atz ( %) and lma ( %). in vivo acute toxicity has been studied in mice; the results proving the fact that the drug doses loaded in the nps are not toxic. atz-lam plga nps seemed to be the most promising nanosystems. efavirenz-efv, one of the most used nnrtis in clinical practice, is a poorly water-soluble drug, and the incorporation in different drug delivery nanosystems being a solution for this drawback. patel et al. [ ] obtained nanosuspensions-ns based on povidone polymer-polyvinylpyrrolidone (pvp) k , poloxamers steric stabilizer ( and ) and an anionic electrostatic stabilizer (sodium lauryl sulphate, a steric stabilizer-sls). compared with the drug alone, an important improvement of saturation solubility has been noticed for the ns with efv. the incorneporation of efv in ns increased the absorption of the drug in rat intestine in situ, and very important the oral bioavailability in the studies on albino rabbits. lactoferin used to prepare nps loaded with atz [ ] , as described above, has been used also to encapsulate efv [ ] , based on the same preparation technique: sol-oil chemistry. compared with free efv, the encapsulation of the drug in nps showed a reduced toxicity to peripheral blood mononuclear cells, jurkat t cells and b -f cells, an increased anti-hiv- activity and improved oral bioavailability and pharmacokinetic profile in studies on rats. atv. low brain permeability and antiretroviral drug resistance are two of the most important disadvantages of atv. this pis drug has been encapsulated in slns and studied as nanosystems for brain delivery using hcmec/d as a blood-brain barrier in vitro model, hcmec/d being human brain microvessel endothelial cell line. average atv had an important increase regarding the cellular uptake once delivered through average nanosystems (around nm) [ ] . sqv, another anti-hiv pis, has been incorporated in poly(ethylene oxide)-modified poly (ε-caprolactone) (peo-pcl) nps [ ] by a solvent displacement process. human monocyte/ macrophage (mo/mac) cell line-thp- has been used for in vitro cellular uptake assay. the drug has been successively released intracellular and a meaningful uptake of the sqv-peo-pcl has been noticed. nfv, used in hiv- and hiv- treatment as pi, is a promising drug that can be used also for other grave medical disorders like cancer [ ] . some studies have showed the ability of different types of nps loaded with nfv to activate latent hiv and to restrict viral spread in vitro. kovochich et al. [ ] showed that lipid nps-lnps incorporated with bryostatin- , a protein kinase c activator (lnp-bry), can be loaded with nfv (lnp-bry-nfv), and proved the above mentioned abilities on j-lat full length cells ( . ). tang et al. [ ] have prepared nps based on poly(lactic-co-glycolic acid)-polyethylene glycol diblock copolymers and anti-cd ro antibody conjugated with suberoylanilide hydroxamic acid (saha) and nfv and tested theirs in vitro properties on ach- cells. more complex studies have been performed by venkatesh et al. [ ] plga nps loaded with nfv have significantly enhanced the oral bioavailability of the drug studied in vivo in new zealand rabbits, a reduced frequency of dosing being needed in this case. the literature data reports also several studies where combinations of pis drugs have been incorporated in nanosystems and pre-clinically evaluated. duan et al. [ ] have included separately atv and drv in lnps, but only atv-lnps proved to form stable drug-lipid concentrations. based on these results, the authors have developed lnps containing atv and rtv and also lnps containing atv + rtv + tenofovir (tfv-an hiv nrtis), the last ones being prepared in a large volume for a preliminary primate pharmacokinetic study. after lnps subcutaneously administration, the three drugs have been detected in plasma for seven days. enf is a fusion inhibitor that is incapable to cross the cerebrospinal fluid. fiandra et al. [ ] proved that by including it into a nanosystem composed from magnetic nanoparticles (mnp) synthesized by solvothermal decomposition in organic solvent followed by fluorescent labelled pma coating could solve enf drawback. in vitro model (co-colture of rbmvecs and astrocytes) and in vivo model (balb/c mice) studies proved that nanoconjugated enf could penetrate bbb. mcv, an entry inhibitor acting as a ccr co-receptor antagonist, has been also included in some nanosystems in order to increase its oral bioavailability. solid drug nanoparticles-sdns, containing wt % mvc and % some polymer/surfactant excipients have been prepared using the emulsion-template freeze drying technique. monolayers of caco- have been used as a human gut in vitro model in order to study the absorption behaviour of mvc sdns and in vivo oral pharmacokinetics of the mvc solid drug nanoparticles (sdns) has been analysed on a rat model. both studies indicated an advanced permeability of the mcv nps (based on pva and sodium , -bis( ethylhexoxy)- , -dioxobutane- -sulfonate (aot) excipients) correlated with the normal drug [ ] . bowman et al. [ ] proved that small organic monovalent molecules conjugated to aunps acts as fusion inhibitors in vitro, while vijayakumar et al. [ ] proved that aunps alone acts as entry inhibitors. moreover, integrase inhibitor, ral, has been functionalized with a thiol group in order to link au-nps. in vitro cellular uptake has been tested on macrophages, human brain microendothelial cells and primary peripheral blood mononuclear cells, the results suggesting that ral-pmba-au-nps penetrate the cells and also can exhibit antiviral activity. in vivo studies performed by injection of ral-pmba-au-nps in female adult balb mice tail proved that the studied nps could cross the bbb [ ] . nanomedicine represents a fast-revolutionizing field that faces rapidly and constantly progress assessed by the numerous nanodrugs that have entered clinical practice and also by even more being investigated in clinical trials. table presents the approved antiviral nanomedicines, from which half are vaccines. [ ] according to singh et al. [ ] review from and also to available information on the respective websites there are still several nanomedicines under evaluation, namely: • fluquit (stp ) from sirnaomics inc. currently under preclinical evaluation, a polymer-based nanotherapeutic that incorporates sirna and targeting the h n (avian flu), h n (swine flu) influenza, and newly emerging h n ; and cervisil (stp ), a nanobased drug candidate, which incorporates sirna for the treatment of hpv and hpv ; • dermavir from genetic immunity, a synthetic pathogen-like nanomedicine that incorporates single plasmid dna expressing hiv antigens that assemble to hiv-like particles; dermavir vaccine completed phase i/ii randomized, placebo-controlled, dose-finding, double-blinded, multicenter study to assess the safety, tolerability and immune response in hiv- -infected adults who are currently receiving anti-hiv treatment (number nct ) [ ] ; • doravirine (mk- ), from merck, a novel, next generation nnrti described as solid drug nanoparticle formulation tested for hiv; currently doravirine completed the pharmacokinetic trial of the bioavailability of four mk- nano formulations in healthy adults (number nct ) [ ] ; • lipid nanoparticles of arb- tkm-hbv containing three rnai therapeutics for hbv genome targeting from arbutus biopharma; in the company completed the phase a, single blind, randomized, placebo controlled, study evaluating the safety, anti-viral activity, and pharmacokinetics (pk) following multiple doses of intravenous arb- (number nct ) [ ] . as discussed above, nanotechnology started to be a critical player in the antiviral therapy. as mentioned by ross et al. [ ] , nanotechnology frees the current therapy payloads in terms of delivery across biological complex barriers, and could resolve the low bioavailability drawback as already stated in section . nanomaterials impart many physical, chemical and biological advantages [ , ] such as: ( ) small particle size in order to facilitate drug delivery through biological barriers, ( ) large surface area to volume ratios to ensure large drug payloads, ( ) tunable surface charge to facilitate cellular entry across the negatively charged cellular membrane, ( ) biomimetic properties which result in intrinsic antiviral assets, ( ) ability to anchor targeting moieties to increase specificity to desired cell types, tissue or other compartments, ( ) improved solubility and pharmacokinetic and/or pharmacodynamics properties translated in longer time to allow greater accumulation, controlled and sustained release, ( ) enhanced efficiency gained either by drug molecules entrapment to protect them from physiologically hostile media, or by using surface conjugation to target drugs to specific tissues, ( ) reduced toxicity and ( ) multifunctionality by combining several beneficial features in a stable construct, designed to simultaneously stimulate the replication of latent virus and deliver an antiviral to the activated cell [ ] . several limitations were also acknowledged such as: ( ) degradation, for example nanoparticles are degraded in the gut following oral administration, or fail to penetrate the mucus barrier and are thus minimally absorbed [ ] , ( ) undesired interactions with biological molecules that leads to opsonization, uptake by macrophages and reduced plasma half-life [ ] , ( ) non-specifically absorption that may induce apoptosis and disrupt cell membrane and adverse immunological responses [ ] , ( ) large dimension for renal clearance therefore cannot be degraded within the body, and are accumulated, leading to toxicity [ ] , and ( ) scaling up issues and high costs. in our perspective, the "ideal" nanocarrier for efficient antiviral delivery must take into considerations several key factors namely: clinical outcome, since patients need safe, effective, targeted, available and affordable therapy, as they are our inspiration; • from the clinical perspective, the future antiviral candidates should improve the efficacy of the fused/encapsulated drug, reduce the intake frequency and time, restrict adverse side effects and reduce therapy costs; • design consideration for the nanoplatforms that will allow targeted delivery of the drugs in sustained released manner and improves efficacy, safety and patient convenience; therefore, from a chemist point of view, hybrid nanosystems can gather all the necessary features in terms of composition, shape and size by overcoming limitations of individual systems and offers greater advantages. starting with the composition, the chosen materials should be biodegradable, biocompatible, and non-toxic, for example polymers are very attractive since they offer the possibility for chemical modifications over the surface or backbone. in addition to these advantages, the second component from the hybrid architecture (in the shape of potential liposomes) should offer besides advanced barrier penetration, higher encapsulation efficiency for the intended drug, which in combination with the polymeric piece will be able to modulate the release kinetics, the stability and prolong drug release. when thinking about the shape, we have in mind targeting capabilities as impact. as we already know, the shape is linked with size and surface charge and density, therefore a complex puzzle that must be solved. the surface charge and density should be carefully chosen during the nanoplatforms design through the surface modification possibility. the ideal candidate here from our perspective is peg due to its versatility to exhibit various charges, shapes and sizes but also to enhance tolerability, reduce clearance, and lengthen circulation time. the size influences the biodistribution and the uptake rate therefore the "nominee" has to be in the submicron size range, recommended to be under nm. taking into consideration the performance indicators of nanomedicine, we claim that the development a personalized nanomedicine is possible via a synergistically approach. since the development of "best" viral carriers involves a multidisciplinary team, virologists should be directly implicated in the development, offering specialized support on the following matters: identification of differentially expressed moieties virus cells for targeted delivery, elucidation of the type of desired targeted and the response from the host cells to nanodelivery platforms. therefore, multidisciplinary research-oriented efforts have to be related also to system biology by exploring machine learning for process optimization and pharmacology in order to introduce best appropriate combination of therapeutic agents. in , an interdisciplinary team of virologists and biochemists, which developed low-cost and "cell-friendly" nanogels that can efficiently prevent viral infections, addressed these challenges [ ] . here, the flexible, nontoxic and broad-spectrum nanogels based on dendritic polyglycerol sulfate mimic cellular surface receptors where several viral families bind. the designed nanogels can multivalently interact with viral glycoproteins, shield virus surfaces, and efficiently block infection since they act as robust inhibitors for these viruses. when thinking about the translation into the clinical practice, the nano-based future antiviral therapy must follow a specific flow-chart, starting with the optimization and scale-up practices according to the good manufacturing practice, the elaboration of suitable regulatory guidelines and finishing with the development of cost-effective and high quality formulations available worldwide. taken into consideration all these enhanced features, the road to clinical practice still has many addressed issues in order to provide effective and safe antiviral nano-formulations to patients. treating or improving treatment success rate for viral diseases are fundamental responsibilities. the established potential and boosted progress of nanotechnology in antiviral therapy development generates great expectations for new therapeutic innovative strategies for attacking or eradicating viral disorders. at present, studies explored numerous and diverse nano-platforms including nanoparticles, liposomes, micelles, with different compositions, size, with single or combined entrapped drugs that may serve as potential antiviral drug delivery transporters. these nano-based systems have exhibited versatile features to improve the identified current therapy drawback. however, the clinical use of a nano-based antiviral formulation to date based on our knowledge has turned out just a few approved or under clinical trials nanoformulations, mainly vaccines, 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nanogels exhibit broad-spectrum antiviral activity by blocking virus entry this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -si pb authors: jouneau, s.; poineuf, j.-s.; minjolle, s.; tattevin, p.; uhel, f.; kerjouan, m.; le hô, h.; desrues, b. title: which patients should be tested for viruses on bronchoalveolar lavage fluid? date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: si pb bronchoalveolar lavage (bal) is a major diagnostic tool in lung diseases, including viral respiratory infections. we aimed to better define the situations where viral tests should be performed on bal fluid (balf). we retrospectively studied all cases where viral tests [immunofluorescence, immunocytochemistry, viral culture, and/or polymerase chain reaction (pcr)] were performed on balf during a period of year ( ) in our institution. we compared the characteristics of patients with virus-positive versus virus-negative balf. of the balf samples sent to the microbiology laboratory, underwent viral tests. of these, ( %) were positive and identified viruses: herpes simplex virus (hsv)- (n = ), cytomegalovirus (cmv, n = ), epstein–barr virus (ebv, n = ), human herpesvirus (hhv)- (n = ), respiratory syncytial virus (rsv, n = ), rhinovirus (n = ), and adenovirus (n = ). the variables associated with positive viral tests on univariate analysis were immunosuppression [human immunodeficiency virus (hiv), corticosteroids > mg/day for ≥ weeks, or other immunosuppressive therapy], ground-glass attenuations on computed tomography (ct) scanning, late-onset ventilator-associated pneumonia (vap), and durations of (i) hospital stay, (ii) intensive care unit (icu) stay, and (iii) mechanical ventilation before bal (p < . for each comparison). on multivariate analysis, only immunosuppression [odds ratio (or) . , % confidence interval (ci) [ . – . ], p < . ] and ground-glass attenuations (or . , % ci [ . – . ], p = . ) remained associated with virus-positive bal. none of the viral tests performed on balf for the initial assessment of diffuse infiltrative lung disease (n = ) was positive. pcr improved the diagnostic yield of viral tests on balf by %. testing for viruses on balf should be mostly restricted to immunocompromised patients with acute respiratory diseases and/or patients with unexplained ground-glass attenuations on ct scanning. bronchoalveolar lavage (bal) is a major diagnostic tool for infectious lung diseases, especially in immunocompromised patients [ ] . viral agents play an important role as etiologies of pneumonia in immunocompetent and immunocompromised hosts [ ] [ ] [ ] [ ] , and may be involved in a substantial proportion of asthma and chronic obstructive pulmonary disease (copd) exacerbations [ ] [ ] [ ] [ ] . the development of molecular biology techniques, including polymerase chain reaction (pcr), has dramatically increased the yield of viral tests for various respiratory diseases [ , , , ] . however, most studies have been performed in specific settings, with highly selected patients. hence, the yield of viral tests on bal fluid (balf) remains poorly characterized in routine practice. a key dilemma with the development of new tests is the relatively high cost associated with their non-discriminated use. to better inform the use of viral tests in patients with respiratory diseases, we performed an observational, retrospective study in our institution, with three aims: (i) to assess the diagnostic value of viral tests on balf in routine practice; (ii) to analyze the characteristics of patients with virus-positive balf; and (iii) to identify the factors predictive of positive viral tests in balf. pontchaillou university hospital is a , -bed tertiary-care hospital which serves as a referral center for the area of rennes, france. all adults (≥ years of age) who had viral test(s) performed on balf during the year were included. cases were identified through the computerized database in the virology department. data were extracted from this database and from the medical records using a standardized questionnaire. the protocol was approved by our institutional review board (rennes university hospital ethics committee, project approval number . ), and informed consent was waived. the data collected included demographic information and co-morbidities. alcohol abuse was defined as daily consumption > units/day for men or > units/day for women. patients were classified as immunocompromised if they were infected with human immunodeficiency virus (hiv), were on systemic corticosteroids ≥ mg/day for at least weeks [ ] , or were on any immunosuppressive drug on admission. medical charts were checked for the following signs: fever > °c, cough, purulent sputum, hemoptysis, chest pain, dyspnea, and crackles on chest examination. radiographic patterns prior to bal were defined by the type of consolidation (alveolar or interstitial, nodules, micronodules, ground-glass attenuations, bronchiectasis, or excavation). other data collected included any antimicrobial treatment received during the week preceding bal, mechanical ventilation, intensive care unit (icu) admission, and in-hospital mortality. bal was performed according to the guidelines [ , ] , under local anesthetic (lidocaine spray): - ml of sterile saline solution was instilled - times into the distal bronchial tree, either at the site where radiographic abnormalities predominated or in the middle lobe in cases with diffuse radiographic pattern. balf specimens were aliquoted and immediately transported to laboratories. appropriate staining was carried out for the direct identification of bacteria, mycobacteria, fungi, and parasites. cultures for bacterial identification were inoculated under standard aerobic conditions on four different media, as well as on specific media for mycobacterium spp., when indicated. for the usual respiratory pathogens, the bacterial count was considered to be significant when quantitative culture yielded > cfu/ml of specimen. in addition, tests for atypical pneumonia agents were performed at the request of the physician in charge, on balf (i.e., pcr, immunofluorescence, specific staining, and culture on appropriate media) and by serology. the first sample of balf was used for viral tests, as it is the most likely to contain significant numbers of epithelial cells. the sample was mixed (volume : ) with viral transport medium, i.e., minimum essential medium enriched with sorbitol ( %), bovine serum albumin, streptomycin, vancomycin, colimycin, and amphotericin b. balf was first analyzed by immunofluorescence (if) with a panel of monoclonal antibodies against respiratory syncytial virus (rsv), a and b influenza viruses, - parainfluenza viruses, and adenovirus, as well as herpes simplex viruses (hsv)- and - , in immunocompromised patients. in parallel, balf was inoculated onto mrc- and llc-mk monolayer cells, and also onto mdck cells during the influenza season. in immunocompromised patients, an immunocytochemistry (icc) for cytomegalovirus (cmv) was performed. the detection of viral genomes was carried out according to the clinical context and a request from the physician in charge. hsv- , hsv- , cmv, varicella zoster virus (vzv), epstein-barr virus (ebv), and human herpesvirus (hhv)- were detected using a commercial method (herpes consensus®, argène, france). in addition, cmv, hsv- , hsv- , ebv, hhv- , and influenza a and b were detected with in-house pcr, adapted from previously described methods [ ] . neither pcr testing on nasopharyngeal swab/aspirates nor viral serology tests were routinely performed during the study period in our institution. statistical analyses were performed using sas software . (sas institute inc., cary, nc, usa). the results are presented as the mean ± standard deviation, with the range in parentheses. we compared the characteristics of patients with viruspositive versus virus-negative balf using student's t-test (n≥ ) or the mann-whitney test (n< ) for quantitative variables, and fisher's exact test for categorical variables. multiple comparisons were performed using analysis of variance with a bonferroni post hoc correction. multivariate analysis was performed using logistic regression models, after adjustment for the duration of stay before balf. variables with a p-value < . in univariate analysis were entered into the multivariate analysis. values of p< . were regarded as significant. in , balf samples were sent to the microbiology department in our institution. of these, viral tests were ordered in balf, from patients (fig. ) . the mean age of the patients was . ± . years (range, . - . ) and the male-to-female ratio was . ( / ). symptoms on admission included dyspnea ( . %), cough ( . %), fever ( . %, with a mean body temperature of . ± . °c (range, . - . ), purulent sputum ( . %), and hemoptysis ( . %). chest radiographic findings included consolidations ( . %), nodules ( . %), and excavation ( . %). chest computed tomography (ct) scanning, performed in patients, indicated ground-glass attenuations ( . %), micronodules ( . %), and bronchiectasis ( . %). for procedures ( . %), bal was performed in an icu, including procedures performed under mechanical ventilation. the mean volume of sterile saline serum instilled during the bal procedure was ± ml (range, . the use of viral transport medium was adequate for % of samples. of the bal investigated for viruses, ( . %) identified at least one virus, for a total of viruses (mean, . viruses per virus-positive balf). viral species identified, and the yields of the four techniques used, are detailed in table . of note, . % of viruses were members of the herpesviridae family. the most frequent virus associations were hsv- +hhv- (n ) and cmv + ebv (n ). one hiv-infected patient with interstitial pneumonia had three herpes viruses identified in the bal: cmv (icc), hsv- (viral culture), and ebv (pcr). pcr, performed in balfs, was positive in cases ( . %). of these, pcr was the only test positive for virus in cases, confirmed the diagnosis documented by other techniques in cases (hsv- , n ; cmv, n ; ebv + cmv, n ) and identified a virus different to that documented by other techniques in eight cases. had pcr not been used, the diagnostic yield would have been %. hence, pcr improved the diagnostic yield of viral tests on balf by %. testing for bacteria and mycobacteria was performed in balfs ( . %) and was positive in balfs comparisons between virus-positive and virus-negative balf are detailed in table (univariate analysis). the variables significantly associated with positive viral tests on univariate analysis were immunosuppression (i.e., hiv infection, corticosteroids > mg/day for ≥ weeks, and/or other immunosuppressive therapy), ground-glass attenuations on chest ct scans, late-onset ventilator-associated pneumonia (vap), and durations of (i) hospital stay, (ii) icu stay, and (iii) mechanical ventilation before bal was performed (p< . for each comparison). on multivariate analysis, after adjustment for the duration of stay before bal, only immunosuppression [odds ratio (or) . , % confidence interval (ci) [ . - . ] , p< . ) and ground-glass attenuations (or . , % ci [ . - . ], p . ) remained significantly associated with virus-positive bal. retrospective analysis of medical charts allowed us to classify indications for the viral analysis of balf into eight categories (table ) and to estimate the diagnostic yield of viral tests in each subgroup. striking differences were observed: for example, the proportion of virus-positive balf was . % in immunocompromised patients, as compared to . % in immunocompetent patients (p< . ). none of the bal performed for the initial assessment of diffuse infiltrative lung disease was virus-positive. associations between viral analysis of bronchoalveolar lavage (bal) and outcomes ( . %) received an antiviral agent: aciclovir (n ), ganciclovir (n ), valganciclovir (n ), or foscarnet (n ). icu patients with virus-positive balf were more likely to be treated than non-icu-patients with virus-positive balf ( % vs. %, p . ). there was no significant association between antiviral treatment and outcome in patients with virus-positive bal. this observational study evaluated the diagnostic yield of viral tests on balf when requested by the physician in charge. of the consecutive balf tested for viruses, ( %) were positive. previous studies have estimated the diagnostic yield of viral tests from balf in different settings, and the proportion of virus-positive balf ranged from to . %, depending on the population studied (e.g., immunocompromised, icu patients) and the viral techniques used (e.g., pcr, icc, if, culture) [ - , , ] . a broad range of respiratory diseases have been associated with viral infections. hence, testing for viruses in balf may be considered in patients with a wide spectrum of clinical and radiological abnormalities [ , ] . however, our study suggests that viral tests are unlikely to return positive except in two, nonexclusive, situations: (i) immunocompromised patients; (ii) bilateral ground-glass attenuations on ct scan. in this study, the vast majority of viruses detected belong to the herpesviridae family, mainly hsv- ( . % of all [ ] . cmv replication was observed in the plasma of onethird of cmv-seropositive patients with vap after - days of mechanical ventilation, and lung involvement was documented by cmv-related cyto-pathogenic effect in - % of patients [ ] [ ] [ ] . all these studies found that hsv and cmv in balf are associated with increased morbidity and/ or mortality. however, the causal relationship between hsv or cmv in balf and patient outcomes cannot be ascertained from these observational studies. among the rapid tests currently available, pcr is one of the most valuable, the results being available within hours with high sensitivity, especially in immunocompromised patients [ ] . multiplex pcr tests, with their ability to detect several viruses in one set, may be particularly interesting in the diagnostic workup of acute respiratory diseases suspected to be of viral origin [ ] [ ] [ ] . in our study, the diagnostic yield of viral tests would have dropped from to %, had pcr tests not been performed. this advocates for the systematic use of pcr techniques for viral tests in balf, in accordance with previous studies [ , ] , in the situations where viruses may reasonably be suspected (i.e., acute lower tract respiratory disease in immunocompromised patients and/or patients with unexplained bilateral ground-glass attenuations on ct scan). on the other hand, the initial assessment of immunocompetent patients with interstitial lung disease should not include any viral test on balf, as previously reported in patients with interstitial fibrosis [ ] . during the exacerbation of idiopathic fibrosis, viral tests on balf may be of higher value, although recent papers have questioned their clinical significance [ , ] . this study has limitations related to its retrospective, monocentric, and observational design, as investigations on balf were not protocolized, and the request for viral testing was left to the discretion of the physician in charge. firstly, the retrospective review of medical charts identified a significant proportion of patients who were unlikely to suffer from viral infections, and for whom viral tests should not have been performed. in contrast, among patients not included in this study as no viral test was requested, there probably were patients who would have benefited from viral tests on balf. however, the comparison of the patients who had balfs tested for viruses in and a random selection of patients who had balf samples not tested for viruses during the same year found that the only significant differences were immunodepression ( % vs. %, p< . ) and ground-glass attenuations on ct scan ( % vs. %, p< . ). this suggests that clinicians are aware of the situations most likely to be associated with the presence of viruses, and that they appropriately select the patients in whom viral tests are more likely to return positive. lastly, pcr was performed in only of balf investigated for viruses. more systematic use of pcr tests in these patients may have increased the proportion of viruses identified. secondly, the identification of virus in balf during the diagnostic workup of a respiratory disease does not systematically imply that the virus is responsible for the disease and that the patient will improve with appropriate antiviral treatment. despite these limitations, this observational study allowed us to identify categories of respiratory diseases where viral tests are very unlikely to return positive (e.g., initial assessment of immunocompetent patients with interstitial pneumonia or pulmonary micronodules). as a consequence, indications for viral tests in balf were dramatically reduced for these patients in our institution. in conclusion, testing for viruses on balf should be mostly restricted to acute lower tract respiratory disease in immunocompromised patients and/or patients with unexplained groundglass attenuations on ct scan, especially when pcr tests are used. acknowledgments the authors thank jean-marc malecot for his statistical advice. the authors declare that they have no conflict of interest. ards acute respiratory distress syndrome a patients were classified as immunocompromised if they were infected with human immunodeficiency virus (hiv), were on systemic corticosteroids ≥ mg/day for at least weeks, or were on any immunosuppressive therapy b clinical or radiological deterioration in immunocompetent patients with infiltrative lung disease (n ), chronic cough (n ), post-surgical atelectasis (n ), pulmonary abscess (n ), unexplained hyperleukocytosis under mechanical ventilation (n ), initial assessment for pulmonary nodules (n ), systemic granulomatosis (n ), or screening before bone marrow allograft in a patient with ill-defined pulmonary abnormalities (n ) a persistent challenge: the diagnosis of respiratory disease in the non-aids immunocompromised host virological diagnosis in community-acquired pneumonia in immunocompromised patients viral community-acquired pneumonia in nonimmunocompromised adults lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults acute exacerbations of copd: identification of biological clusters and their biomarkers role of viral respiratory infections in asthma and asthma exacerbations importance of viral and bacterial infections in chronic obstructive pulmonary disease exacerbations infections and asthma committee, environmental and occupational respiratory diseases interest section risk of infectious complications in patients taking glucocorticosteroids technical recommendations and guidelines for bronchoalveolar lavage (bal) diagnostic flexible bronchoscopy. recommendations of the endoscopy working group of the french society of pulmonary medicine amplification of the six major human herpesviruses from cerebrospinal fluid by a single pcr respiratory viruses and severe lower respiratory tract complications in hospitalized patients viral pneumonia thin-section ct findings in hematopoietic stem cell transplantation recipients with respiratory virus pneumonia comparison of initial high resolution computed tomography features in viral pneumonia between metapneumovirus infection and severe acute respiratory syndrome herpes simplex virus in the respiratory tract of critical care patients: a prospective study monitoring of herpes simplex virus in the lower respiratory tract of critically ill patients using real-time pcr: a prospective study herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation cytomegalovirus reactivation in critically ill immunocompetent patients cytomegalovirus infection in critically ill patients: a systematic review active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-pcr: feasibility study evaluation of a multiplex reverse transcriptase pcr elisa for the detection of nine respiratory tract pathogens role of flexible bronchoscopy in immunocompromised patients with lung infiltrates herpesviruses detection by quantitative real-time polymerase chain reaction in bronchoalveolar lavage and transbronchial biopsy in lung transplant: viral infections and histopathological correlation viral infection in acute exacerbation of idiopathic pulmonary fibrosis acute exacerbations of idiopathic pulmonary fibrosis key: cord- -jmjolo p authors: pulliam, juliet r. c.; dushoff, jonathan title: ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: jmjolo p understanding viral factors that promote cross-species transmission is important for evaluating the risk of zoonotic emergence. weconstructed a database of viruses of domestic artiodactyls and examined the correlation between traits linked in the literature to cross-species transmission and the ability of viruses to infect humans. among these traits-genomic material, genome segmentation, and replication without nuclear entry-the last is the strongest predictor of cross-species transmission. this finding highlights nuclear entry as a barrier to transmission and suggests that the ability to complete replication in the cytoplasm may prove to be a useful indicator of the threat of cross-species transmission. previous studies have compared emerging human pathogens to nonemerging human pathogens and looked for characteristics typical of those considered to be emerging [ ] [ ] [ ] . to ask which characteristics predict host jumps requires a different approach. specifically, we must examine the pool of other hosts' pathogens that a target species regularly encounters. from this pool we can compare the characteristics of microbes that are able to infect the target host versus those that manifest no evidence of an ability to infect the target host. molecular characteristics that facilitate cross-species transmission are likely to be substantially different between viruses, bacteria, and protozoa, because of large differences in the pathobiology of these different taxa. here, we focus on cross-species transmission of viral infections and examine the effects of characteristics that are described in the literature as expected to affect the ability of a viral group to infect a novel host species: genome segmentation, genomic material, and site of replication. the ability to rapidly explore genetic state space is expected to increase the probability of a host jump, so we expect that viruses with rna genomes will have a higher probability of jumping than viruses with dna genomes [ , ] and that viruses with segmented genomes will have a higher probability of jumping than viruses with nonsegmented genomes [ ] . complex interactions with a host's cellular machinery, on the other hand, are expected to decrease the probability of a host jump, so we expect that viruses that are able to complete replication in the cytoplasm will have a higher probability of jumping than viruses requiring nuclear entry [ ] . to examine the effects of these characteristics, we should choose a target species that will maximize the chance that viral infection due to cross-species transmission events will have been detected; the obvious choice is humans. likewise, we should minimize differences in exposure of the target host to infectious virions produced by the source hosts. humans have regular contact with all potentially infectious bodily fluids of domestic food animals; we thus ensure that the target species has contact with all viral groups infecting the source hosts by analyzing the pool of viral species known to infect sheep, goats, cattle, and pigs. methods. we constructed a database containing taxonomic and molecular data on known viruses of domestic artiodactyls. to determine which viruses to include in the database, we searched the primary literature for references documenting infection of these species with all recognized species in all viral genera known to infect mammals. for each viral species infecting sheep, goats, pigs, or cattle, we then searched the literature to determine whether human infections have been documented (see table a in appendix a, which appears only in the electronic edition of the journal). viruses dependent on coinfection with other viral species, known to be maintained through continuous transmission within humans (see appendix a), or for which documented instances of artiodactyl infection resulted from human-to-animal transmission or experimental infection were excluded from the database. all literature searches were performed between january and february using web of science. the database contains information on the molecular characteristics hypothesized to influence the potential of a virus to cross host species: site of replication (x sr ; whether replication is completed in the cytoplasm or requires nuclear entry), genomic material (x gm ; rna or dna), and segmentation of the viral genome (x seg ; segmented or nonsegmented). these characteristics are conserved at the family level, and classifications were made on the basis of standard reference books [ , ] . we used a combination of hypothesis testing and modelbased prediction to analyze the database. hypothesis testing allowed us to determine how likely it was that the observed patterns were due to chance, whereas model-based prediction allowed us to determine what trait or set of traits was the best predictor of a livestock virus's ability to infect humans and to estimate the probability that a particular virus species would be able to jump host species, given knowledge of the traits of interest. computer code and data are available at http://lalashan .mcmaster.ca/hostjumps/ or from the authors. to determine the statistical significance of the effect of each trait on zoonotic transmission independent of the other traits of interest, we performed a series of randomization tests. for a particular trait, we held the values of the other traits and the ability of the viral species in the database to infect humans constant and permuted the values of the trait of interest within each subset defined by the other traits (thereby preserving the full cross-correlational structure of the data with regard to the viral traits) , times. the p value was given by the proportion of permutations that allowed the model to predict outcomes as well as or better than the model that was constructed using the observed data, and an ␣ level of % was used to determine the statistical significance of results. we compared model fit by use of a logistic regression model that predicted the ability to infect humans as a function of replication site, genomic material, and segmentation. the logistic model was fit in the r statistics package [ ] , and fits were compared on the basis of likelihood. because the traits examined are conserved at the family level for all species in our database, treating species as independent may bias our results. we therefore repeated our analysis at the genus and family levels. permutations of the data set were constructed by permuting the values of the trait under consideration at the taxonomic level examined and assigning species within a genus (or family) the corresponding trait value after permutation. p values were calculated as in the species-level analysis. to examine the magnitude and relative importance of the effects that the molecular characteristics of interest have on the ability of the viral species in the database to infect humans, we developed a set of logistic regression models. each model included some combination of viral traits as independent variables and the ability to infect humans as the dependent variable. traits not having a statistically significant effect on the ability of livestock viruses to infect humans were still considered for modelbased predictions, because sample sizes were limited and small-but real-effects may not be detected via hypothesis testing. we estimated parameter values for each model in r and compared models using akaike's information criterion adjusted for small sample size (aic c ) [ ] . results. a total of viral species were found to infect the livestock species of interest and meet other criteria for inclusion in the database. of these, species (representing genera in families) fulfilled the criteria for inclusion in the analysis. the effect of site of replication was significant at all taxonomic levels examined (p Ͻ . , p ϭ . , and p ϭ . for the species, genus, and family levels, respectively), with nearly half of the virus species completing replication in the cytoplasm able to infect humans. neither genomic material nor segmentation showed a significant effect on the ability of livestock viruses to infect humans at any taxonomic level. logistic regression model comparisons are summarized in table . the models are given in order as ranked by aic c . figure compares the observed data with the results of the best model. the best model included site of replication as the only variable (odds ratio, . [ % confidence interval, . - . ), and the top models were the that included site of replication. each of these models showed a positive correlation between replication in the cytoplasm and the ability to infect humans, as expected. segmentation appears in models , , and , and all models showed a positive correlation between having a segmented genome and the ability to infect humans. genomic material appears in models , , , and . again, all models showed a correlation in the expected direction. it is interesting to note that both of the viral species that caused major pandemics in humans in the th century (hiv and influenza virus a) require nuclear entry for replication. because influenza virus a infects domestic artiodactyls but was excluded from our database because it is maintained through continuous transmission in humans, we confirmed the robustness of our results to this exclusion; we also confirmed that our findings were robust to the inclusion of viral species for which human infection data were based solely on serology (see table b in appendix b, which appears only in the electronic edition of the journal). discussion. our analyses indicate that viral species infecting domestic artiodactyls are more likely to infect humans if they complete replication in the cytoplasm without nuclear entry. the observed effect of cytoplasmic replication on host-jumping ability is not surprising given the complex molecular pathways regulating nuclear entry. viral species that are unable to complete replication in the cytoplasm require intracellular transport from the site of penetration, targeting of the nucleus through nuclear localization signals, and importation of genetic material, proteins, and/or whole virions through the nuclear pore complex [ ] . the combination of molecular mechanisms governing this chain of events is likely to be highly host specific, because of strong selective pressure against admission of foreign particles into the nucleus. to date, discussion of barriers to viral replica-tion has largely focused on receptors for cellular entry. the concentration on this aspect of the viral life cycle exists for substantive reasons. first, the inability to enter a cell obviously precludes viral replication; second, several well-documented viral host jumps have been shown to occur after point mutations that modify interactions between viral particles and cellular receptors [ ] [ ] [ ] . the effect of nuclear entry seen in our data set emphasizes that cellular entry, while a necessary step, is insufficient for completion of the viral life cycle. the ability to produce genetic diversity is the factor most widely discussed as expected to increase viral host-jumping ability [ , [ ] [ ] [ ] ] . although the observed effects of genomic mate- note. x sr , x gm , and x seg are variables indicating the molecular characteristics of a viral species (see methods). ln(ᐉ ) is the log likelihood of the best-fit parameter combination for a given model. k is the no. of model parameters for a given model. aic c is the value of akaike's information criterion with small sample size correction for each model; thus, ⌬aic c is the difference in aic c value between a given model and the best model (i.e., the model with the lowest aic c value). w i is the akaike weight of the model. ␤ seg , ␤ gm , and ␤ sr are regression coefficients for genome segmentation, genomic material, and site of replication, respectively. ␤ i represents the estimated intercept for the best-fit parameter combination for each model. rial and segmentation were not statistically significant, our data do not necessarily contradict this expectation. the hypothesized effect of segmentation, in particular, may be obscured in our data set by a combination of the small number of viral species with segmented genomes and the absence of segmented dna viruses. on the other hand, the lack of predictive power associated with genomic material and segmentation in our data set may indicate that consideration of these traits alone is insufficient to capture the potential to generate useful genetic diversity. the degree to which the pool of viruses infecting domestic artiodactyls is typical of all potentially zoonotic viral species is uncertain. other pools of viral species should be examined to determine the generality of our results. similarly, further studies should examine whether the observed patterns hold for crossspecies transmission of viruses to other target host species, including wildlife and domestic animals. given the rapid rates at which ecological relationships between species are changing as a result of anthropogenic landscape changes, global warming, and globalization of both human and animal populations, the development of indicators of the risk of cross-species pathogen transmission is an increasingly important goal. as humans, domestic animals, and wildlife are brought into contact with species from which they were formerly isolated, they inevitably encounter the pathogens that these species carry. the finding that the ability to complete replication in the cytoplasm is the best predictor of zoonotic transmission and that nearly half of domestic artiodactyl viruses that are able to complete replication in the cytoplasm can infect humans suggests that cytoplasmic replication will be a useful indicator of the ability of a newly encountered virus species to jump hosts, an essential prerequisite to epidemic or pandemic emergence [ ] . it should be noted, however, that the present analysis focused exclusively on the ability to infect the target host, and the viral traits influencing this step in the emergence process may differ from those that predispose a virus to cause severe disease in a novel host as well as from those that facilitate transmission within a novel host species. diseases of humans and their domestic mammals: pathogen characteristics, host range, and the risk of emergence risk factors for human disease emergence host range and emerging and reemerging infectious diseases evolvability of emerging viruses viral host jumps: moving toward a predictive framework virus taxonomy: eighth report of the international committee on taxonomy of viruses the springer index of viruses r: a language and environment for statistical computing. vienna: r foundation for statistical computing in: model selection and multimodel inference: a practical information-theoretic approach viral entry into the nucleus the natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor structure of sars coronavirus spike receptor-binding domain complexed with receptor a single amino acid in the pb gene of influenza a virus is a determinant of host range molecular constraints to interspecies transmission of viral pathogens origins of major human infectious diseases key: cord- -kqcx lrq authors: ladner, jason t.; beitzel, brett; chain, patrick s. g.; davenport, matthew g.; donaldson, eric; frieman, matthew; kugelman, jeffrey; kuhn, jens h.; o’rear, jules; sabeti, pardis c.; wentworth, david e.; wiley, michael r.; yu, guo-yun; sozhamannan, shanmuga; bradburne, christopher; palacios, gustavo title: standards for sequencing viral genomes in the era of high-throughput sequencing date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: kqcx lrq thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. however, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. we also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques. v iruses represent the greatest source of biological diversity on earth, and with the help of high-throughput (ht) sequencing technologies, great strides are being made toward the genomic characterization of this diversity ( ) ( ) ( ) . genome sequences play a critical role in our understanding of viral evolution, disease epidemiology, surveillance, diagnosis, and countermeasure development and thus represent valuable resources which must be properly documented and curated to ensure future utility. here, we outline a set of viral genome quality standards, similar in concept to those proposed for large dna genomes ( ) but focused on the particular challenges of and needs for research on small rna/ dna viruses, including characterization of the genomic diversity inherent in all viral samples/populations. our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques. despite the small sizes of viral genomes, complications related to limited rna quantities, host "contamination," and secondary structure mean that it is often not time-or cost-effective to finish every genome, and given the intended use, finishing may be unnecessary ( ) . therefore, we have used technology-agnostic criteria to define five standard categories designed to encompass the levels of completeness most often encountered in viral sequencing projects. each viral family/species comes with its own challenges (e.g., secondary structure and gc content); therefore, we provide only loose guidance on the depth of sequence coverage likely required to obtain different levels of finishing. in reality, a similar amount of data will generate genomes with different levels of finishing for different viruses. to alleviate any reliance on particular aspects of the different sequencing technologies, we have made two assumptions that should be valid in most viral sequencing projects. the first assumption is a basic understanding of the genomic structure of the virus being sequenced, including the expected size of the genome, the number of segments, and the number and distribution of major open reading frames (orfs). fortunately, genome structure is highly conserved within viral groups ( ) , and although new viruses are constantly being uncovered, the discovery of a novel family or even genus remains relatively uncommon ( ) . in the absence of such information, the defined standards can still be applied following further analysis to determine genome structure. the second assumption is that the genetic material of the virus being described can be accurately separated from the genomes of the host and/or other microbes, either physically or bioinformatically. depending on the technology used, it is critical that the potential for crosscontamination of samples during the sample indexing/bar coding process and sequencing procedure be addressed with appropriate internal controls and procedural methods ( ) . for a summary of the proposed categories for whole-genome sequencing of viruses, see fig. and table . the "standard draft" category is for whole shotgun genome assemblies with coverage that is low and/or uneven enough to prevent the assembly of a single contig for Ն genome segments. genomes in this category are likely to result from samples with low viral titers, such as clinical and environmental samples, or to be those containing regions that are difficult to sequence across (e.g., intergenic hairpin regions) ( ) . to distinguish standard drafts from targeted amplification of partial viral sequences, standard drafts should contain at least contig for each genomic segment and should be prepared in a manner that allows the possibility of sequencing the vast majority of a virus's genome. to avoid the inclusion of small pieces of genomes as "drafts," there needs to be some type of minimum cutoff for breadth of coverage. therefore, we suggest that at least a majority (Ն %) of the genome be present for a set of sequences to be considered a draft genome. high quality (hq). genomes should be considered high quality if no gaps remain (i.e., a single contig per genome/segment), even if one or more orfs remain incomplete due to missing sequence at the ends of segments. an hq genome can often be achieved with modest levels of ht sequencing coverage (~ to ϫ) or through sanger-mediated gap resolution of an sd. coding complete (cc). the "coding complete" category indicates that in addition to the lack of gaps, all orfs are complete. this level of completion is typically possible with high levels of ht sequencing coverage (Ͼ ϫ) or may require the use of conserved pcr primers targeting the ends of the segments. complete. a genome is complete when the genome sequence has been fully resolved, including all non-protein-coding sequences at the ends of the segment(s). this is typically achieved through rapid amplification of cdna ends (race) or similar procedures. finished. this final category represents a special instance in which, in addition to having a completed consensus genome sequence, there has been a population-level characterization of genomic diversity. typically this requires~ to , ϫ coverage (see below). this provides the most complete picture of a viral population; however, this designation will apply only for a single stock. additional characterizations will be necessary for future passages. population-level characterization. ht sequencing technologies provide powerful platforms for investigating the genetic diversity within viral populations, which is integral to our understanding of viral evolution and pathogenesis ( , ) . population-level characterization requires very high levels of ht sequencing coverage ( , ); however, the exact level will depend on the background error profiles of the sequencing technology and the desired level of sensitivity. as an example, wang et al. ( ) determined that for pyrosequencing data,~ ϫ coverage is necessary to identify minor variants present at % frequency with . % confidence, and~ , ϫ coverage is needed for variants with a frequency of . %. targeted amplification of the viral genome is often necessary to achieve these coverage requirements. due to the modest sequence lengths of most ht technologies, the state of the art for population-level analysis has been the characterization of unphased polymorphisms. however, single-molecule technologies, with maximum read lengths of Ͼ kb, are opening the door for complete genome haplotype phasing ( ) . identification of contaminants or adventitious agents. after isolation, viruses are often maintained as stocks, which are propagated within host cells in tissue culture and thus amplified and preserved for future use. despite careful laboratory practices, it is possible for these stocks to become contaminated with additional microbes. contaminating microbes are often detrimental to subsequent applications such as vaccine development or the testing of therapeutics, making it imperative to monitor the purity of viral stocks. ht sequencing provides a powerful method for not only detecting the presence of contaminants within a sample but also for identification and characterization of any contaminants. the level of sequencing required for contamination analysis is dependent on the desired sensitivity, with more sequencing required to ensure detection of contaminants present at very low levels. for most approaches, hq-level sequencing should be sufficient. depending on the intended applications, analysis may need to be repeated after further passaging to ensure that no additional contaminants have been introduced. description of novel viruses. despite the rapidly growing collection of viral sequences, the description of novel viruses is likely to remain an important aspect of viral genome sequencing ( , , ) . this is true in part because viruses evolve rapidly and are capable of recombining to form novel genotypes ( , ) . it is also true that most of the viruses that are currently circulating remain uncharacterized ( ) . particularly lacking are representatives from groups that are not currently known to infect humans or organisms of economic importance. it would be imprudent, however, to continue to ignore these uncharacterized reservoirs of diversity, because it is difficult to predict the source of future emerging diseases ( ) ( ) ( ) . additionally, with the current suite of primarily sequence similarity-based pathogen identification tools, the ability to detect novel pathogens is wholly dependent on highquality reference databases ( ) . there is a trend toward requiring a complete genome sequence when a description of a novel virus is being published, and we agree that this is a good goal; however, the amount of time and resources required to complete the last to % of a viral genome is often cost and time prohibitive for projects sequencing a large number of samples, and in most cases the very ends of the segments are not essential for proper identification and characterization. therefore, for the majority of viral characterization projects, we recommend, at a minimum, a cc genome. this will ensure a complete description of the viral proteome and will allow accurate phylogenetic placement. molecular epidemiology. one of the most common and important applications for viral genomes is in the study of viral epidemiology, which encompasses our understanding of the patterns, causes, and effects of disease. early studies of molecular epidemiology targeted small pieces of viral genomes; however, this type of analysis is likely to miss important changes elsewhere in the genome. therefore, there has been a strong focus in recent years toward the sequencing of "full" viral genomes. institutes such as the broad institute and the j. craig venter institute (jcvi) have been instrumental in breaking ground in the collection of large numbers of good-quality viral sequences. their newly identified genomes typically fall within our cc category. this is likely to remain the gold standard for studies involving a large number of genome sequences, especially when some samples come from lowtiter clinical samples, often necessitating amplicon-based sequencing methods. cc genomes allow for interrogation of changes throughout the coding portion of the viral genome and often include partial noncoding regions. in the absence of highthroughput race alternatives, the time and resources required to complete hundreds or thousands of genomes are likely to continue to outweigh the potential information gained from completing the terminal sequences. countermeasure development. advancements in our capabilities to sequence viral genomes are changing the way we counteract global pandemics and acts of bioterrorism. there are two important aspects of countermeasure development that can benefit strongly from the availability of genome sequences and ht sequencing data: the detection of the infectious agent and the treatment of the disease caused by the agent. taxonomic classification and detection through dna/rna-based inclusivity assays (i.e., using techniques such as pcr to detect the presence of a pathogen) can be designed using fragmented and incomplete genomes (e.g., sd and hq sequences). fully resolved orfs (cc) further enable the development of immunological assays, such as enzyme-linked immunosorbent assays (elisa) and immunofluorescence assays (ifa), for protein-based detection, and obtaining a complete genome opens the door to a plethora of additional downstream applications, including the design of exclusivity tests, the establishment of reverse genetics systems, and the design of robust forensics protocols. however, for effective development and testing of animal models, therapeutics, vaccines, and prophylactics, it is necessary to obtain a complete picture of the variability present within both the challenge stock and postinfection populations, thereby necessitating finished genomes. in these medical applications, it is also important to demonstrate the absence of adventitious agents. in addition to standardizing the vocabulary of viral genome assemblies, it is also critical for researchers to routinely provide raw sequencing reads. without these, it is impossible for others to independently verify the quality of an assembly. data repositories such as genbank already provide a platform for depositing ht sequencing reads, but this is not a requirement for the submission of a genome, nor is this option typically utilized. wider analysis of data will ultimately result in higher-quality assemblies. it is worth considering broader implementation of a wiki-like, crowdsourcing strategy to genome assembly, similar to the annotation strategies that have been adopted for specific genomes of high interest ( , ) . this approach would allow multiple parties to work on genome assembly and annotation at the same time and would provide instant updates for the entire community to evaluate and utilize in their own research. our primary goal here is to initiate a conversation. the rate at which viral genomes are being sequenced is only going to increase in the coming years, and without some standardization, it will be impossible for these valuable resources to be utilized to their full potential. we present these categories as a starting point, with the goal of adjusting and refining them over time as our capabilities and needs continue to change. crystal ball. the viriosphere: the greatest biological diversity on earth and driver of global processes metagenomic analysis of coastal rna virus communities the search for meaning in virus discovery genome project standards in a new era of sequencing next generation sequencing of viral rna genomes . virus taxonomy. ninth report of the international committee on taxonomy of viruses human viruses: discovery and emergence double 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middle east respiratory syndrome coronavirus: the "knowns" and "unknowns relationship between domestic and wild birds in live poultry market and a novel human h n virus in china computational tools for viral metagenomics and their application in clinical research web apollo: a web-based genomic annotation editing platform pseudomonas genome database: improved comparative analysis and population genomics capability for pseudomonas genomes key: cord- -r jl authors: bhuvaneshwar, krithika; song, lei; madhavan, subha; gusev, yuriy title: vigen: an open source pipeline for the detection and quantification of viral rna in human tumors date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: r jl an estimated % of cancers worldwide are associated with infectious causes. the extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using human transcriptome (rna-seq) data from tumor samples. we present an open source bioinformatics pipeline vigen, which allows for not only the detection and quantification of viral rna, but also variants in the viral transcripts. the pipeline includes major modules: the first module aligns and filter out human rna sequences; the second module maps and count (remaining un-aligned) reads against reference genomes of all known and sequenced human viruses; the third module quantifies read counts at the individual viral-gene level thus allowing for downstream differential expression analysis of viral genes between case and controls groups. the fourth module calls variants in these viruses. to the best of our knowledge, there are no publicly available pipelines or packages that would provide this type of complete analysis in one open source package. in this paper, we applied the vigen pipeline to two case studies. we first demonstrate the working of our pipeline on a large public dataset, the tcga cervical cancer cohort. in the second case study, we performed an in-depth analysis on a small focused study of tcga liver cancer patients. in the latter cohort, we performed viral-gene quantification, viral-variant extraction and survival analysis. this allowed us to find differentially expressed viral-transcripts and viral-variants between the groups of patients, and connect them to clinical outcome. from our analyses, we show that we were able to successfully detect the human papilloma virus among the tcga cervical cancer patients. we compared the vigen pipeline with two metagenomics tools and demonstrate similar sensitivity/specificity. we were also able to quantify viral-transcripts and extract viral-variants using the liver cancer dataset. the results presented corresponded with published literature in terms of rate of detection, and impact of several known variants of hbv genome. this pipeline is generalizable, and can be used to provide novel biological insights into microbial infections in complex diseases and tumorigeneses. our viral pipeline could be used in conjunction with additional type of immuno-oncology analysis based on rna-seq data of host rna for cancer immunology applications. the source code, with example data and tutorial is available at: https://github.com/icbi/vigen/. an estimated % of cancers worldwide are associated with infectious causes. these infectious agents include viruses, bacteria, parasites and other microbes. examples of viruses include human papilloma viruses (hpvs) in cervical cancer, epstein-barr virus (ebv) in nasopharyngeal cancer, hepatitis b and c in liver cancer (hbv and hcv), human herpes virus in kaposi sarcoma (ks); human t-lymphotrophic virus- (htlv- ) in adult t cell lymphocytic leukemia (atl) and non-hodgkin lymphoma; merkel cell polyomavirus (mcv) in merkel cell carcinoma (acs, ) . bacteria such as helicobacter pylori have been implicated in stomach cancer. parasites have also been associated with cancer, examples are opisthorchis viverrini and clonorchis sinensis in bile duct cancer and schistosoma haematobium in bladder cancer (acs, ) . detection and characterization of these infectious agents in tumor samples can give us better insights into disease mechanisms and their treatment (hausen, ) . vaccines have been developed to help protect against infection from the many cancers. but these vaccines can only be used to help prevent infection and cannot treat existing infections (acs, ) . there are several screening methods widely used to detect viral infections, especially for blood borne viruses including hbv, hcv, hiv and htlv. these include the enzyme linked immunosorbent assay (elisa or eia) (yoshihara, ) , chemluminescent immunoassay (chlia), indirect fluorescent antibody (ifa), western blot (wb), polymerase chain reaction (pcr), and rapid immunoassays . elisa and wb test detects and measures antibodies in serum taken from the patient's blood, and are typically prescribed after certain symptoms are observed in the patient. there are several challenges in detection of viruses in tumors including loss of viral information in progressed tumors and limited or latent replication resulting in low transcription of tumors (schelhorn et al., ) . the extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using human transcriptome data from tumor samples. the popularity of next-generation sequencing (ngs) technology has exploded in the last decade. ngs technologies are able to perform rapid sequencing, and in a massively parallel fashion (datta et al., ) . in recent years, applications of ngs technologies in clinical diagnostics have been on the rise fda complete list of donor screening assays for infectious agents and hiv diagnostic assays (accessed march , ) . available online at: https:// www.fda.gov/biologicsbloodvaccines/bloodbloodproducts/approvedproducts/ licensedproductsblas/blooddonorscreening/infectiousdisease/ucm .htm abbreviations: hbv, hepatitis b virus; hcv, hepatitis c virus; herv k , human endogenous retrovirus k ; tcga, the cancer genome atlas; hcc, hepatocellular carcinoma; nafld, nonalcoholic fatty liver disease; hep b, hepatitis b; hep c, hepatitis c; hepb + hepc, coinfected with both hepatitis b and c virus; hbsag, hepatitis b surface antigen; hbeag, hepatitis b type e antigen; ngs, next-generation sequencing; rna-seq, whole transcriptome sequencing; bam, binary version of sequence alignment/map format; cds, coding sequence; cox ph, cox proportional hazard; hbx, viral gene x; sts, sequence-tagged sites; ncbi, national center for biotechnology information; gff, general-feature-format. (barzon et al., ; byron et al., ) . amongst the various ngs technologies, whole-transcriptome sequencing, also called rna-seq, has been very popular with methods and tools being actively developed. exploring the genome using rna-seq gives a different insight than looking at the dna since the rna-seq would have captured actively transcribed regions. every aspect of data output from this technology is now being used for research, including detection of viruses and bacteria (khoury et al., ; salyakina and tsinoremas, ; wang et al., ) . they are also independent of prior sequence information, and require less starting material compared to conventional cloning based methods, making them powerful and exciting new technologies in virology (datta et al., ) . these high throughput technologies give us direct evidence of infection in the tissue as compared to elisa-based assays, which only proves presence of infection somewhere in the human body. rna-seq technology has hence enabled the exploration and detection of viral infections in human tumor samples. this technology also enables detection of variants in viral genome, which have been connected to clinical outcome (moyes et al., ; downey et al., ) . in recent years, us regulators approved a viral based cancer therapy (ledford, ) , proving that the study of viruses in the human transcriptome has biomedical interest, and is paving the way for promising research and new opportunities. in this paper, we present our pipeline vigen to not only detect and quantify read counts at the individual viral-gene level, but also detect viral variants from human rna-seq data. the characterization of viral variants helps enable better epidemiological analysis. the input file to our pipeline is a fastq (wikipedia, ) file, so our vigen pipeline can be extended to work with genomic data from any ngs technology. our pipeline can also be used to detect and explore not only viruses, but other microbes as well, as long as the sequence information is available in ncbi . we applied our vigen pipeline to two case studies as a proof of concept -a dataset of cervical cancer patients, and a set of liver cancer patients, both from the tcga collection. we first applied the pipeline to the transcriptome of cervical cancer patients to see if we are able to detect the human papilloma viruses. we also performed additional in-depth analyses on a small focused study of liver cancer patients. in this cohort, we performed viral-gene quantification, viral-variant extraction and survival analysis. from our analyses, we show that we were able to successfully detect the human papilloma virus among the tcga cervical cancer patients. we compared the vigen pipeline with two metagenomics tools and demonstrate similar sensitivity/specificity. we were also able to quantify viraltranscripts and extract viral-variants using the liver cancer dataset. this enabled us to perform downstream analysis to give us new insights into disease mechanisms. in addition to the two case studies, we have made available an end-to-end tutorial demonstrated on a publicly available we also provided step-by-step instructions on how to run our vigen pipeline on this sample data, along with the code at https://github.com/icbi/vigen/ and demonstrated the detection of hbv transcripts in this sample. this allows other users to apply this pipeline to explore viruses in their data and disease of interest. we are currently implementing the vigen pipeline in the seven bridges cancer genomics cloud . there are a number of existing pipelines that detect viruses from human transcriptome data. of these, very few pipelines offer quantification at the gene expression level. a comprehensive comparison of these pipelines is provided in table . our goal was not to compete with these other tools, but to offer a convenient and complete end-to-end publicly available pipeline to the bioinformatics community. to the best of our knowledge there are no publicly available pipelines or packages that would provide this type of complete analysis in one package. customized solutions have been reported in the literature however were not made public. in the future, our plan is to package this pipeline and make it available to users through bioconductor (lawrence et al., ) , allowing users to perform analysis on either their local computer or the cloud. in this paper, we applied our vigen pipeline to two case studies as a proof of concept -a dataset of cervical cancer patients, and a set of liver cancer patients, both from the tcga collection (nci, ) . we first applied the pipeline to the transcriptome of cervical cancer patients to see if we are able to detect the human papilloma viruses. we also performed additional in-depth analyses on a small focused study of liver cancer patients afflicted with hepatitis b virus. in this cohort, we perform viral-gene quantification, viral-variant extraction and survival analysis. the results from these analyses allowed us to compare experimental and control groups using viral-gene expression data and viral-variant data, and give us insights into their impacts on the tumor, and disease mechanisms. in the following sections, we describe the vigen pipeline, and the two case studies. the vigen pipeline includes major modules. figure shows an image of our vigen pipeline. in module (labeled as "filtered human sample input"), the human rna sequences were aligned to the human-reference genome using the rsem (li and dewey, ) tool. one of the outputs of rsem includes sequences that did not align to the human genome (hence the name "filtered human sample input"). these un-aligned sequences were taken and aligned to the viral reference file using popular alignment tools bwa (li and durbin, ) and bowtie (langmead and salzberg, ) . in module (labeled as "unfiltered human sample input"), the rna seq sequences were directly aligned to the viral reference using bowtie without any filtering. the reason for using two methods to obtain the viral genomes in human rna-seq data (module and module ) was to allow us to be as comprehensive as possible in viral detection. the aligned reads from module and were in the form of bam files (center-for-statistical-genetics, ), from which read counts were obtained for each viral genome species (referred to as "genome level counts") using samtools idxstats or picard bamindexstats tools. using the genome level counts, we estimated the number of reads that covered the genome, a form of viral copy number. viral copy number was defined as in equation below: viral copy number = number of mapped reads × read length genome length only those viral species with copy number more than a threshold are selected for the next module. the bam files from module and (from bowtie and bwa) were input into module (referred to as "viral gene expression level analysis"), which calculated quantitate read counts at the individual viral-gene level. we found existing rnaseq quantification tools to be not sensitive enough for viruses, and hence developed our own algorithm for this module. our in-house algorithm used region-based information from the general-feature-format (gff) files of each viral genome, and the reads from the bam file. it created a summary file, which had a total count of reads within or on the boundary of each region in the gff file. this is repeated for each sample and for each viral gff file. at the end, a matrix is obtained where the features (rows) are regions from the gff file, and the columns are samples. the read count output from module (viral gene expression module) allowed for downstream differential expression analysis of viral genes between case and controls groups. the source code for our in-house algorithm, written using the r programming language (r core team, ), has been made public at available at github.com/icbi/vigen. the bam files from module and (from bowtie ) were also input to module to detect mutations in the transcripts from these viruses (referred to as "viral rna variant calling module"). the bam files were first sorted coordinate-wise using samtools ; pcr duplicates were removed using tool picard , then the chromosomes in the bam file were ordered in the same way as the reference file using picard. the viral reference file was created from combining all known and sequenced human viruses obtained from ncbi . because viral variants are known to be low frequency, we have selected a variant calling tool varscan (koboldt et al., ) , which allows detection of low-frequency variants (spencer et al., ) . low quality and low depth variants were flagged, but not filtered out, in case these low values were due to low viral load. once the variants were obtained, they were merged to form a multi-sample vcf file. only variants that had a variant in two or more samples were retained. plink was used to perform case-control association test (fishers exact test) to compare groups. the vigen pipeline is easy to implement because our pipeline incorporates existing best practices and tools available. for module , we developed our own algorithm for viral-gene quantification. the major motivation for this paper was to build on existing viral detection tools, and to build a quantification tool in order to quantify, explore and analyse the genes detected in viruses. the source code for the in-house algorithm, along with a tutorial on how to execute the code on sample data has been made public at https://github.com/icbi/vigen/. since access to tcga raw data is controlled access, we could not use this dataset to create a publicly available tutorial. so we used a publicly available rna-seq dataset to demonstrate our pipeline with an end-to-end workflow. we chose one sample (srr ) from publicly available hbv liver cancer rna-seq dataset from ncbi sra (http://www. ncbi.nlm.nih.gov/bioproject/prjna ). this dataset is also available through ebi sra (http://www.ebi.ac.uk/ena/data/view/ srr ). the dataset consisted of hbv liver cancer patients, and adjacent normal liver tissues. we downloaded the raw reads for one sample, and applied our vigen pipeline to it and were able to successfully detect hbv transcripts in this sample. a step-by-step workflow that includes -description of tools, code, intermediate and final analysis results are provided in github: https://github.com/icbi/vigen/. this tutorial has also been provided as additional file . we were interested in exploring all viruses existing in humans. so we first obtained reference genomes of all known and sequenced human viruses obtained from ncbi ( viruses) and merged them into one file (referred to as the "viral reference file") in fasta file format (wikipedia, ) . this file has been shared in our github page. cervical cancer is caused by the human papilloma virus (hpv). this dataset consisted of cervical cancer patients in the tcga data collection. these samples were primary tumors from either cervical squamous cell carcinoma or endocervical adenocarcinoma where rna-seq data was available. we applied our vigen pipeline on these samples using the seven bridges platform (https://cgc.sbgenomics.com). among the cervical cancer patients, patients had virus detection confirmed by pcr or other lab methods and made available through the clinical data. so we used this information from the patients to estimate the sensitivity and specificity of our vigen pipeline. this dataset consisted of liver cancer patients in the tcga data collection. of these patients were afflicted with hepatitis b virus (labeled "hepb"), while the rest of the patients had a co-infection of both hepatitis b and c viruses (labeled "hepb+c"). information about viral presence was obtained from "viral hepatitis serology" attribute from the clinical information. we first applied the vigen pipeline on the samples, using the globus genomics platform (bhuvaneshwar et al., ) . once the viral genomes were detected, we then chose only the high abundance viral species for the gene quantification step and viral variant detection steps (module and respectively). we then performed a focused analysis on this dataset. we used the viral-gene expression read counts, to examine the differences between "dead" and "alive" samples. the dead/alive status of the samples was obtained from the clinical data and refers to patients in the cohort that died or not from cancer. we performed this analysis on the patients in the hepb only group to prevent any confounding with the hepb+hepc group. out of hepb patients, were alive (baseline group), and dead (comparison group) as per the clinical data. the analysis was performed using a bioconductor software package called edger (robinson et al., ) in the r programming language (http://www.r-project. org). cox proportional hazards (cox ph) regression model (cox and oakes, ) was then applied to look at the association of viral-gene expression data with overall survival. thie cox model was applied on all samples in the cohort (i.e., hep b and hepb+hepc) samples to maximize power. we also compared the dead and alive samples at the viral rna variant level in the hepb group using a tool called plink to see if it can add valuable information to the tumor landscape in humans. we used our vigen pipeline to detect viruses in the rna of human cervical tissue and obtained viral copy number for each species. we used a threshold copy number of as a "positive" viral detection for both hpv- , hpv- and hpv- viruses. based on this criterion, hpv- was detected in % of the samples, hpv- in % of the samples and hpv- in . % of the samples (figure ). the threshold copy number limit that defines a "positive" detection is one of the parameters of the software which could be set by the user depending on the specifics of the experiment. we obtained the clinical data for this tcga cervical cancer cohort from the cbio portal (cerami et al., ) . among the patients, patients had virus detection confirmed by pcr figure | the hpv viruses detected in cervical cancer patients using the vigen pipeline. frontiers in microbiology | www.frontiersin.org or other lab methods and made available through the clinical data. out of the patients, patients had the hpv- virus, patients had hpv- , and the rest had other hpv viruses. so we used this information from the clinical data to estimate the sensitivity and specificity of our vigen pipeline. we got a sensitivity of % and specificity of % for hpv- detection ( table a) ; and a sensitivity of % and specificity of % for hpv- detection (table b ). we applied our vigen pipeline (modules and ) on the rnaseq data from the tcga liver cancer tumors, and obtained genomelevel read counts for each viral species. we used a threshold copy number of to define a positive detection of the hepatitis b virus. once the viral genomes were detected, we short-listed the high abundance viral species for the viral-gene quantification step and viral-variant detection steps (module and respectively). high abundance was defined as those virus species that were detected in at-least samples. in addition to hepatitis b and c viruses, several other viruses came up in this short list including human endogenous retrovirus k (herv k ) and others. a complete list is provided in table . to get a more detailed overview of the viral landscape, we applied module of the vigen pipeline to the liver cancer dataset. this allowed us to quantify viral-gene expression regions in the rna of liver tumor tissues. we then used those results to examine the differences between dead and alive samples. it is known that these patients were afflicted with the hepatitis b virus and hence many of the differentially expressed regions were from this viral genome. but as we know, other viruses also coexist in humans. this was confirmed by the presence of differentially expressed viral-regions from other viruses. the differentially expressed regions that were significant among the results are shown in tables a,b. table a lists only the differentially expressed regions from hepatitis b virus and table b shows the differentially expressed regions from other viruses. from the differential expression analyses, the two most informative results were ( ) a region of the hepatitis b genome that produced the hbeag and hbcag proteins were overexpressed in the dead patients and ( ) another region of the hepatitis b genome that produced hbsag protein was overexpressed in the alive patients. in detail, we saw several important findings as described below: (a) region nc_ . _cds_ _ of the hepatitis b genome was . times overexpressed (log fold change = + . ) in dead patients. this region contains gene c that produces pre-code protein external core module ) , were the same. we collated the significant common results (p-value ≤ . ) in tables a,b . among these results, we saw several missense and frameshift variants in gene x of the hepatitis b genome (nucleotide ), gene p ( , , ) , and a region that overlaps gene p and pres (nucleotides , , , ) . all these variants were found mutated more in the cases than controls. other significant common results included variants in gene c (nucleotide , ) and variants in pres region (nucleotide positions , and ) ( table a ). in addition, there were two missense variants that were common among the top results, but not significant (p-value = . ). they were variants in the x gene of the hepatitis b genome (nucleotides and ) ( table a) . among the significant common results to both, were a few variants of the human endogenous retrovirus k complete genome (herv k ). these include nucleotide positions , , and . these map to frameshift and missense mutations in the putative envelope protein of this virus (q _gp , also called "env") ( table b) . (c) the overall model is significant with p-value < . from the log rank test (also called score test). the table is sorted based on annotation. annotation includes gene name, protein name, etc., separated by commas, multiple annotations separated by semi-colon. table a shows variants in the hepatitis b virus only while table b shows variants in other species. (shows only common results between two possible analysis steps). frontiers in microbiology | www.frontiersin.org the seven bridges team used two metagenomic tools,centrifuge (kim et al., ) and kraken (wood and salzberg, ) , to detect hpv viruses on the same cohort of tcga patients (bridges, ; malhotra et al., ) , and shared the results with us. they used an abundance of . as a positive viral detection (bridges, ; malhotra et al., ) . we compared vigen with kraken and centrifuge in terms of the percentage of samples where the species was detected ( table ) . we can see that the results are in the same range for all three tools. we also estimated the sensitivity and specificity of these tools using the same patients and compared with that of the vigen pipeline. the centrifuge tool had a sensitivity of % and specificity of % for hpv- detection; and a sensitivity of % and specificity of % for hpv- detection. the kraken tool had a sensitivity of % and specificity of % for hpv- detection; and a sensitivity of % and specificity of % for hpv- detection (detailed in additional file ). it shows that our vigen pipeline was able to match the sensitivity and specificity of centrifuge tool and surpassed that of kraken (detailed in additional files , ). we used our vigen pipeline to get genome-level read counts obtained from viruses detected in the rna of human liver tissue. in our results, hbv was detected in % of the samples. this is similar to earlier analyses of tcga liver cancer cohort study (khoury et al., ; tang et al., ; the cancer genome atlas research network, ) , which detected the hbv virus in and % (with typically low counts range) of cases respectively. it has also been reported that the viral gene x (hbx) was the most predominately expressed viral gene in liver cancer samples (tang et al., ) which is in concordance with our findings where the peak number of reads were observed for gene x region of the hbv genome. to get a more detailed overview of the viral landscape, we examined the human rna-seq data to detect and quantify viral gene expression regions. we then examined the differences between dead and alive samples at the viral-transcript level on the hepatitis b sub-group (tables a,b) . from the differential expression analyses, the two most informative results were ( ) a region of the hepatitis b genome that produced the hbeag protein was overexpressed in the dead patients and ( ) another region of the hepatitis b genome that produced hbsag protein was overexpressed in the alive patients. presence of hbeag or hbcag is an indicator of active viral replication; this means the person infected with hepatitis b -jensen et al., ; liang, ). so our results, showing that antigens hbeag and hbcag were overexpressed in dead patients compared to alive patients makes sense, indicating that these patients never recovered from acute infection. the results also indicate a higher level of hbsag in the alive patients compared to the dead patients. the highest levels of hbsag in the virus are known to occur in the "immunotolerant phase." this pattern is seen in patients who are inactive carriers of the virus i.e., they have the wild type dna, and the virus has been in the host for so long, that the host does not see the virus as a foreign protein in the body, and hence there's no immune reaction against the virus. in this phase, there is known to be minimal liver inflammation and low risk of disease progression (park, ; tran, ; locarnini and bowden, ) . this could explain why we saw higher level of hbsag in the alive patients compared to the dead patients. also among the significant results were three regions from the human endogenous retrovirus k (herv k ) genome (with negative log fold change) that were overexpressed in the alive patients. two of these regions were sequence-tagged sites (sts) and the third region was in the gag-pro-pol region that has frameshifts. herv could protect the host from invasion from related viral agents through either retroviral receptor blockade or immune response to the undesirable agent (nelson et al., ) . overall, we found that our results from viral-gene expression level make biological sense, with much of the results validated through published literature. we performed variant calling on the viral data to see if it can add valuable information to the tumor landscape in humans. we then compared the dead and alive samples at the viral-variant level on the patients in the hepatitis b sub-group. among the significant results (tables a,b) included variants in gene c (nucleotide , ) and variants in pres region (nucleotide positions , and ). the gene c region creates the pre-capsid protein, which plays a role in regulating genome replication (tan et al., ) . the mutation in the position lies in a known cpg island (ranging from to ), whose methylation level is significantly correlated with hepatocarcinogenesis (jain et al., ) . mutations in pres are associated with persistent hbv infection, and emerge in chronic infections. the pres and pres regions are known to play an essential role in the interaction with immune responses because they contain several epitopes for t or b cells (cao, ) . mutations in the / positions of the x gene are known to be associated with greater risk of hcc (cao, ; wang et al., ) , and is independent of serum hbv dna level (wang et al., ) . this mutation combination is also known to be associated with hepatitis b related acute-on-chronic liver failure (xiao et al., ) . it is predicted that mutations associated with hcc variants are likely generated during hbv-induced pathogenesis. the a t/g a combined mutations was shown to be a valuable biomarker in the predicting the risk of hcc (cao, ; wang et al., ) ; and are often detected about years before the diagnosis of hcc (cao, ). among the significant common results to both, were a few variants of the human endogenous retrovirus k complete genome (herv k ). these variants map to frameshift and missense mutations in the putative envelope protein of this virus (q _gp , also called "env"). studies have shown that this envelope protein mediates infections of cells (robinson and whelan, ) . herv k is a provirus and is capable of producing intact viral particles (boller et al., ) . studies have shown a strong association between herv-k antibodies and clinical manifestation of disease and therapeutic response (moyes et al., ; downey et al., ) . it is hypothesized that retroviral gene products can be "reawakened" when genetic damage occurs through mutations, frameshifts and chromosome breaks. even though the direct oncogenic effects of hervs in cancer are yet to be completely understood, it has shown potential as diagnostic or prognostic biomarkers and for immunotherapeutic purposes including vaccines (downey et al., ) . we compared various viral detection pipeline using the several criteria (table ) . our pipeline provides similar functionality as the tools listed in table for the detection of viruses from human rnaseq data; but also has an advantage of enabling gene-level expression analysis and quantification, as well as variant analysis of viral genomes in a single open source publicly available package. one limitation of our vigen pipeline is that it is dependent on sequence information from reference genome. this makes it challenging to detect viral strains where reference sequence information is not known. in the future, we plan to explore de novo assembly incorporating more sophisticated methods like hidden markov models (hmm) (alves et al., ) . this would enable us to provide in-depth analysis of strain pathogenicity in the context of clinical outcome. in recent years, us regulators approved a viral based cancer therapy (ledford, ) , proving that the study of viruses in the human transcriptome has biomedical interest, and is paving the way for promising research and new opportunities. we show that our vigen pipeline can thus be used on cancer and non-cancer human ngs data to provide additional insights into the biological significance of viral and other types of infection in complex diseases, and tumorigeneses. our viral pipeline could be used in conjunction with additional type of immuno-oncology analysis based on rna-seq data of host rna for cancer immunology applications. detection and characterization of these infectious agents in tumor samples can give us better insights into disease mechanisms and their treatment (hausen, ) . with the decreasing costs of ngs analysis, our results show that it is possible to detect viral sequences from whole-transcriptome (rna-seq) data in humans. our analysis shows that it is not easy to detect dna and rna viruses from tumor tissue, but certainly possible. we were able to not only quantify them at a viral-gene expression level, but also extract variants. our goal is to facilitate better understanding and gain new insights in the biology of viral presence/infection in actual tumor samples. the results presented in this paper on two case studies are in correspondence with published literature and are a proof of concept of our pipeline. this pipeline is generalizable, and can be used to examine viruses present in genomic data from other next generation sequencing (ngs) technologies. it can also be used to detect and explore other types of microbes in humans, as long as the sequence information is available from the national center for biotechnology information (ncbi) resources. this pipeline can thus be used on cancer and non-cancer human ngs data to provide additional insights into the biological significance of viral and other types of infection in complex diseases, and tumorigeneses. we are planning to package this pipeline and make it open source to the bioinformatics community through bioconductor. the tcga liver cancer dataset was used in the analysis and writing of this manuscript. the data can be obtained from https:// cancergenome.nih.gov/. since access to tcga raw data is controlled access, we could not use this dataset to create a publicly available tutorial. so we looked for publicly available rna-seq dataset to demonstrate our pipeline with an end-to-end workflow. we chose one sample (srr ) from publicly available liver cancer rna-seq dataset from ncbi sra (http://www. ncbi.nlm.nih.gov/bioproject/prjna ). this dataset is also available through ebi sra (http://www.ebi.ac.uk/ena/data/view/ srr ). the dataset consists of liver cancer patients, and adjacent normal liver tissues. we downloaded the raw reads for one sample, and applied our vigen pipeline to it. a step-by-step workflow that includes -description of tools, code, intermediate and final analysis results are provided in github: https://github.com/icbi/vigen/. project name: vigen project home page: https://github.com/icbi/vigen/ operating system(s): the r code is platform independent. the shell scripts can run on unix, linux, or ios environment programming language: r, bash/shell other requirements: n/a license: n/a any restrictions to use by non-academics: n/a infections that can lead to cancer genseed-hmm: a tool for progressive assembly using profile hmms as seeds and its application in alpavirinae viral discovery from metagenomic data applications of next-generation sequencing technologies to diagnostic virology rapid identification of non-human sequences in high-throughput sequencing datasets a case study for cloud based high throughput analysis of ngs data using the globus genomics system human endogenous retrovirus herv-k is capable of producing intact viral particles identifying viral sequences in tcga data using kraken and centrifuge translating rna sequencing into clinical diagnostics: opportunities and challenges clinical relevance and public health significance of hepatitis b virus genomic variations available online at the cbio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data second-generation plink: rising to the challenge of larger and richer datasets virusseq: software to identify viruses and their integration sites using next-generation sequencing of human cancer tissue analysis of survival data next-generation sequencing in clinical virology: discovery of new viruses human endogenous retrovirus k and cancer: innocent bystander or tumorigenic accomplice? infections causing human cancer comprehensive dna methylation analysis of hepatitis b virus genome in infected liver tissues landscape of dna virus associations across human malignant cancers: analysis of , cases using rna-seq centrifuge: rapid and sensitive classification of metagenomic sequences varscan : somatic mutation and copy number alteration discovery in cancer by exome sequencing pathseq: software to identify or discover microbes by deep sequencing of human tissue fast gapped-read alignment with bowtie software for computing and annotating genomic ranges cancer-fighting viruses win approval rsem: accurate transcript quantification from rna-seq data with or without a reference genome fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools viralfusionseq: accurately discover viral integration events and reconstruct fusion transcripts at single-base resolution hepatitis b: the virus and disease hepatitis b surface antigen quantification: not what it seems on the surface enabling scalable and rapid metagenomic profiling of the transcriptome with the seven bridges cancer genomics cloud the distribution of the endogenous retroviruses herv-k and herv-k in health and disease the cancer genome atlas demystified. human endogenous retroviruses r: a language and environment for statistical computing infectious entry pathway mediated by the human endogenous retrovirus k envelope protein edger: a bioconductor package for differential expression analysis of digital gene expression data viral expression associated with gastrointestinal adenocarcinomas in tcga high-throughput sequencing data sensitive detection of viral transcripts in human tumor transcriptomes performance of common analysis methods for detecting lowfrequency single nucleotide variants in targeted next-generation sequence data immunosuppressive treatment of hbsag-positive chronic liver disease: significance of hbeag the interface between hepatitis b virus capsid proteins affects self-assembly, pregenomic rna packaging, and reverse transcription the landscape of viral expression and host gene fusion and adaptation in human cancer comprehensive and integrative genomic characterization of hepatocellular carcinoma immune tolerant hepatitis b: a clinical dilemma using small rna deep sequencing data to detect human viruses virusfinder: software for efficient and accurate detection of viruses and their integration sites in host genomes through next generation sequencing data detection of hepatitis b virus a t/g a mutant by amplification refractory mutation system fasta format kraken: ultrafast metagenomic sequence classification using exact alignments hepatitis b virus genotype b with g a and a t/g a mutations is associated with hepatitis b related acute-on-chronic liver failure kb and yg designed the pipeline. kb and ls implemented the pipeline. kb and yg wrote the manuscript with editorial comments from sm. this work was funded by the lombardi cancer center support grant (p ca ). the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. additional file | vigen github tutorial.additional file | detailed results from analysis of tcga cervical cancer patients.additional file | output from kraken and centrifuge shared by the seven bridges team. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © bhuvaneshwar, song, madhavan and gusev. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - u pdpk authors: gonzalez‐scarano, f.; tyler, kenneth l. title: molecular pathogenesis of neurotropic viral infections date: - - journal: ann neurol doi: . /ana. sha: doc_id: cord_uid: u pdpk classical virologists defined a number of viruses that affect the nervous system and identified tissue tropism, extraneural replication, and viremia as important parameters that determine whether viral infections will affect the central nervous system. molecular techniques are expanding this knowledge by permitting us to relate specific genes and gene products to two defined phenotypes: neuroinvasion and neurovirulence. two converging situations make this knowledge particularly useful: ( ) the development of antiviral drugs and subunit vaccines, which mandate that pathogenesis be related to specific regions of the viral genome; and ( ) the expanding problem of central nervous system infections in immunodeficient states. the recent revolution in the biological sciences has had a tremendous impact on virology, which benefited early from the technological advances in molecular biology. viruses are now used to study gene expression and receptor-ligand interactions and to express cloned genes in eukaryotic systems. it has nevertheless remained easier to study viral infections in vitro rather than viral-host interactions in whole organisms. recently a number of significant insights into the molecular and genetic basis for the pathogenesis of viral infections of the nervous system have been reported by different groups. in this review we will highlight some of the major findings in the field. viral pathogenesis is the process by which a virus causes disease in a susceptible host. pathogenesis can be analyzed in terms of a series of stages (see ) and ) for reviews). to cause systemic illness, a virus must first enter the host animal, undergo primary replication at a site near its portal of entry, and then ultimately spread to distant target tissues, such as the central nervous system (cns). by definition, all animal viruses are intracellular pathogens, and the process of replication must commence with entry into a susceptible cell. an infecting animal virus faces two main blocks to penetration of the cns or any other specific target organ: ( ) a variety of barriers prevent the free access of viruses to target cells, and ( ) even when these barriers are ineffective, only certain cell types will support the internalization and replication of a particular virus. it is useful to think of the capacity of a virus to establish a lethal infection within the cns as the property of neumirulence and the ability to penetrate the cns after inoculation and growth at a peripheral site as the property of neuroinvasiveness. experimentally, it is easy to bypass the barriers to cns infection (by intracerebral inoculation, for example), and therefore each of these properties can be tested for independently. a major goal of much of the current work in virology has been the correlation of viral properties such as neurovirulence and neuroinvasiveness with specific viral genes or proteins or, when the systems have been more advanced, with specific regions of these genes andor proteins. each of the potential entry routes into the organismskin, mucosal membranes, gastrointestinal (gi) tractis utilized by viruses capable of eventually infecting the cns. the specific entry point will be determined by the biology and physicochemistry of the virus as well as by the need for vectors like mosquitoes, ticks (arboviruses), or mammals (rabies). for a number of neurotropic viruses, the physical barrier provided by the skin is breached by the bite of an animal or arthropod vector, or through the use of contaminated needles or other foreign bodies. many other neurotropic viruses enter the host via natural portals such as the respiratory and gi tracts. entry via these routes may be direct (via contaminated saliva, for example) or through aerosols. the molecular mechanisms that influence the capacity of viruses to become aerosolized and their subsequent stability are unknown. most nonenveloped viruses (poliovirus, reovirus) appear to lose infectivity in conditions of low (< %,) relative humidity. for example, aerosolized poliovirus shows a lo -fold drop in infectivity when kept for hour at % relative humidity (when compared with % humidity) { ]. conversely, the infectivity of aerosols of most enveloped viruses is not notably altered by changes in the relative humidity, although in general enveloped viruses are more sensitive to environmental inactivation. the g i tract presents formidable chemical barriers to the entry of viruses. the mechanisms by which agents such as those present in the gi tract (e.g., acids, bile salts, proteolytic enzymes) affect viral structure, and hence determine the ability of a virus to survive transit through the local environments present at entry sites such as the gi tract, are gradually becoming understood. in the case of nonenveloped viruses, the outer capsid proteins appear to be the major determinants of viral stability to a wide variety of physicochemical agents. for example, rhinoviruses are rapidly inactivated at acidic p h levels, whereas other picornaviruses, such as polio, are not. one practical consequence of this is that rhinoviruses do not survive transit through the stomach and hence rarely produce disease outside the limited confines of the respiratory tract. exposure of rhinoviruses to an acidic p h results in the loss of the viral capsid protein v p . this in turn produces a "hole" in the virus particle through which the viral nucleic acid leaks out, leaving a noninfectious "empty" viral capsid. interestingly, the same type of structural alteration can occur in polioviruses exposed to certain adverse physical conditions, which again emphasizes the role of the viral capsid in determining virion stability [ , . another example of the role of outer capsid proteins in determining viral stability can be seen in the reoviruses. these viruses, like the enteroviruses, are neurotropic viruses whose natural portal of entry is via the gi tract. the three serotypes of reovirus show profound variations in their in vitro sensitivity to a number of physicochemical agents, including temperature, ph, alcohol, and detergent solutions. studies using reassortant viruses containing genes derived from "sensitive" and "resistant" serotypes of virus (see fig. for an analogous experiment) allowed specific genes to be identified and associated with particular phenotypes. sensitivity to alkahne p h and guanidine mapped to the viral s gene, sensitivity to high temperature and the detergent sodium dodecyl sulfate mapped to the s gene, and sensitivity to phenol and ethanol mapped to the m gene. in each case these genes encode proteins that are components of the outer cap-sid of the virus, indiccating again the importance of these proteins in determining susceptibility to environmental conditions [ ., . just as they differ with respect to sensitivity to p h changes, viruses vary in their response to proteolytic enzymes. some viruses are extremely sensitive to trypsin-foot and mouth disease virus demonstrates a drop in infectious titer upon treatment with it-and others, like influenza, need the effects of a trypsin-like protease to become infectious e , . the human rotaviruses, which are enteric pathogens, show dramatic enhancement of infectivity in the presence of trypsin, which cleaves the v p outer capsid protein . reoviruses also differ in their sensitivity to proteolytic digestion with enzymes such as chymotrypsin [ , . genetic studies have shown that the viral m gene, which encodes an outer capsid polypeptide (mlc), is responsible for determining sensitivity or resistance to proteolysiij by chymotrypsin [ ] . the effects of proteolytic enzymes on the infectivity of ortho-and paramyxoviruses have been investigated extensively. in the case of influenza virus, a trypsin-like protease present in host cells cleaves the hemagglutinin protein into two smaller peptides, ha and ha , held together by a tlisulfide bond. the cleavage and subsequent removal (of an arginine residue exposes the hydrophobic n h terminal of ha , which is essential for the fusion function of this virus (see below) [ }. if influenza virus is grown in cells that lack this peptidase activity, h a cleavage does not occur, and, though the virus still binds to cellular receptors, it is not infectious [ , , , . enveloped viruses-those that incorporate a lipid bilayer on their out'er surface-are particularly sensitive to inactivation b y bile salts, which dissociate the lipoprotein components of the viral envelope. this may explain why enveloped neurotropic viruses only rarely enter the h :jt via the gi tract (the coronaviruses, which include the neurotropic mouse hepatitis virus, are a known exception to this rule). the neurotropic viruses that commonly use the gi portal of entry are all nonenvel.oped and therefore insensitive to the action of bile salts. many viruses, such as those that infect the gi and upper respiratory tracts, attack target cells that are near the point of entry. for those agents, infection of contiguous cells satisfies the requirement of their life cycle. except in special circumstances , viruses that infect the cns rnwt reach it after entering the body at a distant site. in general, this means primary replication must occur in a target cell outside the cns, and then the virus must reach the cns by either the bloodstream (hematogenous spread) or via nerves (neural spread). it is ( ) cns target cell, ( ) the efficiency of spread after the primary infection of these target cells, and ( ) the success of the host's immune response that determine whether a virus will be neuroinvasive. incidentally, under most circumstances this combination of factors reduces the incidence of successful cns infections, making these a small proportion of the total number of infections caused by viruses, even those that are traditionally considered neurotropic. most neurotropic viruses reach their target tissue through the bloodstream. arboviruses, for example, are inoculated directly into the subcutaneous tissue or bloodstream, then replicate in extraneural tissues such as muscle or regional lymph nodes , ). figure is a schematic diagram of the spread of an arbovirus from the blood to the nervous system. following primary replication, the virus is released into the bloodstream, where it is transported in the plasma. for a viremia to serve as a source of dissemination of virus to target organs, it must be of adequate magnitude and duration. studies with a variety of arboviruses have shown that the degree and persistence of viremia is directly related to the ability to penetrate the cns c , ). the viruses of the california encephalitis group, which are transmitted by mosquito species endemic to the midwestern united states and to upper new york state, are responsible for the majority of the cases of arboviral encephalitis in this country. their rna genome is segmented (a quality they share with the influenza viruses, arenaviruses, and rotaviruses and reoviruses), and different "pieces" can be recombined artificially in the laboratory. by taking genomic segments from neuroinvasive and noninvasive strains, one can ask the question: which genome segment is responsible for the ability to penetrate the cns? figure summarizes such an experiment, in which viruses representing neuroinvasive and noninvasive strains were used to coinfect cells. "reassortant" viruses, containing genomic elements from two different strains, were thus generated and then tested for their ability to penetrate the murine cns. using this approach, it has been determined that the gene coding for the envelope glycoproteins cosegregates with the phenotypic property of neuroinvasiveness, although the genes coding for the other structural proteins of the virus can modulate this effect ). the same line of reasoning can be extended by using viral mutants that have changes in a single protein product. one frequently used approach, primarily with rna viruses, is the selection of antigenic variants with monoclonal antibodies c , , , ) . in this ap- proach, a virus mutant present in the initial stock at low concentration is amplified by neutralizing the wildtype virus with a monoclonal antibody (fig ) . the resultant virus variant contains a mutation in the protein against which the neutralizing antibody is directed, which in most instances is the protein responsible for attachment to cellular receptors. that mutation can usually be mapped to one or two amino acids which, being altered, prevent binding of the mutant virus by the monoclonal antibody. monoclonal antibody variants have been used to map the antigenic sites of the influenza hemagglutinin , , and have been used successfully to define important regions of the cellular binding proteins of rabies virus, reovirus, coronaviruses, and the california serogroup-all cns pathogens. antigenic variants of the california serogroup obtained with a single monoclonal antibody have altered neuroinvasiveness; if injected directly into the cns their pathogenesis is not altered. thus, the property of neuroinvasiveness can be specifically associated with a single protein in this system . by sequencing the genome of the variant virus and comparing this sequence with that of the wild-type parent, it may be possible to show that the property of neuroinvasiveness is associated with specific regions within the glycoprotein molecule, as has been done in other systems f ). besides arboviruses, other agents, like poliovirus, also use the bloodstream as their primary route to the nervous system. in polio the organ of primary repli-cation is the gut, yet it is release into the bloodstream that determines whether the characteristic myelitis will occur. in addition to plasma viremia, some viruses, including human irnrnunodeficiency virus (hiv), are transported in the bloodstream in association with lymphocytes or macrophages f , . following hemsttogenous spread, neuroinvasive agents penetrate the cns through the choroid plexus or through the endothelial cells. the lack of endothelial or choroid plexus tissue culture systems has made it difficult to analyze .in detail the molecular mechanisms of penetration of these tissues. classic investigations by pasteur and colleagues on rabies virus, and b,y goodpasture and colleagues on herpesviruses clearly established that neurotropic viruses could spread to the cns via nerves [ ] . more recent investigations with a number of viruses including rabies, polio, and herpes have provided abundant confirmation and more detailed insights into the process of neural spread , , , , . however, until recently it wm not possible to identify the role of specific viral genes in influencing the capacity of viruses to spread through nerves. in the case of rabies virus, after an initial period of replication in skeletal muscle, the virus concentrates at the neuromuscular junction in close proximity to neuromuscular and neurotendinous spindles. the virus then enters nerve terminals in proximity to these sites and is transported to the dorsal root ganglia and spinal cord [so, . rabies virus mutants with alterations in the virion glycoprotein (selected with monoclonal antibodies, as described for california encephalitis virus) appear to have altered capacity to enter certain types of nerve fibers - . these findings suggest that the rabies glycoprotein may play an important role in viral entry into and transport within cells. reovirus type has also been shown to spread from peripheral tissue tc) the cns via nerve cells. furthermore, the microtubule-associated system of fast axonal transport has been implicated in this spread, because experimental infection of the cns following footpad inoculation of mice is inhibited by low concentrations of colchicine. selective inhibitors of slow axonal transport had no effect on spread to the cns { . like the california encephalitis viruses, reoviruses have a segmented genome (double-stranded rna in this case). experiments similar to the one outlined in figure can be used to generate reassortant viruses. unlike type reovirus, type reoviruses spread to the cns via a hematogenous route. tyler and collaborators exploited this fact to test reassortants of type and type viruses and again ask the question: which gene segmeiit accounts for the different patterns of spread in these two strains? these reassortant vi- ism, because they confer resistance to proteolysis, dehydration, and p h changes. in most instances, this has been determined through a combination of genetic methods, usually involving either reassortant viruses or selected antigenic variants. in the next section we discuss the role of these proteins in determining tropism at the cellular level. ruses, containing gene segments from both type and type viruses, have clearly shown that the property of spread can be associated with the gene that codes for the sigma polypeptide-an outer protein that functions as the viral hemagglutinin and cell attachment protein. additional insights into the molecular basis for neural spread have recently come from the study of herpes simplex virus recombinants. for these experiments, two strains (hsv- , ; hsv- , ), which differ in their ability to spread to the mouse cns after corneal inoculation, were utilized to generate recombinant viruses [ ] . the region of the hsv- genome which codes for a number of proteins including the gb glycoprotein, a nucleocapsid protein (p ), and a dna-binding protein (icp- ), as well as the dna polymerase, was found to be important in the spread of viruses from cornea to cns. in summary, the available evidence all points to the conclusion that, in several different viral systems, the envelope proteins (in the case of enveloped viruses) or the capsid proteins (in the nonenveloped agents) are the major determinants of spread to the nervous system. the outside proteins naturally play a very important part in other aspects of penetration into the organ- the process of entry of viruses into typical mammalian cells is outlined in figure . experimentally and biologically it can be divided into two main steps: ( ) binding to the plasma membrane, and ( ) penetration and uncoating. many of the concepts regarding virus binding to plasma membranes have developed from the pioneering work of brown and goldstein on the low-density lipoprotein receptor {zs}. in this model, macromolecules destined for the cytoplasm first interact with cellular receptors. the interaction with these receptors is quite specific and accounts for the restriction of unwanted macromolecules from the cells. viruses bind to the plasma membrane of susceptible target cells through specific receptors which may be proteins (hiv), lipids (vesicular stomatitis virus), or contain sialic acid (reovirus, influenza) [ , . in some instances, neurotropic viruses have been thought to bind to pharmacological receptors. this has been best demonstrated for reovirus type , which in some tissues appears to bind to the beta-adrenergic receptor [ , lo] , but it has also been suggested for rabies virus, which may use the acetylcholine receptor as its entry point [ , . to a large extent, the specificity of this virus-receptor interaction can dictate the pathogenesis of a particular agent. a topical example is the virus that causes the acquired immunodeficiency syndrome (aids), now termed hiv c . this virus binds to the t molecule, a membrane protein of unknown function which defines a particular subclass of lymphocytes many viruses can infect cells if their nucleic acid genome is introduced directly into them-in the absence of any viral polypeptides. introduction of infectious dna clones has been used to bypass cellular receptors in infections with hiv. in such an experiment a variety of cell types can be infected, not necessarily only those containing the appropriate membrane receptor [ . another well-known receptor-virus interaction involves the influenza hemagglutinin, which binds to receptors containing sialic acid. unlike the hiv system, where knowlege of the interaction between the receptor and the virus is still at a descriptive stage, the relationship of the influenza hemagglutinin to its receptor has been characterized to the single amino acid level, because the molecular structure of the influenza hemagglutinin has been determined through x-ray crystallography c , ) . the hemagglutinin molecule is present as a trimer on the virion surfaces. this trimer contains a "pocket" that appears to function as the site of attachment of the receptors present on susceptible cells. viruses selected from different hosts (avian and equine, for example), which have naturally different enzymatic activities and slightly different sugar residues on their sialic acid molecules, have variations in the amino acids surrounding this pocket [ , . thus, even though the target cells are presumably the same, small changes in the receptor-binding site of influenza virus may account for species specificity. different technology has been used to identify the receptor-binding site of rhinoviruses. the large number of rhinovirus serotypes has made production of a general vaccine impractical. the majority of serotypes use a single specific receptor present on the surface of infectable cells. this receptor was initially identified through the use of a polyclonal antibody, then by monoclonal antibodies ell]. the virus itself has subsequently been crystallized c , and a large cleft or "canyon" present on each icosahedral surface is thought to be the receptor-binding site, by virtue of its relationship to antibodies capable of neutralizing the virus. drugs are now being designed to block this cleft. although rhinoviruses are not neurotropic, they form part of the same family (picornaviridae) as poliovirus, and many of the features of the viral topology are being extended to that group as well as to footand-mouth disease c and theiler's virus, other members of the picornaviridae. in summary, a necessary condition for the entry of viruses into all cells, and nerve cells in particular, is the availability of specific receptor molecules on the sur-face of such cells. some receptors are undoubtedly present in all cns cells, and viruses that utilize such may cause a generalized infection, or panencephalitis. in the cns, the pattern of illness a virus produces is determined in large part by the specific regions of the brain that are infected and by the population of cells in the affected regions that are injured or destroyed. some viruses, like jc papovavirus, which is responsible for progressive multifocal leukoencephalopathy, infect astrocytes an'd oligodendroglia while largely sparing neurons, whereas herpes simplex infects all cell populations. striking differences also occur in the topographical distribution of viral injury to the cns. rabies virus causes pathology in the cerebellum, hippocampus, and limbic areas, whereas polio affects the motor nuclei in the cortex, brainstem, and anterior horns of the spinal cord c , . many of these specific topographic ;associations have been postulated to be the result of specific receptor-binding interactions. after a virus has bound the appropriate receptors, the process of internalization can proceed in one of two ways: ( ) viruses can fuse with the plasma membrane, discharging their contents directly into the cytosol, or ( ) viruses may be internalized through the endocytotic pathway. fusion at the plasma membrane has been observed for viruses like herpes simplex and coronaviruses, both of which are capable of forming giant syncytia during routine infection. presumably there are no special requirements for internalization under these circumsi:ances, and if the appropriate receptors are present the virus will appear within the cell. other viruses are not capable of fusing directly with the cell membrane, and for those the endocytotic pathway (outlined in figure ) is the method of internalization. this pathway, which again resembles the mechanism of interrlalization of low-density lipoproteins [ , involves ithe collection of receptor-ligand complexes at regions of the membrane characterized by electron-dense material at their cytoplasmic side. the viruses are then transported within endocytotic vesicles, which have been determined to have a mildly acidic ph , . for many enveloped virusesthose viruses that inc:orporate a lipid bilayer into their structure-it has been shown that exposure to such a low p h leads to alterations in the conformation of the envelope proteins c . again using the influenza hemagglutinin as a model, such a change probably brings to the surface stretches of hydrophobic amino acids normally buriecl within the viral protein. the hydrophobic residues interact with the membrane of the endosome, fusing the viral envelope to it and releasing the contents of the virus into the cytoplasm, where the process of replication can then begin. in influenza and related viruses, a host protease is responsible for the cleavage step that prepares this portion of the hemagglutin for this exposure (from a precursor protein). this proteolysis is essential for replication and accounts for some of the host specificity of the viruses, as noted above. in fact, epidemics of influenza in avian rpecies have been ascribed to the ease of proteolysis in different strains. for nonenveloped viruses (poliovirus, rhinovirus), there is also evidence that a ph-dependent step may be important in infection, but the process has not been observed clearly, as it has been in the enveloped agents. the degree of acidity that can elicit the changes in the viral proteins necessary for fl ion to occur varies for different viruses. because of the inherent difficulties of such measurements, the endosomal p h has not been determined for a large number of cell lines (and for none of the cns cell lines). however, one can easily theorize that the relationship between the viral requirements and the acidity of these cellular organelles must influence the potential for infection of a particular cell type. in fact, the ability to fuse has been correlated with the encephalitogenic potential of bunyaviruses and mumps virus strains , ) . once the contents of the viral envelope (genome and proteins necessary for replication) have been discharged into the cytosol, the process of viral replication can begin. it is likely that for most nononcogenic rna viruses the specificity of the virus-target interaction is determined prior to this step. an efficiently replicating virus, like vesicular stomatitis virus, for example, is probably capable of initiating replication of its genome in any cell line that it can penetrate. however, not all proteins or genome segments may be reproducible in any given system, and for lymphocytic choriomeningitis virus it appears that restriction at the segment level may be responsible for the outcome of infection { . for many dna viruses like herpes simplex and varicella-zoster, persistence is clearly associated with the arrest of replication. such mechanisms are highly specific for different families. recently the role of regulatory signals in the enhancement of replication has become a prominent area of investigation. signals within the viral genome that stimulate gene transcription or translation appear to play an important role in determining viral host range and tissue tropism in certain systems . these signals, variously termed enhancers, promoters, and transactivators depending on their position and orientation, are capable of stimulating the transcription of genes. some of these enhancers determine tissue tropism (for example, in polyoma virus), whereas others are active in all tissues (simian virus {svi- , herpes simplex) { , , , . transcriptional activators are genes whose products can activate the transcription of other genes. the viruses associated with aids have prominent transactivating activity, and they may also have regulatory activities at posttranscriptional events. in any case this kind of "tat" activity can greatly amplify the production of viral gene products and may be responsible for increased cytopathological changes. in some instances it may be responsible for host specificity. tat activity had previously been shown to be an important feature of sv- , an experimentally important virus that is related to jc virus, the etiological agent for progressive multifocal leukoencephalopathy. this kind of activity may be important in the selectivity that this virus shows for glial cells and could play a role in a relationship between hiv and jc. in several experimental models, the relative contribution of jc virus regulatory sequences in conferring tissue specificity has been studied ( , . it has been argued that the jc virus promoter may contribute substantially to neurovirulence. outcome of infection the final determinant of pathogenesis is the outcome of infection of the host cell. four major pathways exist: ( ) death of the cell; ( ) alteration of its growth pattern and change into a cancerous phenotype (transformation); ( ) persistence of infection without obvious cellular change; and ( ) persistence of infection with alteration of specialized cellular functions (luxury functions). neurotropic viruses can be responsible for any of these final results. transformation is a highly intricate subject and will not be discussed further. interested readers should refer to general reviews ( , . cytopathic effect significant enough to stop cellular metabolic activity is obviously the most common outcome of viral infection in all acute and some subacute viral infections. for the most part, morphological evidence has been utilized as the primary way of determining cell death. at a molecular level, mechanisms of viral cytopathology include the inhibition of production of host cell dna, rna, or protein. for some rna viruses it is known that interference with the transfer of a methylguanosine "cap"-m'gpppn-to host messenger rna (mrna) occurs in order to direct the host machinery to translate viral proteins preferentially. for influenza viruses and bunyaviruses (california encephalitis), the stolen cap is essential for transcription of the viral rna to proceed efficiently [ , }. except for these and a few other examples, the specific virally mediated mechanism for inhibition of cellular metabolism is unknown. much has been written about the role of the host immune response in mediating cell death. the immune response is frequently capable of harming the host instead of protecting it from viral infection as has been clearly demonstrated for a variety of neurotropic agents, including rabies, lymphocytic choriomeningitis virus, coronaviruses, and others (for review, see )). there has been a recent surge of interest in the role of viruses in inducing cell membrane molecules involved in the recognition of foreign antigens. these h- antigens are generally expressed at a low level in neural cells, but they increase in the presence of certain viral infections and interferons . it has been postulated that the presence of these antigens on neural cells may make them a better target for cellular immunity directed at viral components and thus increase the possibility of virally triggered immunopathology. although this work is clearly just beginning, the availability of nucleic acid probes for the mrna's coding for these antigens should make it possible to study potential immunopathological mechanisms at a molecular level. similarly, the wealth of information now available about the t-cell receptor molecules { ) should allow exploration of the genetic correlation with immunopathological diseases. because of the complexity of the work involving virally induced immunopathology, we will not discuss it further here. particularly when dealing with clinical specimens, it can be very useful in terms of analyzing the state of the latent viral dna in experimental infections. although considerable effort has been spent on this question, to date there is no clear indication whether herpes simplex virus is integrated into host dna or is present as an "episome" in latently infected ganglia [ . work by fraser and co-workers indicates that the dna is present in a form distinct from that of the isolated virions , and personal communication). similar techniques can also be used to demonstrate that varicella-zoster virus is present latently in human dorsal root ganglia, and probably reactivated to cause zoster dermatitis . this particular herpesvirus has not been cultured in explants of such ganglia. when the histological features so allow, the particular cell type that is latently infected may be identifiable-in varicella-zoster virus the genome is thought to be harbored exclusively by neurons of the dorsal root ganglia. because of the lack of a suitable experimental animal, the state of the varicella-zoster virus latent genome is even less known than that of herpes simplex virus. persistent infection with alteration of lux-ury functions. oldstone and co-workers have recently introduced the concept that some persistent viral infections may lead to the alteration of cellular function without any morphological change. in young mice infected with lymphocytic choriomeningitis virus, changes of growth and glucose metabolism are associated with persistent infection of the pituitary ). such effects are subjelct to significant variation, depending on the age and strain of the mice as well as the viral strain. however, when present, this persistent infection of the mouse cns is associated with the absent expression of the viral glycoproteins. the synthesis of these glycoproteins may be decreased, allowing the virus to escape detection by the cellular immune mechanisms. summary a variety of factors affect the ability of viruses to infect cells within the cns. neurotropic viruses must penetrate the external barriers that protect the organism, replicate in an appropriate peripheral site, and then spread through either the bloodstream or through nerves to the cns itself. genetic analysis in several systems has pointed to lrhe external proteins as the main determinants of neuroinvasiveness and neurovirulence. however, other factors, including host enzymes, activating factors, and the replicative machinery of the virus itself, must come into play. ultimately, the balance between the viral machinery and the host immune system will determine the outcome of any infection. supported by tida ns (to f.g-s.) and physician-scientist award a (to k. l. t.) and by the william p. anderson foundation. f.g. . is a harry weaver neuroscience scholar of the national multiple sclerosis society, and k. l. t. is an alfred p. sloan research fellow. we thank neal nathanson for his helpful comments. presented in part at a conference sponsored by the american society for neurologic investigation, chicago, il, october , . production of acquired immunodeficiency syndrome associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone isolation of a tlymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids). science - i danger of accidental person-to-person transmission of creutzfeldt-jakob disease by surgery specific 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key: cord- -iwfidoer authors: urayama, syun-ichi; takaki, yoshihiro; nunoura, takuro title: flds: a comprehensive dsrna sequencing method for intracellular rna virus surveillance date: - - journal: microbes environ doi: . /jsme .me sha: doc_id: cord_uid: iwfidoer knowledge of the distribution and diversity of rna viruses is still limited in spite of their possible environmental and epidemiological impacts because rna virus-specific metagenomic methods have not yet been developed. we herein constructed an effective metagenomic method for rna viruses by targeting long double-stranded (ds)rna in cellular organisms, which is a hallmark of infection, or the replication of dsrna and single-stranded (ss)rna viruses, except for retroviruses. this novel dsrna targeting metagenomic method is characterized by an extremely high recovery rate of viral rna sequences, the retrieval of terminal sequences, and uniform read coverage, which has not previously been reported in other metagenomic methods targeting rna viruses. this method revealed a previously unidentified viral rna diversity of more than complete rna viral genomes including dsrna and ssrna viruses associated with an environmental diatom colony. our approach will be a powerful tool for cataloging rna viruses associated with organisms of interest. viruses are the universal genetic elements associated with all three domains of life ( ) , and virus-host interactions impact on the status of life and surrounding ecosystems ( ) . historically, viruses are most often recognized as pathogens ( ) , and, thus, have been studied in the field of medical and crop science. recent advances in high-throughput sequencing technologies have enabled us to identify not only viruses associated with diseases, but also those present in natural environments including oceans ( ) and soil ( ) . although these sequencing technologies have opened a new era in virus identification ( ) , a limited number of methods have been established for virus enrichment and library construction. the diversity and distribution of viruses in non-viral nucleic acid-dominant environments, such as the intracellular environments in which viruses actually replicate, still remain unclear due to technical difficulties ( ) . the development of a new procedure for effective virus enrichment and library construction is required in order to understand the full spectrum of diverse viruses. rna sequencing (rna-seq) is a popular method in rna virus metagenomics and is widely used for rna virus identification ( ) . purification and library construction methods have been established for rna viruses at the extracellular stage ( , , ) . however, the viral read ratio of intracellular rna viruses (rna viruses at the intracellular stage) in the rna-seq library is typically < % because mrna and rrna are dominant in the total rna fraction extracted from biological samples ( ) . therefore, the enrichment of viral rna is essential for maximizing sensitivity in the identification of novel viruses. the physical enrichment of viral particles and nuclease digestion of non-viral nucleotides has been employed to increase the viral read ratio; however, a relatively low abundance of viral reads is still observed in most studies ( ) . these techniques are only applicable to specific rna viruses because not all rna viruses form viral particles ( ) . in addition, difficulties are associated with capturing terminal rna sequences in an efficient and effective manner ( ) and obtaining uniform coverage using the rna-seq method. sample preparation methods for effective viral rna-seq are still inadequate and the sequence information generated is biased and incomplete. in an attempt to resolve these issues, an environmental viral metagenomic approach targeting intracellular long doublestranded rna (dsrna) has recently been examined ( , , , ) . intracellular dsrna consists of the genomes of dsrna viruses and replicative intermediates of single-stranded rna (ssrna) viruses, and, thus, long dsrna is known as an rna virus-specific molecule and molecular marker for rna virus infection and replication ( ) . therefore, a metagenomic analysis targeting intracellular long dsrna theoretically retrieves dsrna and ssrna viruses, except for ssrna retroviruses, which do not form dsrna in the replicative stage. in addition, it is possible to eliminate non-viral nucleic acids such as mrna and rrna, which dominate rna-seq reads, by dnase i, s nuclease, rnase, or column chromatography ( ) . however, previous studies have reported technical issues with the purification of dsrna and library construction. random priming for the reverse transcription of dsrna does not enable the terminal sequences of the dsrna molecule to be determined or eliminate significant contamination by non-viral sequences. the heterogeneous sequencing depth in certain viral genome segments is also an issue associated with this method ( , , , ) . although the full-length cdnas of dsrna viruses may be obtained using loop primers that are ligated to the dsrna terminal ends for reverse transcription ( ) , this method is only useful for short dsrna viruses. therefore, to the best of our knowledge, this method has not yet been applied to a viral metagenomic analysis. we herein established a novel strategy to obtain full-length rna virus sequences with extremely high efficiency by applying a short dsrna full-length cloning method ( ) for physically fragmented dsrnas. the improved method, named flds (fragmented and loop primer ligated dsrna sequencing), was applied to a diatom colony in a tide pool and revealed previously unidentified rna viruses. our results indicate that the diversity of environmental rna viruses has been underestimated due to the technical limitations in identifying entire rna viromes in cellular organisms, and this technique will be a powerful tool for cataloging rna viruses associated with organisms of interest. mycelial plugs of magnaporthe oryzae strain s- -ii a, naturally infected with magnaporthe oryzae chrysovirus strain a (mocv -a) ( ) were incubated in . % yeast extract and % glucose liquid broth (yg broth) with reciprocal shaking ( rpm) at °c for weeks in the laboratory of prof. teraoka (tokyo university of agriculture and technology). colonies of a diatom on tidal rocks in tokyo bay ( . ° n, . ° e) were sampled in april . after washing with distilled water, the colonies were stored at - °c. dsrna was purified as described by okada et al. with a few modifications ( , ) . briefly, the microbial sample was disrupted in liquid nitrogen in a mortar and total nucleic acids were manually extracted. dsrna was purified twice through a micro-spin column (empty bio-spin column; bio-rad laboratories, inc., hercules, ca, usa) containing cellulose powder (cellulose d; advantec, tokyo, japan) to obtain pure dsrna. the dsrna eluted from cellulose powder in mq water was treated with dnasei (amplification grade, invitrogen, carlsbad, ca, usa) and s nuclease (invitrogen) in nuclease buffer ( mm ch coona, . mm mgcl , . mm znso , and mm nacl) and was then incubated at °c for h. the final concentrations of ch coona, mgcl , znso , and nacl were adjusted to mm, mm, mm, and mm, respectively. dsrna was purified using an rneasy mini kit (qiagen, valencia, ca). a one-tenth volume of × shortcut buffer and × mncl provided with shortcut rnase iii (neb japan, tokyo, japan) was added to the dsrna solution and fragmented by ultrasound at °c in snap-cap microtubes using a covaris s (woburn, ma, usa). the fragmentation conditions were as follows; run time s, peak power . w, duty factor . %, and cycles/burst. fragmented dsrna was divided into two equal volumes, and maintained at °c with or without shortcut rnase iii (neb). dsrnas were then purified using a zymoclean gel rna recovery kit (zymoresearch, orange, ca). note that dsrna purification from m. oryzae was carried out in the laboratory of prof. teraoka. the pc -t loop primer ( ′-p-gga tcc cgg gaa ttc ggt aat acg act cac tat att ttt ata gtg agt cgt att a-oh- ′) was ligated to fragmented dsrna as described by potgieter et al. ( ) , and dsrna was then purified using the minelute gel extraction kit (qiagen). after the addition of dmso at a final concentration of % (v/v), dsrna was denatured at °c for min and snap-frozen in ice-water slurry. rna was reverse transcribed into cdna from the ligated loop primer region using the superscript iii first-strand synthesis system (invitrogen). after excess and hybrid rnas were removed ( ) , cdna was desalted and concentrated using the minelute pcr cleanup kit (qiagen). primary cdna strands were re-annealed by lowering the temperature from to °c, as described previously ( ) . second strand dna polymerization was performed using kod-plus neo (toyobo, osaka, japan) with a primer complementary to the partial sequence of the pc -t loop primer, pc ( ′-ccg aat tcc cgg gat cc- ′) ( ) . after heat activation of kod-plus neo in the reaction mixture provided at °c for min, template cdna was added and incubated at °c for min. after the reaction, cdna was amplified under the following conditions: °c for min, (for mocv -a) or (for diatom colony) cycles of °c for s, and °c for min. small cdna and primer dimers were removed using the . × spriselect reagent kit (beckman coulter, brea, ca, usa) according to the left side size selection procedure in the manufacturer's protocol. total rna was isolated from a diatom colony using the trizol plus rna purification kit (invitrogen) according to the manufacturer's protocol. the rna fraction was treated with dnase i (takara, otsu, japan). double-stranded cdna was synthesized from μg of total rna with random primers ( -mers) using a primescript double strand cdna synthesis kit (takara). the resultant cdna was quantified using a qubit dsdna hs kit. ultrasound was used to fragment cdna in snap-cap micro-tubes at °c using a covaris s (woburn, ma, usa). the fragmentation conditions were as follows; run time s, peak power . w, duty factor . % and cycles/burst. the illumina library was constructed with kapa hyper prep kit illumina platforms (kapa biosystems, woburn, ma, usa). the quantity of the library was evaluated using the kapa library quantification kit (kapa biosystems). each bp of the paired-end sequences of each fragment were determined with the illumina miseq platform (san diego, ca, usa). raw sequence reads were processed with the clc genomics workbench (clc bio, aarhus, denmark). adaptor and primer sequences were trimmed, and low quality sequence regions were removed with default parameters. phix sequences derived from control libraries and experimentally contaminated sequences (< . % of total reads) were also removed using a mapping tool. the consensus sequences of viral contigs were obtained de novo exclusively with the clc genomics workbench (clc bio), and assemblies were manually examined and extended using the tablet viewer ( ) . using the mapping tool, each contig was confirmed to be constructed with at least × sequence coverage, × average coverage, and , bp in length. in cases of dominant reads (more than reads) that stopped in the same position around the ends of contigs, the position was recognized as a terminal end. the predicted terminal ends of the viral genome segments were also confirmed by the presence of adjacent pcr primer sequences next to the predicted terminal sequence, except for cases of contigs with a poly(a) tail. contigs with - % nucleotide identity with other contigs were classified as the genome types of the same species. contigs with > % nucleotide identity were assigned as the same genome type and only major contigs were used in further analyses. sequences were compared against the ncbi non-redundant nucleotide and amino acid (aa) databases using blastn-plus and blastx-plus, respectively ( ), and then classified by megan . . . ( ) . a sequence analysis was performed using genetyx-mac software version . . (genetyx corp., tokyo, japan) and genetyx software version . . (genetyx). most full-length small subunit rrna sequences in the diatom colony were reconstructed from rna-seq reads with emirge ( ). multiple alignments based on the deduced aa sequences of putative rna-dependent rna polymerase (rdrp) genes in dsrna contigs were obtained using clustalx . ( ) and mega software ( ) . phylogenetic analyses were conducted using mrbayes . . ( ) with the model of aa substitution, rtrev+i+g+f, selected by prottest . ( ), as judged by the akaike information criterion ( ) . bayesian analyses with the covarion parameter were run with one run and four chains for , , generations. the data sets supporting the results of this study are available in the genbank database repository (accession nos. ddbj: ap -ap ) and short read archive database (accession no. ddbj: dra and dra ). the novel dsrna purification and library construction method, named flds, consists of cellulose column chromatography, the physical fragmentation of dsrna, cdna synthesis using a loop primer, and the pcr amplification of cdna (fig. ). the purification of dsrna was achieved by the repeated affinity purification of dsrna using cellulose powder and the enzymatic removal of ssrna and dna. purified dsrnas were fragmented using ultrasound to retrieve all types of dsrna viruses in order to apply the previously reported full-length dsrna cloning method using a loop primer ( ) . the full-length dsrna cloning method requires overlapped cdnas synthesized from both terminal ends for further cdna amplification, and was only applicable to short dsrna molecules. reverse transcription was initiated from the ligated loop primer on both ends of the dsrna fragment. cdna was then thermally denatured to allow annealing of single-stranded cdna with the complementary sequence in the ′ terminal region. the single-stranded regions of annealed cdna were filled in with dna polymerase. the doublestranded cdna derived from dsrna was amplified by pcr with a single primer (pc ) in order to obtain sufficient cdna to construct a sequencing library. mycelial mocv -a was used to test the feasibility of this method. since pcr amplicons were not observed in the dsrna-specific rnaseiii-treated sample prior to reverse transcription, most of the amplicons (cdna) were likely to have been derived from dsrna (fig. s ). the results of the sequencing analysis indicated that . % of total reads were derived from the mocv -a genome (table s ). five contigs obtained by de novo assembly were identical to the entire region of the mocv -a genome segments attained using a conventional cloning and sequencing method ( , ) with > . % identity (table s ) . read mapping on mocv -a genomes (fig. s ) showed that the sequence coverage of terminal regions was generally higher than that of the central regions of each segment with few exceptions. no obvious relationship was observed between read coverage and gc content (fig. s ) . these results indicated that flds effectively enriched dsrna reads, thereby allowing the retrieval of complete genome sequences including terminal regions without the requirement for the additional rapid amplification of cdna ends (race). gel electrophoresis showed that the total long dsrna fraction from the diatom colony contained at least ten dsrna segments, whereas genomic dna and rrna were the predominant in total nucleic acids (fig. ) . total dsrna extracted from g of the diatom colony was analyzed using the flds method. pcr amplicons were not observed in the dsrnaspecific rnaseiii-treated sample prior to reverse transcription (fig. s ) . as a result of de novo assembly and manual extension, we obtained composite viral contigs (table and table s ). more than . % of reads were mapped to these contigs (table ) as in the case of the model experiment described above. both terminal ends of of the viral contigs were identified and recognized as full-length viral genome segments. the terminal sequences of the full-length segments were used to identify segment compositions for some of the viral species because terminal sequences are highly conserved between segments in some dsrna viral genomes for viral rna replication and/or encapsidation ( ) . based on aa sequence similarities (e-value ≥ × - ) in the predicted protein-encoding sequences (cdss), the number of genome segments in related viruses, and terminal conserved sequences in each segment of a single virus, we identified viral putative composite genomes out of full-length viral segments. sequence similarities between the putative viral composite genomes were used to classify them into putative viral species, and each of the two genome types was identified in three species (table ) . seventeen dsrna and two ssrna viral species were identified and named diatom colony-associated dsrna virus - (dcadsrv- - ) and diatom colony-associated ssrna virus - (dcassrv- - ) ( table ). since ssrna viruses form an rna duplex as an intermediate in genome replication, these contigs were most likely derived from replicating ssrna viruses ( ) and not from contaminant ssrna. an additional seven full-length viral segments with predicted cdss were also identified; however, we were unable to determine the combination of their segments or reconstruct viral genomes based on information from previously reported viruses. thus, these viral segments were assigned as diatom colony-associated virus-like rna segments (dcavlrs- - ). total rna from the diatom colony was also investigated using shotgun rna-seq in order to determine the active organisms of the colony and the abundance of viral rna genomes in total rna. sequence reads derived from rrna were identified using emirge ( ) . the results of the analysis revealed that % of all trimmed reads were rrna sequences, while . % of all reads showed more than % identity to s and s rrna from the diatom achnanthes brevipes. in addition, . and . % of reads belonged to the other diatom genus cylindrotheca and chlorophyte genus cladophora, respectively. the relative abundance of the rrna reads was shown in table s . only . % of reads from total rna-seq was mapped on the major viral contigs obtained using flds with a read mapping algorithm in the clc workbench ( table ) . comparisons of the relative read frequencies of each major viral contig between total rna-seq and flds revealed that flds achieved . - . -fold enrichment for each viral contig ( . mean) (fig. ) . flds also had apparent advan-tages in uniform read coverage and efficiency for retrieving terminal sequences (fig. ) . sequence reads for ssrna viruses in flds were also more abundant than when rnaseq was used for four out of five ssrna contigs. in addition, by de novo assembly, only six partial viral contigs were obtained using rna-seq, and no viral contigs specific for total rna-seq were found. accordingly, we concluded that flds is more efficient than total rna-seq for the detection and identification of rna viruses, with the exception of retroviruses, which theoretically cannot be identified using flds. a phylogenetic analysis of viral rna replicases (rnadependent rna polymerase; rdrp) presented the phylogenetic relationship between viral genomes from a diatom colony and known rna viruses (fig. s ) . viruses belonging to the family totiviridae harbor non-segmented dsrna genomes and form isometric virions that infect either fungi or protozoa ( ) . thirteen composite genomes of totiviridaerelated viruses were identified and classified into four clades distinct from the five characterized genera of totiviridae (clades a-d in fig. s a ). clade c was the sister clade of the proposed genus "trichomonasvirus" and clade d included ustilaginoidea virens rna virus (uvrv ). in general, - ribosomal frameshift signals [the xxxyyyz motif ( ), in which xxx may be any three identical nucleotides, yyy may be either aaa or uuu, and z may be a, u, or c] or + ribosomal frameshift signals [cccuuuu ( ) or uccuuucgu ( )] were located in the upstream region of the nd cds, and were used in the expression of overlapping viral genes such as the pol (rdrp) of totivirus and leishmaniavirus. these regions were examined in an attempt to better classify the identified viruses. however, as in the case of uvrv , - or + ribosomal frameshift signals were not found in any of the totiviridae genomes obtained in this study. cdss in the predicted totiviridae virus-like segments dcavlrs- and dcavlrs- showed significant similarities with the gag (coat protein; cp) and pol (rdrp) of known totiviruses, respectively. totiviridae genomes consist of a single genome segment that encodes the two essential cdss, whereas dcavlrs- and - lacked pol and gag, respectively. these two segments harbored nine identical ′-terminal nucleotide sequences, which were distinguishable from the other identified terminal viral sequences. genomic features implied that dcavlrs- and - may be parts of a bisegmented viral genome. rdrp in dcadsrv- segment showed significant homology with that in fox picobirnavirus, a member of the picobirnaviridae, although dcadsrv- was phylogenetically distinct from the known picobirnaviridae viruses (fig. s b) . picobirnaviruses are small, non-enveloped, bisegmented dsrna viruses that infect animals and humans ( ) . the genome structure of dcadsrv- was similar to that of the known picobirnaviridae ( ) . dcadsrv- was classified into the genus deltapartitivirus of the family partitiviridae based on the predicted rdrp sequence (fig. s c) ( ) . to date, all of the alphacryptoviruses have been identified from plants including the angiosperm, gymnosperm, and chlorophytes ( ) . rrna sequences belonging to the streptophyta, including land plants, have not yet been detected by an rna-seq analysis, whereas cladophora sp. of the chlorophyta, a sister division of streptophyta, were detected (table s ). the cdss of dcadsrv- and a few viral contigs showed significant homology with viruses belonging to the endornaviridae (dsrna), naranviridae (ssrna), or hypoviridae (ssrna), whose virion formation has not yet been observed. in the ssrna viral population, rdrp in dcassrv- presented a close relationship with border disease virus-bd (e-value = × - ), a member of the genus pestivirus of the family flaviviridae, which consists of the arthropodborne pathogens of humans and other animals. the genome size and cds structure of dcassrv- ( . kb) were similar to those of flaviviridae ( . - . kb) ( ) , and the phylogenetic tree of rdrp indicated that dcassrv- was not classified into the three known flaviviridae genera (fig. s d) . a phylogenetic analysis of rdrp in dcassrv- suggested that the rna virus was classified into the genus mitovirus, which has a non-segmented ssrna genome, infects the mitochondria of fungi, and lacks viral particles (fig. s e) . the presence of multiple uga codons suggested that the putative coding strand of dcassrv- was likely to be translated in mitochondria. the genome size of dcassrv- ( . kb) was larger than those of the known mitoviruses ( . - . kb) ( ) . this study revealed the presence of novel rna viruses associated with a diatom colony and inferred the unexpected evolutionary relationship between environmental viruses and pathogenic animal viruses. among the rna viral genomes obtained in this study, some dominant populations showed greater similarities to fungal viruses than to known diatom viruses; however, several ssdna and ssrna viruses have already been identified from marine diatoms ( , ) . we cannot exclude the possibility that these viral genomes were derived from fungi associated with a diatom colony, but it is more likely that they came from the major components of a diatom colony because of their high abundance in the rna viral metagenomic library. since extracellular viral particles have been a major target of virus surveillance and isolation, information on intracellular viruses in microorganisms is very limited ( , ) . therefore, the accumulation of knowledge on intracellular rna viruses infecting diverse host organisms is essential for understanding the evolution and distribution of rna viruses. flds revealed full-length and some partial composite viral rna genomes associated with a diatom colony by de novo assembly. these were classified into five dsrna (totiviridae, endornaviridae, picobirnaviridae, cystoviridae, and partitiviridae) and four ssrna (flaviviridae, narnaviridae, virgaviridae, and hypoviridae) virus families. to the best of our knowledge, this is the largest number of full-length genome sequences of novel rna viruses identified in one metagenomic library. the viral rna community successfully detected in this study consisted of dsrna viruses with or without virion formation and ssrna viruses detected as replicative intermediates. our results suggest that flds has the potential to detect a wide range of rna viruses, excluding retroviruses. several studies have been performed using metagenomic analyses targeting dsrna with next-generation sequencing technology. in these studies, viral read abundance reached a maximum of . % ( ) . in contrast, flds provided extremely high viral read abundance. the improvement in viral read rates with flds was likely derived from [ ] a combination of repeating cellulose powder column chromatography and subsequent enzymatic treatment, [ ] the fragmentation and efficient thermal denaturation of dsrna prior to cdna synthesis, and [ ] the selective duplex formation of dsrna-derived cdna prior to pcr amplification. furthermore, flds also presented advantages in reconstructing complete genome sequences including terminal regions, which are difficult to obtain using rna-seq and random priming methods ( ). the complete sequences of viral rna segments are beneficial for the identification of rna virus segments, particularly in cases in which coding cdss did not show significant similarities with viral cdss in databases. the application of a fulllength dsrna cloning method using a pc -t loop primer ( ) to fragmented dsrna enabled us to determine the terminal regions of long dsrna genomes. since t rna ligase requires a ′ phosphoryl-terminated nucleic acid donor (pc -t loop primer) and ′ hydroxyl-terminated nucleic acid acceptor for ligation activity, dsrna fragments with ′ terminal phosphate were not used as substrates. the terminal structures of dsrna fragmented by ultrasound have not been reported. however, in the case of dsdna fragmented by ultrasound, double-strand breaks occur preferentially in ′-cpg- ′ dinucleotides, and the phosphate group is at the ′ side of g in the products ( ) . in this study, fragmented dsrnas were successfully converted into cdna and amplified. taking this into consideration, dsrna fragmentation using ultrasound with covaris s also produced ′ hydroxylterminated fragments. furthermore, the lack of any modifications to the ′ hydroxyl-terminal of viral rna genomes ( ) also allowed us to retrieve the terminal regions of the rna viral genome. total rna-seq is considered to be a less-biased method for identifying rna viruses despite the very low abundance of viral reads in general. in the present study, flds enriched the viral rna reads by > -fold that with total rna-seq (table ) . notably, flds produced significantly more ssrna viral reads than total rna-seq; however, flds only has the ability to detect ssrna viruses at the replicative stage. moreover, flds showed more uniform read coverage than rna-seq. these results indicate that flds is more effective than total rna-seq for revealing all rna viruses in cellular organisms. prottest: selection of best-fit models of protein evolution deep sequencing analysis of rnas from a grapevine showing syrah decline symptoms reveals a multiple virus infection that includes a novel virus a new approach to determining whole viral genomic sequences including termini using a single deep sequencing run mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal blast+: architecture and applications deep sequencing analysis of viruses infecting grapevines: virome of a vineyard metagenomic analysis of coastal rna virus communities optimized approaches for the sequence determination of double-stranded rna templates analysis of double-stranded rna from microbial communities identifies double-stranded rna viruslike elements metagenomic analysis of rna viruses in a fresh water lake plant viral doublestranded rna 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virology. frameshifting to pa-x influenza this research was supported in part by a grant-in-aid for scientific research ( ) from the ministry of education, culture, sports, science and technology of japan. we would like to thank hiromitsu moriyama, shinsuke kawagucci, yukari yoshida-takashima, and mitsuhiro yoshida for their fruitful discussions and valuable suggestions. we also thank tohru teraoka at tokyo university of agriculture and technology allowing us the use of equipment. key: cord- - b yz f authors: holder, benjamin p.; simon, philippe; liao, laura e.; abed, yacine; bouhy, xavier; beauchemin, catherine a. a.; boivin, guy title: assessing the in vitro fitness of an oseltamivir-resistant seasonal a/h n influenza strain using a mathematical model date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: b yz f in , the a/brisbane/ / (h n ) seasonal influenza virus strain acquired the oseltamivir-resistance mutation h y in its neuraminidase (na) gene. although previous studies had demonstrated that this mutation impaired the replication capacity of the influenza virus in vitro and in vivo, the a/brisbane/ / h y oseltamivir-resistant mutant completely out-competed the wild-type (wt) strain and was, in the – influenza season, the primary a/h n circulating strain. using a combination of plaque and viral yield assays, and a simple mathematical model, approximate values were extracted for two basic viral kinetics parameters of the in vitro infection. in the st gali-mdck cell line, the latent infection period (i.e., the time for a newly infected cell to start releasing virions) was found to be – h for the wt strain and more than h for the h y mutant. the infecting time (i.e., the time for a single infectious cell to cause the infection of another one) was between and min for the wt, and less than min for the h y mutant. single-cycle viral yield experiments have provided qualitative confirmation of these findings. these results, though preliminary, suggest that the increased fitness success of the a/brisbane/ / h y mutant may be due to increased infectivity compensating for an impaired or delayed viral release, and are consistent with recent evidence for the mechanistic origins of fitness reduction and recovery in na expression. the method applied here can reconcile seemingly contradictory results from the plaque and yield assays as two complementary views of replication kinetics, with both required to fully capture a strain's fitness. influenza is the most important respiratory disease in terms of mortality and morbidity. each year, between and million severe cases and , to , deaths due to seasonal influenza are reported worldwide [ , ] . cyclic pandemics due to antigenic shifts constitute an important threat [ ] as was demonstrated by the swine-origin pandemic of [ ] . since vaccines for novel influenza virus strains require approximately months to develop and produce [ ] , antivirals remain the first line of defense. there are only two classes of antivirals approved for treatment of influenza [ ] . the adamantanes, such as amantadine and rimantadine, are ineffective against b-type viruses [ ] and have recently become ineffective against most a/h n and some a/h n viruses due to a mutation in the m gene [ ] . the neuraminidase inhibitors (nai), which include zanamivir and oseltamivir, were approved a decade ago and have shown excellent activity against all influenza a subtypes and b viruses [ ] . a recent rapid increase in resistance to oseltamivir, however, has become a cause for concern. the h y mutation in the neuraminidase (na) gene (h y in n numbering), first described in [ ] , is the most frequent mutation associated with oseltamivir-resistance in the n subtype, but it had long been thought to critically reduce viral fitness [ ] . with a location on the framework residue of the enzyme catalytic site [ ] , the mutation has been shown to cause a reduced affinity for the substrate in enzyme activity assays [ , ] , an impaired viral fitness in vitro [ ] [ ] [ ] , and up to a -fold reduction in transmission efficiency in ferrets [ , ] . for these reasons, strains carrying the h y mutation were not thought to be a great concern for public health [ , ] . during the - influenza season, however, the a/ brisbane/ / -like (h n ) h y mutant emerged and rapidly disseminated worldwide in the apparent absence of antiviral pressure [ , [ ] [ ] [ ] . recently, our group performed a study on the replicative capacities of the a/brisbane/ / h y mutant strain where we showed that its fitness, based on in vitro and animal studies, was similar to that of its wild-type (wt), oseltamivir-susceptible, counterpart [ ] . these observations, and those of others [ ] , correlate with the clinical situation encountered in the - season where almost % of the a/h n viruses isolated in north america and europe were resistant to oseltamivir due to the h y mutation [ , , ] . recent work suggests that the origin of both the fitness reduction conferred by the h y mutation and the unique fitness of the a/brisbane/ / mutant strain is found in the virus na activity and surface expression. specifically, the reduction of na activity conferred by the h y mutation has been associated with a reduced expression of surface neuraminidase, possibly due to defects in the folding of the molecule or its transport through the cellular membrane [ ] . it has been shown, however, that two other mutations in the na gene (v m and r q) can provide a compensatory effect by increasing na surface expression, and that these two substitutions indeed occurred in the evolution of the h n seasonal strain between and [ ] . the neuraminidase of contemporary (a/brisbane/ / -like) strains susceptible to oseltamivir have shown a higher affinity for the substrate in na activity assays than older h n seasonal strains (e.g., a/new caledonia/ / and a/solomon islands/ / ); contemporary oseltamivir-resistant strains (with the h y substitution) have shown a decrease in that affinity, but remain above the level of older strains [ ] . thus, it seems that these pre-existing mutations led to an over-expression of na and provided a favorable environment for the appearance of the h y mutation. the eventual dominance of the h y mutant may be due to a better balance between the hemagglutinin (ha) and na activity [ , ] . it remains an open question, however, precisely how these mechanistic changes lead to viral fitness changes. the answer to this should be found in the details of the infection kinetics in the interaction of virus and cell. in this paper, we present a method to extract the values of viral kinetics parameters, specific to a particular strain, from parallel experiments of plaque and viral yield assays. our previous study [ ] assessed the in vitro replicative capacity and fitness of pairs of wt influenza virus strains and their h y mutant counterparts by use of viral yield assays and by qualitatively comparing plaque sizes. here, we show that it is possible to use these experimental measures to quantitatively characterize the kinetics parameters responsible for the replicative efficiency of influenza virus strains. as a proof of concept, we apply our method for extracting the viral kinetics parameters to the oseltamivir-susceptible/-resistant pair of a/brisbane/ / influenza virus strains in order to determine how the known genotypic differences in these two strains map to quantitative changes in the viral kinetics parameters characterizing their replicative efficiency. the kinetics parameters extracted through our method suggest that the h y mutant has weaker na activity compared to its wt counterpart -confirmed by na activity assays -which manifests itself as a longer phase of latent infection before viral release -confirmed by single-cycle viral yield experiments. however, the results also indicate that this longer latent infection period for cells infected by the h y mutant is compensated for by a shorter infecting time required for that cell, once releasing virions, to successfully infect other cells. in order to obtain two complementary views of the infection kinetics for the a/brisbane/ / wt and h y mutant strains, virus growth over time was observed in two different in vitro systems: the viral plaque assay and the multiple-cycle viral yield assay. viral plaque assays. figure shows representative plaques for the wt and h y mutant strains of a/brisbane/ / (h n ) viruses at each time point. the average plaque radius of each strain over time, calculated by averaging three independent experiments of three such wells at each time point, is shown in figure . the plaque growth is characterized by an initial delay where no growth is observed, followed by a period of linear increase of the plaque radius over time. after h, the rate of plaque growth declines and the linear approximation is no longer valid. the growth attenuation could be due to a number of factors including a hardening of the overlay, a depletion of nutrients required for viral production and cell maintenance, and the widespread destruction of the cell monolayer leading to holes and irregularities disrupting and limiting further growth. the plaque assay is a long-standing and standard technique in virology [ , ] and plaque sizes have been used in many in vitro studies to qualitatively evaluate the phenotypes of various viruses [ , , ] . plaque assays are often used for strain comparison and, in that context, plaque diameters at a single time point are reported. these plaque diameters are then typically used to conclude that, for example, if the plaques observed at h for strain a are larger than those for strain b, then strain a must have a higher replicative fitness than strain b. however, one should question whether such conclusions are valid and robust to experimental variability. looking at figure , one can see that at h, the plaque radius of both a/brisbane/ / wt and its h y mutant counterpart are comparable in size. yet at h, the wt strain has significantly larger plaques than the h y mutant, and the situation is reversed at h. thus, relying on single time point measurements for comparing strains can be misleading. here, instead, we exploit the fact that the average plaque growth is approximately linear in time between and h. this allows us to extract a novel measure, the plaque velocity, which is the slope in the linear regression of the plaque radius to the , and h time points. the plaque velocity, unlike the plaque radius at a given time point, is a robust measure in that it takes into account plaque radius at several time points and is not affected by differences in the length of the delay period which precedes the period of linear growth. using this method, the measured plaque growth was more rapid for the wt than the h y mutant, with a plaque velocity of : + : d cell =h compared to : + : d cell =h, where d cell is the diameter of one cell. thus, using plaque velocity alone, it would appear that the wt strain has a replicative fitness advantage over the h y mutant. multiple-cycle viral yield assays. in order to complement the information provided by the plaque assay, namely the plaque velocity, we also conducted multiple-cycle viral yield experiments. the results of these experiments for the wt and h y mutant strains are shown in figure . the kinetics of the viral yield experiments can be broken into two different phases: an exponential growth of virus concentration, characterized by the viral titer growth rate, followed by an exponential decay of virus concentration, characterized by the viral titer decay rate, after the viral titer peak. the viral titer decay rate was the same for both strains at : + : h { . the viral titer growth rate of the h y mutant was : + : h { , slightly greater than that of the wt which was : + : h { . thus, it would appear from the viral titer growth rate alone, that the h y mutant has a replicative advantage over the wt strain, in contrast with the findings using the plaque velocity extracted from the plaque assay alone. this discrepancy between the conclusions drawn from each experimental measure points to a complementarity between the two assays: they appear to emphasize different aspects of viral replication. thus, combining the information provided by these two assays is key to obtaining a complete and consistent picture of what shapes a particular strain's replicative fitness. the plaque growth experiment yields a single experimental measure for each strain: the plaque velocity. the multiple-cycle viral yield assay provides two quantities: the viral titer growth rate, and the viral titer decay rate. it is the goal of this paper to associate these broad experimental measures to the values of fundamental infection parameters, specific to each strain, which quantitatively characterize replicative efficiency. to this aim, we have constructed mathematical models which allow us to simulate each in vitro assay in a computer experiment. the basic mathematical model used here is similar to other within-host models of viral infection [ ] [ ] [ ] [ ] [ ] [ ] . a cell can be in one of four states -target (uninfected), latently infected (infected but not yet releasing virus), infectious (releasing virus) and dead (no longer releasing virus) -and its passage through these states ( figure ) is determined by five infection kinetics parameters. target cells interacting with virus become latently infected at a constant infection rate per virus, b. the average time a cell remains latently infected is called the latent infection period, t l , and the average time a cell releases virus is called the infectious lifespan, t i . virus is produced by infectious cells at a constant viral production rate, p, and this free virus loses infectivity exponentially at a constant rate of viral infectivity loss, c (as is observed in experiments [ ] ). in applying this model, it is assumed that the growth of a particular influenza virus strain in a particular cell line is determined by a single, unique set of values for these five parameters. thus, although the mathematical structure of the models used for each experimental assay is different (see materials and methods for a detailed description of each), the parameter values for a particular virus strain are assumed to be constant from assay to assay. with only three experimentally measured quantities, it would be impossible to uniquely identify all five parameters for a particular virus strain. fortunately, it is possible to reduce the number of parameters considered and obtain unique identification of a few key parameter values. one parameter can be determined immediately from the multiple-cycle viral yield results. the viral titer decay rate, characterizing the decline of the virus concentration after the peak (figure ), corresponds to the slowest of the rate of loss of virus-producing cells and the rate of viral infectivity loss [ ] . since prior in vitro experiments have shown that infectious cell death is nearly complete shortly after the viral titer peak [ , ] , we set the rate of viral infectivity loss, c, equal to the viral titer decay rate. because this decay rate was determined to be : h { for both a/brisbane/ / strains, we have fixed the rate of viral infectivity loss to this value for all simulations. this corresponds to a virion half life of approximately . h, which is consistent with prior measurements for influenza virus in the experimental literature (see, e.g., [ , ] ). having fixed the rate of viral infectivity loss, we are left with four undetermined parameters and two experimental measures. for the experiments considered here, the infection rate per virus, b, and the viral production rate, p, can be combined into a single parameter, leaving only three parameters to be determined. the rationale for this simplification is the fact that, during an infection, the two parameters play equivalent roles: doubling the rate at which virus is produced by cells will have the same effect on new infections as doubling the rate at which virus infects cells. therefore, the only identifiable quantity is the product of the two rates, pb. since their product has units of inverse time squared, we have chosen to express this quantity as a new characteristic time, the infecting time, t inf~ffi ffiffiffiffiffiffiffiffiffiffiffiffiffi = pb ð Þ p , which corresponds to the average time it takes a single virus-producing cell to cause the latent infection of one more (see materials and methods). we are left then with two experimental measures -the viral titer growth rate and the plaque velocity -whose values may depend on three unknown infection kinetics parameters: the infecting time, t inf ; the latent infection period, t l ; and the infectious lifespan of a cell, t i . to determine how each of these parameters affect the infection dynamics, we varied each individually about a base value and measured the effect on the simulated experimental quantities ( figures a and b ). one parameter, the infectious lifespan of a cell, t i , had very little effect on either the plaque velocity or the viral titer growth rate. in the latter case, this parameter was explicitly neglected in the derivation of the growth rate, because earlier viral yield experiments have shown little cell death prior to the peak of the virus concentration (see, e.g., [ , ] ). the fact that, over a wide range of infectious lifespan values, the resulting plaque velocity remained unchanged, is perhaps more surprising. indeed, a shorter infectious lifespan will lead to the earlier appearance of plaques, resulting in larger plaque sizes at any given time. we have shown, however, in earlier work where influenza virus plaques were observed by immunostaining [ ] , that the same plaque velocity can be measured from both the progress of dead cells, as we consider here, and the progress of newly infected cells. this indicates that plaque velocity is established at the advancing edge of an infection wave, and is likely unaffected by cell death in the wake of that wave. in those experiments, the infectious lifespan of a cell appears only as a time-delay between the infected cell plaque growth and the dead cell plaque growth. this has also been observed for the plaques of other viruses [ ] . since the infectious lifespan has little effect on the experiments we consider, and is therefore not identifiable here, we have fixed its value for both strains and for all simulations to value of t i~ h, obtained from the literature ( table ) . this leaves only two parameters, the infecting time, t inf , and the latent infection period, t l , to be determined from our two experimental measures, the plaque velocity and viral titer growth rate. the full dependence of each experimental measure on the two remaining parameters are presented as contour plots in figures c and d . because the plaque velocity and the viral titer growth rate depend on both the infecting time, t inf , and the latent infection period, t l , the experimental measurement of either quantity alone is not sufficient to specify the values of these infection parameters for a given strain. the measurement of both, however, can provide enough information for this specification, provided that the dependence on the parameters is sufficiently different for the two quantities. to demonstrate this concept using the a/ brisbane/ / (h n ) wt and h y mutant strains, we have plotted the experimentally-measured values of plaque velocity and viral titer growth rate as functions of the infecting time and latent infection period, using the model dependence determined above ( figure ). figures a and d show the values of the kinetics parameters most consistent with the measured plaque velocities of the a/ brisbane/ / wt and h y mutant strains, respectively. rather than plot a single line at the average measured value, we have accounted for the error in the measurement of the plaque velocities by plotting regions of contour denoting the one-and two-standard deviations (for a detailed description see materials and methods). we can see that while the plaque velocity does constrain the two parameters to a specific region, that region is too large to allow any useful comparison of the two strains. similarly, figures b and e show the values of the kinetics parameters most consistent with the measured viral titer growth rate. the consistency of a particular pair of parameter values with each of the two experimental measures can be combined by finding the intersection of the two parameter regions. this region of intersection corresponds to those parameter values most consistent with the parallel plaque and viral yield experimental measurements for a particular strain. the extent of these regions, shown in figure c for the a/brisbane/ / wt strain and figure f for the h y mutant strain, is summarized in table . the region of intersection suggests that the latent infection period for the h y mutant (w h) is longer than that of the wt strain ( - h), while the infecting time of the mutant (v min) is much shorter than that of the wt ( - min). in order to test the predictions made in the previous section by applying the mathematical model to parallel plaque and viral yield assays, we performed two additional experimental tests which could provide some qualitative and quantitative confirmation. to independently estimate the latent infection period for the two a/brisbane/ / influenza virus strains, we performed a single-cycle viral yield experiment. single-cycle experiments were performed at an moi of such that most cells would be infected simultaneously and pass through the phases of latency and viral release at the same time. therefore, the observed virus production of the cell culture can be considered roughly proportional to that of an individual cell. the results of two independent experiments for each strain are shown in figure a ; one experiment shows the viral titer over one full day post-infection and the other over only h but with more frequent sampling. for each replicate, the viral titer of each strain was observed to grow rapidly after h postinfection, with the wt viral titer reaching a plateau at approximately h post-infection and that of the h y mutant reaching a plateau between h and h post-infection. although the viral titer data in each replicate followed a relatively smooth curve, the inter-replicate variation was quite large, with peak virus titer varying from | pfu ml to almost pfu ml. it is also notable that all of these peak values were well below the values seen in the multiple-cycle viral yield assay (figure ) , by a factor of * > . both of these features could be explained by the action of a relatively large defective interfering particle population [ , ] within the viral stock, which is not uncommon for the influenza virus [ ] . the delay in the peak of viral titer between the two strains is qualitatively consistent with the model predictions of the previous section: the h y mutant strain appears to have a longer latent infection period than the wt strain. to make this comparison more quantitative, we scaled each experimental data set such that the peak virus was equal to one and then performed a least-squares fit to the full set of normalized data for each virus strain ( figure b ). we utilized a model similar to that used for the analysis of the multiple-cycle viral yield assay, but allowed for a normal distribution of the latent infection period among cells rather than a fixed value for all cells, as assumed previously (see materials and methods). the fitted value of the average latent infection period, t l , was found to be . h for the wt strain and . h for the h y mutant, with fitted values of the standard deviation in the normal distribution, s l , of . h and . h, respectively. these results are summarized in table , along with % confidence intervals determined by fitting bootstrap replicates [ ] . the longer latent infection period predicted for the h y mutant strain, could be the result of poorer na activity. this would also explain the shorter infecting time for the mutant strain in that its virions would more easily bind to new cells with less interference from its na activity. to investigate whether the h y mutant had poorer na activity compared to the wt, we directly measured the enzymatic activity of the na of each virus strain using the through modeling and simulation of two common in vitro experiments, the plaque and viral yield assays, we have extracted we have shown that seemingly contradictory results from the two experiments -plaques of the susceptible strain grow more quickly than the resistant strain, while the reverse is true of their titer growth in viral yield assays -can be considered complementary views of the infection kinetics which allow for the determination of parameter values controlling the replication of each strain. specifically, we have found that the latent infection period of the h y mutant strain -equal to the time elapsed between the successful infection of a cell by a virion and the significant release of virus progeny by the newly infected cell -is much longer than that of the wt strain (by - h). the infectivity of the mutant strain, however, was found to be much higher than the wt, as quantified by the infecting time -equal to the time for a single infectious cell to cause the latent infection of one other, within a completely susceptible cell population. independent single-cycle viral yield assay results lend support to the hypothesis of a longer latent infection period for the mutant strain than the wt, but suggest a more moderate (* h) difference between the two. these results are consistent with the larger na activity of the susceptible (wt) strain compared to the h y mutant, reported here and by others [ ] , and its increased na surface expression [ ] . since neuraminidase is the viral surface enzyme responsible for cleaving the virus from its sialic-acid receptors at the cell surface [ ] , it can be expected that an increase of its expression would lead to more rapid viral release (a shorter latent infection period for the wt strain) but may also hinder the subsequent attachment of virions to other cells, leading to decreased infectivity (longer infecting times). a complete understanding of the viral kinetics requires investigation of the ha/na balance. it has been shown that the a/brisbane/ / -like strains of the - influenza seasons differ from earlier h n seasonal strains by a few amino acid substitutions in the ha gene [ ] , but none of these involve interaction with the receptor and are therefore not likely to have influenced the changes in fitness. we have recently sequenced the entire genomes of our a/brisbane/ / strains, and found three amino acid substitutions in the ha gene for the h y mutant compared to the wt strain. two substitutions, g v and l f, do not involve interaction with the receptor, but the third, a t, lies within the receptor-binding site. this latter substitution has been noted in earlier work in relation to oseltamivir-resistant strains of the influenza virus [ ] and an investigation of its influence on viral kinetics is a necessary direction for future work. mathematical models have been successfully applied previously to characterize the in vivo virus replication kinetics of hiv [ , ] , hepatitis b and c [ , ] , and influenza [ , , ] , as well as in vitro viral yield experiments studying the effects of antiviral drugs [ ] and the optimization of vaccine production [ , ] . models of viral plaques have also been considered [ ] [ ] [ ] , although these were primarily directed at phage growth in an agar suspension of bacteria, a slightly different system than the cell monolayers considered here. the method presented here for the determination of the infection parameters differs from previous mathematical modeling approaches to viral dynamics in that we have considered the explicit dependence of two experimental quantities on the parameters, rather than fitting a full dynamical model to the time-course of an experiment. there are a number of benefits to this approach. first, we have been careful to determine that the two experimental quantities under consideration, plaque velocity and viral titer growth rate, depend on only two unknown infection parameters, the infecting time, t inf , and the latent infection period, t l . this ensures that the problem of parameter extraction is theoretically solvable, which is often not the case when fitting a multi-parameter model to experimental data (see, e.g., [ ] ). second, the experimental quantities themselves are robust and easily measurable in repeated experiments. the viral plaque is formed by the progression of an infection wave across the monolayer of cells [ ] whose constant velocity is determined by the infection kinetics averaged over many thousands of cells. therefore the measured plaque velocity depends on the average interaction of virus and cell, and is insensitive to stochastic effects on a small scale. similarly, the viral titer growth rate is due to the collective infection of thousands of cells and is independent of the details of initial infection (i.e., the precise value of the multiplicity of infection) or the total number of cells in the system. other quantities of in vitro experiments, such as the time and value of the viral peak in a yield experiment, are much more sensitive to experimental details. finally, the method we have applied here is robust to changes in the construction of the mathematical model itself. we have, for example, performed the same analysis of the plaque and yield assays using a stochastic model with more general assumptions about the cell transitions from latently infected to infectious and found nearly identical results (not shown here). it is important to note that the results we present here are preliminary, a proof of concept for the method which requires further verification and refinement. in particular, it would be useful to develop an experimental assay which could measure the infecting time for a given strain, in the same way that single-cycle viral yield experiments give an approximate measure for the latent infection period. it is also of interest to design a set of experiments which may be less expensive and laborious than those presented here, perhaps using fluorescent or photographic observations of cell cultures rather than virus titrations, and which can identify a fuller set of viral kinetics parameters. we are currently designing competition experiments for the a/brisbane/ / wt and h y mutant strains in which the predictions which follow from the parameters extracted here can be tested directly. when verified, the basic method of analyzing parallel plaque and viral yield experiments introduced here should be useful in other contexts. for example, the investigation of other drug-resistant viruses (e.g., that of the pandemic a/h n ), the rapid characterization of fitness for emerging strains, and assays measuring the activity of new antivirals would all be enhanced through the application of our method. the a/brisbane/ / -like (h n ) strains used were the oseltamivir-susceptible a/québec/ / (wt) and the oseltamivir-resistant a/québec/ / (na-h y mutant). these clinical isolates were obtained from two distinct, untreated, immunocompetent patients during the - influenza season [ ] . all experiments were performed on st gali-expressing mdck cells [ ] which over-express the a -( , ) sialic acid receptor predominantly found in the human upper respiratory tract. prior to infection, cells were grown to confluence, achieving an average diameter of * mm (used herein as a unit of length, d cell ). plaque assays were prepared using a semi-solid overlay of . % avicel rc- (fmc biopolymers, newark, delaware, usa) as described by matrosovich et. al. [ ] and stained with crystal violet. six-well plates (corning life sciences, lowell, ma, usa) were infected with + pfu=well, representing a multiplicity of infection (moi) of approximately { , and stained every h for h. the plates were then photographed using a dslr camera (fujifilm s with a mm nikkor macro objective) and the areas of viral plaques were measured using the threshold and analyze particle features of imagej, an nih opensource image analysis software [ , ] . all plaque radii at one timepoint (three independent experiments of three wells each) were averaged and the standard error of the mean was calculated. the radial growth rate was determined by linear regression to the average radii at time points prior to h. multiple-cycle viral yield assays for the a/brisbane/ / wt and h y mutant were performed with moi& { . supernatants were harvested every h for the first h of infection and every h subsequently, then titrated by plaque assay as previously described [ ] . the geometric average and standard deviation was determined from three replicates at each time point. high moi single-cycle yield assays were performed as described by hurt et. al. [ ] . monolayers of st gali-mdck cells were grown to confluence in -well plates and infected with pfu well (moi = ) in ml of infection medium. virus was adsorbed for h at c in a co incubator. the supernatant was then removed and cells were quickly washed once with acidic saline ( . % nacl in water, ph . ) and twice with pbs (ph . ). fresh maintenance medium was added and plates were returned to the incubator. supernatants were harvested every two hours for h ( figure a , experiment ) or every hour for h (experiment ), in duplicate. samples were frozen at { c until titrated in duplicate by plaque assay [ ] . the enzyme kinetics of the neuraminidase was measured in duplicate for each strain as described in [ ] , using the munana reagent ( -methyl-umbelliferyl-n-acetyl neuraminic acid (sigma-aldrich, st-louis, co, #m )). briefly, ml of live viruses diluted to : | pfu ml were incubated at c in opaque black microfluor b cs -well plates (vwr, montreal, qc, # - ) with ml of munana reagent ranging from to mm final concentration and ml of enzyme buffer [ : mix of mm mes ( -[n-morpholino]ethanesulfonic acid) ph . (sigma-aldrich, st-louis, co, #m ) and mm cacl (sigma-aldrich, st-louis, co)]. fluorescence was measured in a viktor multilabel counter (perkinelmer, waltham, ma) every seconds for minutes. the excitation wavelength was nm and the emission wavelength nm with a : nm excitation slit and a nm emission slit. k m and v max were calculated using a homemade excel macro, created following [ ] , and confirmed using the built-in ''enzyme kinetics'' features of the graphpad prism . software (graphpad software, la jolla, ca). plaque growth was simulated using a one-dimensional, timedelayed, partial differential equation (pde) model: where t(r,t) and i(r,t) are the densities of target and infectious cells, respectively, and v (r,t) is the virus concentration. s ( -fold smaller than the stokes-einstein value for a nm particle in c water); the rate of viral infectivity loss was fixed at c~ : h { based on the observed viral titer decay rate for both a/brisbane/ / strains in the multiple-cycle viral yield assays (see results); and the infectious lifespan was held fixed at h (table ) . simulations were initialized with a ''top-hat'' central region of infectious cell density with radius d cell = , and with all other cells in the target state. all fields rapidly take the form of traveling waves t(z), i(z) and v (z), where z~r{vt, with the same velocity, v. the multiple-cycle viral yield assay was modeled using a meanfield, delay-differential system of equations: where t and i are now the number of target and infectious cells, n is the total number of cells, v is the homogeneous virus concentration and all parameters have the same meaning as in the pde model above. these equations can be derived from equation ( ) by assuming spatial homogeneity and integrating over space. an expression for the exponential growth rate, l g , of viral titer in the multiple-cycle yield assay can be derived from this system by assuming that in the early phases of an infection (well before the viral titer peak): the number of target cells is approximately constant t(t)~t &n; there is an exponential growth of infectious cells i(t)~i e lgt and virus v (t)~e lgt with common rate l g ; and infectious cell death can be neglected (t i ??). substituting these expressions into ( ) yields a transcendental equation for the viral titer growth rate: for any values of p, b, t l and c, this equation can be solved numerically for the viral titer growth rate, l g . the assumptions made in deriving the expression require that the viral titer growth rate be measured early in the course of infection, well before the time of peak, when the number of infected cells is small compared to the total number of cells. both the plaque velocity and viral titer growth rate depend on the infection and production rates only through pb. since this quantity has units of inverse time squared (units of virus cancel), it is useful to rewrite the dependence on these rates as a characteristic time, ffiffiffiffiffi ffi pb s . a physical meaning can be ascribed to this quantity by considering equation ( ) in the case of a single infectious cell (i ~ ), within a completely susceptible cell population (t ~n ). if loss of viral infectivity, c, is neglected, the equations can be then integrated to show that t inf~ffi ffiffiffiffi ffi pb s is the time for that single infectious cell to cause the (latent) infection of one more cell. therefore, we call this characteristic time the infecting time. the contour plots in figure were created using the functional dependence of the plaque velocity and viral titer growth rate on the infecting time, t inf , and the latent infection period, t l , as determined by model simulation, along with the experimentally measured values of these quantities and their associated measurement error, under the assumption that these errors are normallydistributed. for example, the function f v , plotted in figure a and figure d , takes values between zero and one, according to where v exp is the experimentally-measured plaque velocity with measurement error s v and v mod (t inf ,t l ) is the theoretical dependence of the plaque velocity determined by model simulation. contours for the one and two-s values are drawn at f v~ : and : . a function on the parameter space, f lg , for the viral titer growth rate is constructed analogously. the product of these two functions is plotted in figures c and f to show the likely regions of viral kinetics parameters controlling growth for each virus strain. in fitting the single-cycle viral yield data, a more biologicallyrealistic model was used which assumes that the set of latent infection periods for a collection of cells is normally-distributed about t l , rather than fixed [ ] . in this model, target cell and virus dynamics are identical to that of equation ( ) where l and i are the number of cells latently infected and infectious, respectively, at the start of the experiment, f l (t) is the probability density function for the latent infection period and p i (t) is the probability that a cell remains infectious for at least a time t after the latent-infectious transition. if a dirac delta function is used for f l (t) and a heaviside step function for {p i (t), then the infectious cell dynamics of equation ( ) are recovered. in the fits to the single-cycle data (figure ), f l (t) was taken to be normal (truncated at t~ and renormalized) with parameters t l and s l ; the function p i (t) was also derived from a normal distribution f i (t) with p i (t)~ { d dt f i (t) , with fixed parameters t i~ h and s i~ h. oseltamivir for treatment and prophylaxis of influenza infection inhibition of neuraminidase inhibitor-resistant influenza virus by das , a novel sialidase fusion protein avian influenza (h n ): a threat to human health severe respiratory disease concurrent with the circulation of h n influenza world health organization h n vaccine task force ( ) vaccine production capacity for seasonal and pandemic (h n 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confidence intervals, and other measures of statistical accuracy influenza virus neuraminidase inhibitors the genetic makeup of amantadine-resistant and oseltamivir-resistant human influenza a/ h n viruses viral dynamics in hepatitis b infection mathematical model of antiviral immune response iii: influenza a virus infection structured model of influenza virus replication in mdck cells mathematical model of influenza a virus production in large-scale microcarrier culture the growth of viral plaques during the enlargment phase replication of viruses in a growing plaque: a reactiondiffusion model time-delayed spread of viruses in growing plaques parameter identifiability and estimation of hiv/aids dynamic models new low-viscosity overlay medium for viral plaque assays a simple and fast method for determining colony forming units assessing the viral fitness of oseltamivir-resistant influenza viruses in ferrets, using a competitive-mixtures model fluorometric assay of neuraminidase with a sodium ( -methylumbelliferyl-a-d-n-acetylneruaminate) substrate advanced biochemistry: protein properties and kinetics (btc ) -course website exploring the effect of biological delays in kinetic models of influenza within a host or cell culture induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells apoptosis: a mechanism of cell killing by influenza a and b viruses ns protein of influenza a virus down-regulates apoptosis impairment of multicycle influenza virus growth in vero (who) cells by loss of trypsin activity mode of action of the anti-influenza virus activity of plant flavonoid, , , -trihydroxy- -methoxyflavone, from the roots of scutellaria baicalensis the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the - oligo(a) synthetase/ rnase l pathway the computational aspects of this work were made possible by the facilities of the shared hierarchical academic research computing network (sharcnet: www.sharcnet.ca) and compute/calcul canada. we wish to thank marc auger for photographing the plaques. key: cord- - e kx authors: monto, arnold s.; fendrick, a.mark; sarnes, matthew w. title: respiratory illness caused by picornavirus infection: a review of clinical outcomes date: - - journal: clin ther doi: . /s - ( ) - sha: doc_id: cord_uid: e kx background: respiratory infections result from invasion of the respiratory tract, mainly by viruses, and are the leading cause of acute morbidity in individuals of all ages worldwide. during peak season, picornaviruses cause % of all episodes of acute nasopharyngitis (the common cold), the most frequent manifestation of acute respiratory infection, and produce more restriction of activity and physician consultations annually than any other viral or bacterial source of respiratory illness. objective: this article reviews the clinical impact and outcomes of picornavirus-induced respiratory infections in specific populations at risk for complications. it also discusses the potential economic impact of the morbidity associated with picornavirus-induced respiratory infection. methods: relevant literature was identified through searches of medline, ovid, international pharmaceutical abstracts, and lexis-nexis. the search terms used were picornavirus, rhinovirus, enterovirus, viral respiratory infection, upper respiratory infection, disease burden, economic, cost, complications, asthma, copd, immunocompromised, elderly, otitis media, and sinusitis. additional publications were identified from the reference lists of the retrieved articles. conclusions: based on the clinical literature, picornavirus infections are associated with severe morbidity as well as considerable economic and societal costs. future research should focus on identifying patterns of illness and the costs associated with management of these infections. new treatments should be assessed not only in terms of their ability to produce the desired clinical outcome, but also in terms of their ability to reduce the burden of disease, decrease health care costs, and improve productivity. respiratory infections are recognized as the leading cause of acute morbidity in individuals of all ages worldwide. in developing countries, the morbidity associated with respiratory infections may be at least as severe as that in industrialized countries, and these infections are the leading cause of death in children under years of age.',* the health interview survey quantifies illnesses that result in disability and/or physician consultation in the us population. based on this survey, the annual incidence of acute respiratory infections in the united states in was %, exceeding the rates of digestive conditions, injuries, and infective/parasitic conditions combined. these infections may result from invasion of the respiratory tract by bacteria, viruses, or, in rare cases, other infectious agents; however, viruses are the most frequently identified pathogens. the viral pathogens primarily associated with acute respiratory infections include picomaviruses, coronaviruses, adenoviruses, paraintluenza viruses, influenza viruses, and respiratory syncytial viruses' (figure ) . picornaviruses are a large group of rna viruses. in terms of causing acute respiratory infection in humans, the most important picornaviruses are the rhinoviruses and enteroviruses. picornaviruses contribute significantly to the incidence of acute respiratory infections. in a study conducted during the autumn, picornaviruses were found to cause % of all episodes of acute nasopharyngitis, which is the most common manifestation of acute respiratory infection. individually, the incidence of acute nasopharyngitis in adults ranges from to cases per year and in children from to cases per year. , although picornavirus-induced nasopharyngitis is often referred to as the common cold, the morbidity associated with these infections should not be trivialized. in fact, because rhinovirus-induced illnesses are so common, they produce more restriction of activity and physician consultations annually than respiratory illnesses caused by other viruses or bacteria.s this article reviews the clinical impact and outcomes associated with picomavirusinduced respiratory infection in specific populations at risk for complications secondary to these infections. it also discusses the potential economic impact of the morbidity associated with picornavirusinduced respiratory infections. relevant literature was identified through searches of medline, ovid, international pharmaceutical abstracts, and lexis-nexis. the search terms used were picnrnuvirus, rhinovirus, enterovirus, vim respiruto~ infection, upper respiratory infection, disease burden, economic, cost, complicutions, asthma, copd, immunocompromised, elderly, otitis media, and sinusitis. additional publications were identified from the reference lists of the retrieved articles. rhinovirus infections are associated with more pronounced clinical manifestations than respiratory infections of other viral etiologies. as many as % to % of human rhinovirus infections result in symptomatic respiratory episodes characterized by rhinorrhea, nasal obstruction, cough, and hoarseness." the median duration of illness in young adults is days; however, symptoms may last up to weeks in one quarter of cases, and the duration of illness may be even longer in children and the elderly. there is a peak in the incidence of rhinovirus infection in early fall and again in mid-to late springjo ( figure ). younger children appear to be more susceptible to viral respiratory infections, as demonstrated by the fact that preschool children can have to respiratory infections per year. i ' in fact, during an outbreak of a new virus strain, over three fourths of the children in a nursery school were infected. also, secondary attack rates in family members have been observed to range from % to %, depending on the immune status of the exposed individual.r the high incidence of viral respiratory infections is most likely due to the large number of rhinovirus types and the fact that immunity is type specific and of short duration. for these reasons among others, the role of rhinoviruses has overshadowed that of enteroviruses, which also contribute to the morbidity of viral respiratory infections. it is estimated that enteroviruses infect between and million people annually. episodes of enterovirusinduced respiratory infection are seen throughout the year; however, the most prominent respiratory syndrome caused by these pathogens occurs in children during the summer months.'" these enteroviral infections typically result in a nonspecific febrile illness complicated by respiratory symptoms. i although viral respiratory infections due to picornaviruses appear to be selflimiting in most healthy adults, they are increasingly implicated in acute infectious exacerbations of illnesses in high-risk populations. technological advances such as polymerase chain-reaction testing have made virus identification more sensitive and readily available. this, in turn, has made it possible to identify specific patient populations (eg, the elderly, infants, and immunocompromised persons), who are particularly susceptible to picornavirus infection and in whom infectious episodes typically significantly increase the use of health care resources. in addition, these advances in technology have confirmed the results of earlier studies, further demonstrating the significance of rhinoviruses in causing or predisposing patients to otitis media and sinusitis and exacerbating other chronic respiratory diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (copd). asthma is estimated to affect > million people ( % of the population) in the united states, resulting in over i million days of restricted activity and , hospitalizations annually. . in , the estimated cost of treating asthma was $ . billion; % of those costs were associated with emergency department visits, hospitalizations, and death. is respiratory infections caused by picornaviruses are believed to be a major contributor to asthma exacerbations, particularly in children. asthmatic children appear to experience a significantly greater number of viral respiratory infections compared with nonasthmatic children. up to % of acute asthma exacerbations in children are thought to be related to viral respiratory infections the most common cause of these infections are the rhinoviruses, which have been recovered from the respiratory tract at the onset of an asthma attack, suggesting that the virus may have been an initiating factor in the attack.*' several studies have identified rhinovirus as the pathogen most commonly associated with asthma in those > years of age.r rakes et a conducted a crosssectional study examining the prevalence of respiratory viruses in children presenting to the emergency department because of wheezing episodes. they found that respiratory viruses were present in % of children < years of age and in % of children between and years of age. rhinovirus was the predominant pathogen isolated in children > years of age ( i%), indicating that the majority of wheezing episodes in this age group may be associated with rhinoviruses. the prevalence of rhinoviruses in asthmatic children was further investigated by johnston et a . " in a -month trial, these authors evaluated children between the ages of and years with the respiratory symptoms of wheezing and/or cough and a decrease in peak expiratory flow rate (pefr). respiratory viruses, two thirds of which were rhinoviruses, were detected in % of patients. viral respiratory infections were implicated in % of episodes with upper respiratory symptoms and % of episodes with reductions in pefr. the median reduction in pefr was l/min, and the median duration of decline in pefr was days. based on their results, the authors reported that % to % of asthma exacerbations in schoolaged children appear to be associated with viral respiratory infections, primarily with rhinoviruses. in only % to % of adult asthmatic patients.*(' however, beasley et a found the prevalence of viral respiratory infections to be % in adults with severe asthma and % in those with mild exacerbation of asthma. nicholson et a conducted a longitudinal trial in asthmatic adults between the ages of and years, evaluating episodes of respiratory illness over years. the average duration of asthma in the patient population was - years. thirty-eight percent of the patients had previously been hospitalized for asthma. the investigators found that % of subjective asthma exacerbations occurred with symptomatic colds and % of symptomatic colds were associated with symptoms of asthma. these findings suggest that acute respiratory infections may be as commonly linked to exacerbations of asthma in adults as they are in children. in this study, clinical episodes of respiratory illness were associated with objective evidence of an asthma exacerbation (decrease in pefr l/min). sixty-three percent of laboratory-confirmed acute respiratory infections were found to be caused by rhinoviruses. of the clinical episodes of respiratory illness associated with an objective asthma exacerbation, patients consulted a general practitioner in % of episodes, required oral steroids in %, required a nebulizer in %, and were prescribed antibiotics in %. in addition to the clinical impact of rhinoviruses in asthma, their impact on medical resource use has also been demonstrated. teichtahl et al*" compared the incidence of respiratory infection in patients admitted to the respiratory medical unit for acute asthma with that in control subjects admitted to the surgical unit for elective surgery. twenty-nine ( %) of the patients who were admitted for asthma exacerbation had evidence of a respiratory infection, compared with ( %) of the control group (p < . ). of the asthma patients with confirmed evidence of a viral infection, ( %) had rhinovirus infections. significant mortality resulting from viral respiratory infections is routinely observed in the elderly. although mortality resulting specifically from rhinoviral respiratory infections has not been demonstrated, considerable morbidity is associated with these infections. in a study by nicholson et a , adults aged between and years were monitored for episodes of viral upper respiratory tract infections. the annual incidence of viral respiratory infections was found to be . per patient. a total of pathogens were identified in ( %) of the episodes for which specimens were available. of the pathogens identified, ( %) were rhinoviruses. of the ( %) patients in whom rhinovirus was the sole pathogen isolated, ( %) patients had lower respiratory tract involvement. the median duration of illness in these patients was days. of the patients with lower respiratory tract involvement, ( %) had to restrict their normal activities (eg, cleaning, grocery shopping), and ( %) were bedridden. a total of i ( %) patients sought medical attention, and antibiotics were prescribed for %. altogether, patients were hospitalized, of whom died from exacerbation of copd by rhinovirus infection. the authors reported that the overall burden of rhinovirus infection was greater than that of the viruses typically associated with significant morbidity and mortality, such as influenza. a second study assessing the impact of rhinovirus infection on the elderly focused on an outbreak of respiratory illness among patients in a long-term care facility.'x specimens from the throat and nasopharynx of patients were obtained and cultured. of the cultures, ( %) were positive for rhinovirus. each of the patients with rhinovirus-positive cultures had upper respiratory tract symptoms, ( %) had systemic symptoms, ( %) had gastrointestinal symptoms, and i ( %) had lower respiratory tract symptoms. more severe disease progression was found in ( %) rhinovirus-infected patients with concomitant copd. five of ( %) patients with copd required pharmacologic bronchodilation, ( %) had to be transferred out of the facility because of declining respiratory function. and ( %) died of respiratory failure. these findings indicate that outbreaks of rhinovirus infections in nursing homes should be considered potentially serious. a study from the s indicated that . % of community respiratory diseases were complicated by otitis media and . % by sinusitis. although such percentages may seem insignificant, the high frequency of respiratory infections means that large numbers of patients are affected. a conservative estimate places the inci-dence of viral respiratory infections in the united states at . infection per person per year." based on these data, an estimated million people in the united states experience a viral respiratory infection each year, with - million of these infections complicated by otitis media and almost million complicated by sinusitis. new technologies that permit more accurate identification of viral pathogens will probably reveal that the frequency of virally induced infections is even higher and that viral pathogens are a precursor in more cases of otitis media and sinusitis than originally estimated; however, further research is necessary. it has been estimated that > % of patients with otitis media also have symptoms of upper respiratory tract infections that are probably the result of a primary viral infection. this correlation is supported by the fact that % to % of patients with otitis media have detectable virus in the nasopharynx, and % have detectable virus in the middle ear. viruses can also complicate the course of recovery for patients with otitis media. in recent studies,"o,"' viruses in the middle ear have been implicated in the apparent failure of antibiotic treatment by complicating the response to treatment. in a study in patients with combined viral and bacterial otitis media,"' the presence of rhinovirus was associated with a higher failure rate of antibiotic therapy than was respiratory syncytial virus, parainfluenza virus, or influenza virus. rhinovirus has been identified as a potential pathogen in - % of cases of community-acquired sinusitis. although there is evidence that these viruses contribute to the development of sinusitis, their exact role remains undefined. in a study by turner et al,"" healthy adults were experimentally infected with rhinovirus. on magnetic resonance imaging, changes were observed in the paranasal sinuses of % of subjects. the relationship between viral infections and sinusitis was further investigated in a study evaluating naturally acquired acute nasopharyngitis.' in this study, sinus abnormalities were detected by computed tomography in over % ( ) of adult patients. after weeks of observation, spontaneous resolution of these abnormalities occurred in almost % ( ) in some patient populations. is although the role of picornaviruses in these more serious diseases of the lower respiratory tract cannot be determined definitively without more extensive sampling of the lower respiratory tract, recent studies have pointed to the importance of picornavirus infection in these disease processes in particular patient populations.'" infants with bronchopulmonary dysplasia are at a significantly increased risk for acute episodes of severe viral respiratory infection. in a study by daily, of ( %) preterm infants with bronchopulmonary dysplasia required rehospitalization, the most common cause being upper respiratory tract infection. whereas some studiesi ,i have indicated that respiratory syncytial virus is the pathogen most commonly isolated in these patients, oth-er@.s have demonstrated that rhinovirus makes a substantial contribution. in fact, because none of these studies used assays suitable for identifying rhinoviruses, the role of these agents may have been underestimated. chidekel et alx studied patients ( cases) with a history of bronchopulmonary dysplasia for an average of months and identified ( %) cases of severe lower respiratory tract infection that were associated with rhinoviruses. of the patients in these cases, ( %) required hospitalization for a mean duration of days, and ( %) were admitted to the intensive care unit. patients with rhinoviral respiratory infections required additional long-term medical therapy, suggesting that rhinoviruses can have lasting clinical implications. patients with cystic fibrosis are also at increased risk for complications of viral respiratory infections. in fact, % to % of pulmonary exacerbations are preceded by viral respiratory infections. collinson et ais recorded episodes of acute nasopharyngitis over months in children with cystic fibrosis aged between months and years, translating into an annual incidence of . %. cultures were available for of the episodes, and the causative pathogen was identified as rhinovirus in ( %) episodes. these viral respiratory infections were associated with acute decreases in pulmonary function, increased rates of disease progression, and a predisposition to bacterial pulmonary infection. lower respiratory tract infections are the most common complication of viral respiratory infection in immunocompromised patients.s in one study,"o the com-monly identified causes of viral respiratory infection in immunocompromised patients included cytomegalovirus, herpes simplex virus, and varicella-zoster virus; however, viruses such as the picornaviruses were also implicated. in this and other studies, it is not certain that the techniques used were capable of detecting rhinovirus and whether these viruses were accurately represented in the study findings. immunocompromised patients are at risk for developing serious or life-threatening disease as a result of a viral infection of the respiratory tract. in the study by rabella et a " both rhinoviruses and enteroviruses were identified as viral pathogens in immunosuppressed patients (ie, patients infected with hiv, bone marrow transplant recipients, organ transplant recipients, patients with hematologic malignancies) with a suspected respiratory infection between january and december . picornavirus infections were associated with respiratory failure in patients, of them requiring mechanical ventilation and the other having a clinical course resulting in pneumonia. in a study conducted at the university of texas m.d. anderson cancer center (mdacc), " cases of picornavirus infection were identified in adult patients with leukemia and bone marrow transplant recipients. more than % of the picornavirus infections tested were found to be caused by rhinoviruses. in several patients, the infection progressed to pneumonia that appeared to be bacterial or fungal in origin. however, some patients had a clinical course consistent with viral pneumonia and died of unexplained interstitial pneumonia. in a -year retrospective follow-up study of blood and bone marrow transplant recipients who had symptoms of up-per respiratory tract infection, rhinovirus was identified as the causative pathogen in all patients. eight ( %) patients developed progressive pneumonia that resulted in death. autopsies on of patients demonstrated interstitial pneumonitis and/or changes of acute respiratory distress syndrome. the deaths were attributed to progressive viral pneumonia on the basis of histologic features and ante mortem isolation of rhinovirus from lower respiratory tract sites. in a prospective study conducted at mdacc " an evaluation of adult bone marrow transplant recipients hospitalized for an acute viral respiratory infection demonstrated that % of the identified infections were due to picornaviruses. of patients in whom rhinovirus had been implicated as the causative pathogen, ( %) developed complicating pneumonia, and ( %) died as a result. findings from autopsies performed on of the patients were consistent with progressive viral pneumonia. a third trial at mdacc investigating acute respiratory illnesses in hospitalized patients with leukemia found that % of viral infections were caused by picornaviruses. together, the results of these studies demonstrate that immunocompromised hosts are at increased risk from serious picornavirus infections, which have the potential to result in death. although the economic impact of picornavirus infections is most pronounced in the at-risk populations described, these infections have a significant economic impact on the general population as well. picornavirus infection is manifested primarily as acute nasopharyngitis. according to estimates from the national center for health statistics > million cases of the common cold required medical attention and resulted in million days of restricted activity in . even more significant from the economic and productivity perspectives, acute nasopharyngitis caused - million lost workdays in adults aged years and million lost school days in children aged < years. also important from an employer's perspective is the fact that employees miss days of work not only when they themselves are ill but when they stay home to care for sick children. productivity is also diminished when employees come to work with a picornavirus-induced respiratory infection. the compromised quality of life associated with these infections in turn has a negative effect on productivity. furthermore, many of the medications currently used for the palliative treatment of respiratory symptoms cause drowsiness, which may further diminish efficiency and productivity in the workplace. finally, because respiratory picornavirus infections are easily transmitted to persons in proximity to infected individuals, other employees are likely to become infected, resulting in additional losses in productivity. because factors affecting productivity and quality of life are increasingly important in today's health care environment, further analyses are needed to better quantify the impact of respiratory picornavirus infections on the overall health care population. to date, the direct use of health care resources associated with viral respiratory illness in the general population has not been investigated comprehensively. the current literature focuses primarily on atrisk populations. these data indicate that use of emergency department and hospital services for exacerbations or compli-cations caused by picomavirus infections is the major factor in the economic impact of respiratory infections within such atrisk subpopulations as asthma patients, the elderly, high-risk patients with diseases of the lower respiratory tract, cystic fibrosis patients, and immunocompromised patients. patients with sinusitis or otitis media are an exception, with physician office visits and antibiotic use accounting for the majority of the economic impact. the economic impact of picornavirus infections is illustrated by findings that asthma exacerbations are linked to rhinovirus infections in % of children and % of adults hospitalized for asthma exacerbations. ", " if these statistics are applied to the average , asthmarelated hospitalizations per year ( . % of which involve children),ls rhinovirus infections are responsible for an estimated , hospitalizations annually. based on these figures and an average cost of $ per hospitalization for an asthma exacerbation, the cost of hospitalizations due to rhinovirus-induced asthma exacerbations would be ~$ million annually. a prospective economic analysis of hospitalizations due to respiratory infections in the asthmatic population remains to be conducted. other markers of the economic impact of rhinoviral respiratory infections in the asthmatic population are not as well defined as the costs associated with hospitalization. factors such as the use of other health care resources (medications and physician office visits), productivity, quality of life, and work and school absenteeism have not been measured to a significant extent, and further evaluation of these variables will be crucial to establishing the true economic burden of viral respiratory infections in the asthmatic population. patients with picornavirus infections who are at high risk for complications because of age (infants, the elderly), lower respiratory tract involvement (patients with copd), or immune status (cystic fibrosis patients, immunocompromised patients) constitute additional populations in which hospital and emergency department use are the greatest contributors to overall costs. again, the literature described in this review illustrates a definite link between picornavirus infections and increased health care use in these populations; however, the overall patterns of resource use and costs of illness have not been defined. in addition to the significant morbidity associated with picornavirus infections in the clinical literature, these infections also have substantial economic and societal implications. because the magnitude of these implications is not yet well defined, future research should focus on identifying the patterns of illness and costs associated with the management of these infections. the ability to determine the costs associated with viral respiratory infections will be particularly important when evaluating new treatments, which should be viewed not only in terms of their ability to produce the desired clinical outcome, but also in terms of their ability to reduce the burden of disease, decrease health care costs, and improve productivity. acknowledgment support for this article was provided by viropharma inc, exton, pennsylvania. the clinical impact of human respiratory virus infections acute respiratory infection in children of developing countries: challenge of the s current estimates from the national health interview survey, . national center for health statistics rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes frequency and natural history of rhinovirus infections in adults during autumn rhinovirus infections in an industrial population. i. the occurrence of illness illness in the home: study of , illnesses in a group of cleveland families viral respiratory infections in the community: epidemiology, agents, and interventions principles and practice oj' infectious diseases the common cold respiratory disease in group day care acute respiratory illness in nursery school children: a longitudinal study of the occurrence of illness and respiratory viruses viral isolation rates during summer from children with acute upper respiratory tract disease and healthy children temporal and geographic patterns of isolates of nonpolio enterovirus in the united states rhinoviruses: important respiratory pathogens surveillance for asthma-united states - centers for disease control and prevention. forecasted state-specific estimates of self-reported asthma prevalence-united states an economic evaluation of asthma in the u.s greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings the incidence of respiratory tract infection in adults requiring hospitalization for asthma viruses as precipitants of asthmatic attacks in children rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses community study of role of viral infections in exacerbations of asthma in l i year old children viral respiratory tract infection and exacerbations of asthma in adult patients respiratory viruses and exacerbations of asthma in adults acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study a rhinovirus outbreak among residents of a long-term care facility report of a workshop on respiratory viral infections: epidemiology, diagnosis, treatment, and prevention bacteriologic failure of amoxicillinclavulanate in treatment of acute otitis media caused by nontypeable haemophilus influenzae association of rhinovirus infection with poor bacteriologic outcome of bacterial-viral otitis media. c/in infecr dis hayden fg. detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds computed tomographic study of the common cold home oxygen therapy for infants with bronchodysplasia: growth and development rhinovirus infection associated with serious respiratory illness in patients with bronchopulmonary dysplasia effects of upper respiratory tract infections in patients with cystic fibrosis community respiratory virus infections in immunocompromised patients with cancer respiratory viral infections in immunocompetent and immunocompromised persons rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients conventional respiratory viruses recov community respiratory virus infections among hospitalized adult bone marrow transplant recipients key: cord- -tv ntug authors: gautam, ablesh; tiwari, ashish; malik, yashpal singh title: bioinformatics applications in advancing animal virus research date: - - journal: recent advances in animal virology doi: . / - - - - _ sha: doc_id: cord_uid: tv ntug viruses serve as infectious agents for all living entities. there have been various research groups that focus on understanding the viruses in terms of their host-viral relationships, pathogenesis and immune evasion. however, with the current advances in the field of science, now the research field has widened up at the ‘omics’ level. apparently, generation of viral sequence data has been increasing. there are numerous bioinformatics tools available that not only aid in analysing such sequence data but also aid in deducing useful information that can be exploited in developing preventive and therapeutic measures. this chapter elaborates on bioinformatics tools that are specifically designed for animal viruses as well as other generic tools that can be exploited to study animal viruses. the chapter further provides information on the tools that can be used to study viral epidemiology, phylogenetic analysis, structural modelling of proteins, epitope recognition and open reading frame (orf) recognition and tools that enable to analyse host-viral interactions, gene prediction in the viral genome, etc. various databases that organize information on animal and human viruses have also been described. the chapter will converse on overview of the current advances, online and downloadable tools and databases in the field of bioinformatics that will enable the researchers to study animal viruses at gene level. viruses are notorious to infect all forms of life ranging from bacteria to chordates. in humans, viruses are known to cause infectious diseases such as influenza, hepatitis, aids, diarrhoea, encephalitis, dengue fever and, more recently, severe acute respiratory syndrome (sars), ebola (singh et al. a) , zika (singh et al. b) , etc. despite the vaccines and treatments for such diseases, morbidity and mortality both occur as a result of the viral infections. viral disease of animals not only affects the production but also is a threat to humans (saminathan et al. ) . a rapid growth in the availability of sequencing methods and a vast amount of viral sequence data have been generated during recent times. thus, it is imperative to decipher this data using more advanced tools such as bioinformatics resources. a large number of bioinformatics tools that can aid in the analysis of viral genomes and develop preventive and therapeutic strategies have been developed for human as well as animal viruses. this chapter will introduce virologists to some of the common as well virus-specific bioinformatics tools that the researches can use to analyse viral sequence data to elucidate the viral dynamics, evolution and preventive therapeutics. analysis of viral sequence involves use of certain tools that are employable on any novel sequence, for example, gene identification, orf identification, functional annotation and phylogeny. however, due to small genome size, viruses have complex methods to maximize the coding potential of genomes and evolution. many viruses utilize overlapping reading frames or translational frameshifts to code for multiple proteins from limited genome sequences. also, higher rates of mutations and recombination between related viruses pose a challenge in accurate phylogenetic and evolutionary analysis of viruses using general-purpose softwares. lately, enormous growth in the volume and diversity of viral sequences in the databases has been seen. now, it has become imperative to organize data of these viral sequences in virus family-specific resources tailored for accurate analysis of a specific virus. one of the most common applications of bioinformatics in virology was to use phylogenetic analysis of the viral isolates to aid in the epidemiological analysis of viral outbreaks. general-purpose phylogeny programs such as phylip (felsenstein ) have been used extensively for the phylogeny and molecular epidemiology of viruses. a comprehensive list of these packages and web servers is maintained by joe felenstein at http://evolution.genetics.washington.edu/phylip/software.html. an open reading frame (orf) is the part of genome that translates into a protein. finding orf is one of the key steps in viral genome analysis. it forms the basis for further analysis such as homologous search, predicting proteins, functional analysis and viral vaccine and antiviral target discovery. if an orf translates a surface protein that is unique to that virus, it may elicit immune responses and could potentially be a vaccine candidate. orf finder by ncbi is a orf prediction program (rombel et al. ) . the program outputs a range of each orfs along with its protein translation in six possible reading frames from the input dna sequence. it can be used to search newly sequenced dna for potential protein encoding sequences and to verify predicted proteins using smart blast or blastp (altschul et al. ). however, the web version of the program is limited to a query sequence length of kb only. a standalone system has no limitation on length but is available only for the linux operating system. neg , a -codon novel orf in segment of influenza virus, was visualized using orf finder (clifford et al. ). using the orf finder in association with the basic local alignment search tool blast, orfs were found in the hz- virus genome (cheng et al. ) . due to small genome size, viruses employ multiple strategies to maximize the coding potential including frameshifts and alternative codon usage. thus, virus-specific programs have been developed to overcome these challenges. genemark (http://opal.biology. gatech.edu/genemark/genemarks.cgi) provides gene prediction tools for viruses (besemer and borodovsky ) . viral genome organizer (vgo) -a java-based web tool -offers identification of gene and orf identification in viral sequences (upton et al. ) . identification of immune epitopes is important in designing new vaccine candidates and in diagnostics. an epitope is the part of an antigen that is recognized by the receptors of immune system components such as antibodies, b cells or t cells. epitopes have been generally classified as either linear or conformational epitopes. t cells recognize linear epitopes, short continuous strings of amino acids derived from protein antigen, presented with mhc class i molecules. b cells and antibodies, on the other hand, recognize conformational epitopes which are formed by interactions of amino acids with multiple discontinuous segments forming a threedimensional antigen (barlow et al. ). owing to the simple linear structure of t cell epitopes, their interaction with receptors can be modelled with high accuracy (delisi and berzofsky ) . a large number of prediction databases and servers thus are available for linear epitope prediction. mhcpep (brusic et al. ) , syfpeithi (rammensee et al. ) , fimm (schonbach et al. ) , mhcbn (bhasin et al. ) and epimhc (reche et al. ) are some of the commonly used t cell epitope prediction programs. immune epitope database and analysis resource (https://www.iedb.org) (vita et al. ) offers the most comprehensive set of tools for epitope analysis for epitope prediction covering hla-a and hla-b for humans as well as chimpanzee, macaque, gorilla, cow, pig and mouse and is one of the few databases that cover such a variety of organisms. since , iedb uses netmhcpan as prediction method. netmhc server uses the artificial neural network method to predict binding of peptides to different alleles from human as well as animals including cattle and pig ( from core). the database also contains curated data for many viruses including influenza and herpesviruses. b cell receptors and epitope interactions are more complex in nature than the linear epitopes for t cells; thus, accuracy of b cell epitopes is relatively low. furthermore, most of the current databases are centred on linear rather than conformational epitopes. bcipep is a tool developed for predicting the linear epitope of b cells (saha et al. ) . epitome is a database of structure-inferred antigenic residues in proteins (schlessinger et al. ) . epitome is especially useful in the prediction of antibodyantigen complex interaction. the database is available at http://www.rostlab.org/ services/epitome/. antijen is an intricate database with entries on both t cell and b cell epitopes. it emphasizes on integration of kinetic, thermodynamic, functional and cellular data within the context of immunology and vaccinology (toseland et al. ) (fig. . a ). three-dimensional prediction of viral proteins can be used to predict the correlation between actual protein structure and antigenic sites, folding surfaces and functional motifs. such structural modelling tools may be implicated to identify and design novel candidates for antiviral inhibitors and vaccine targets. secondary structures may be predicted using the tool predictprotein (http://www.predictprotein.org/) (rost et al. ) . using this online tool, along with secondary structures, solvent accessibility and possible transmembrane helices can be predicted. further, it also provides expected accuracy of prediction methods. swiss-model (http:// swissmodel.expasy.org/) is a popular tool for the prediction of a -d structure of a protein. -d structure prediction programs usually employ homology searching using similar and known protein structures as templates. one of the most commonly used database for such templates is protein data bank (pdb) (reddy et al. ) . output from the swiss-model program includes the template selected, alignment between the query sequence and the template, and the predicted -d model. results of swiss-model are, however, only sent by email (figs. . b, . c, . d and . e). for long, bioinformatic analysis of viruses utilized common bioinformatics tools developed for other organisms. however, analysing viral genomes using general bioinformatics tools could compromise the accuracy and sensitivity of analysis. virus genomes are too small (e.g. < kb) to compute statistics with their codon usage. to maximize the coding potential, viruses work with unusual codon usage patterns comprising of overlapping coding and non-coding functional elements. additionally, viruses also rely on other translational mechanisms such as stop codon read-through, frameshifting, leaky scanning and internal ribosome entry sites. comparative genomic analysis of viruses is complicated by the fact that highly conservative sequences may not be coding for anything. presence of overlapping pairs may be indicated by conservation for the sequences where there is overlapping of cdss and/or non-coding functional elements. novel virus types comprise of new cdss that are different than previously known cdss. there are multiple databases and tools available for analysis of human viruses; however, there are still only a limited number of resources designed specifically for veterinary viruses. in this section, some of the databases and resources useful for the analysis of veterinary viruses are discussed (table . ). viruses are one of the most diversified and dynamic microorganisms. with increasing viral genome sequencing, there was a need to develop bioinformatics tools to compare and analyse the voluminous data. to meet this requirement, one such downloadable software package is base-by-base, which aids in analysis of whole viral genome alignments at single nucleotide level (brodie et al. ). moreover, with the online resource genome information broker for viruses (gib-v), comparative studies can be made using the generic tools such as clustalw, blast and keyword search algorithms (hirahata et al. ). another downloadable web server tool, viroblast, is an exclusive blast tool that can be used for queries against multiple databases (deng et al. ). sequences from a variety of viral strains can be analysed simultaneously using the alvira software, which is a multiple sequence alignment tool that provides graphical representation as well (enault et al. ). furthermore, comparative analysis of genes and genomes of coronavirus can be carried out by using the covdb (coronavirus database) (huang et al. ). the digital resource viralzone is designed specifically to comprehend viral diversity and acquire information on viral molecular biology, hosts, taxonomy, epidemiology and structures (hulo et al. ). the simmonics program was upgraded to the simple sequence editor (sse) software package, wherein the user-given sequences can be aligned and annotated and further can be analysed for diversity and phylogeny (simmonds ) . evolutionary changes in viral genome lead to polymorphisms in their proteins, which in turn result into changes in viral phenotype such as viral virulence, viral-host interactions, etc. the digital database, viralorfeome, not only stores all variants and mutants of viral orfs, but also provides tools to design orf-specific cloning primers (pellet et al. ). further, degenerate primer pairs can be selected and matched to amplify user-defined viral genomes using the online tool prism (yu et al. ). the recent advances in nextgeneration sequencing and technologies have facilitated to study viral population at an advanced level. the viral population biodiversity and dynamics can be studied using the first such tool developed, phaccs (phage communities from contig spectrum), that can analyse the shotgun sequence data to estimate the structure and diversity of phages (angly et al. ) . later on, more tools/resources were developed to analyse viral metagenomics sequences, such as viral informatics resource for metagenomic exploration (virome), viral metagenome annotation pipeline (vmgap) and metavir (lorenzi et al. , roux et al. , wommack et al. . novel viruses can be identified from a pool of specimen types using a specific computational pipeline, virushunter ). the phenomenon of genetic recombination in viruses is responsible for the emergence of new viruses, increased virulence and host range, immune evasion and development of antiviral resistance. this distinct process of viral recombination can be detected by two bioinformatics tools, viz. jphmm (jumping profile hidden (schultz et al. ; routh and johnson ) . the jphmm, a web server, can be used for predicting recombination in hiv- and hbv, whereas virema, a downloadable software, can be used to analyse next-generation sequencing data. additionally, another software called vipr hmm (viral identification with a probabilistic algorithm incorporating hidden markov model) can detect recombinant and nonrecombinant viruses using microbial detection microarrays (allred et al. ). further, viral genome sequences can be searched for degenerate locus of recombination (lox)-like sites by a web server called selox (surendranath et al. ) . a downloadable software, virapops, is a forward simulator that allows simulation of rna virus population (petitjean and vanet ) . with this software, the drastic changes in rapidly evolving rna viruses such as mutability, recombination, variation, covariation, etc. can be simulated to predict their effects on viral populations. seqmap is a tool capable of identifying viral integration sites (vis) from ligationmediated pcr (lm-pcr), linear amplification-mediated pcr (lam-pcr) and nonrestrictive lam-pcr (nrlam-pcr) reactions and mapping short sequences to the genome (hawkins et al. ) . further, vis can also be detected by three more distinct tools, virusseq, viralfusionseq, and virusfinder , li et al. . for more precise vis prediction, all four tools can be employed by virologists. mirnas: a microrna (mirna) is a small, regulatory, non-coding rna molecule that regulates the translation or stability of viral and host target mrnas, thereby affecting viral pathogenesis. this host-viral regulatory relationship can be investigated by a database called vita, capable of curating known viral mirna genes and known/putative target sites of host mirna (hsu et al. ). vita exploits miranda and targetscan to scan viral genomes and determine mirna targets. vita is also capable of annotating the viruses, virus-infected tissues and tissue specificity of host mirnas. subtypes of viruses, for example, influenza viruses, and the conserved regions in various viruses can also be compared using the vita database. viral mirna candidate hairpins can be predicted using the database vir-mir. it serves as a platform to query the predicted viral mirna hairpins (based on taxonomic classification) and host target genes (based on the use of the rnahybrid program) in human, mouse, rat, zebrafish, rice and arabidopsis (li et al. ) . sirna: a sirna is similar to mirna that operates within the rna interference (rnai) pathway. it interferes in expression of specific genes and, therefore, is used in post-transcriptional gene silencing. virsirnadb is an online curated repository that stores experimentally validated research data of sirna and short hairpin rna (shrna) targeting diverse genes of important human viruses, including influenza virus (tyagi et al. , thakur et al. . the current database includes experimental information on sirna sequence, virus subtype, target gene, genbank accession, design algorithm, cell type, test object, method, efficacy, etc. a web-based software, sivirus, is an antiviral srna design software that allows analysis of influenza virus, hiv- , hcv and sars coronavirus (naito et al. ). further, viral sirna sequence data sets can be analysed using the softwares visitor and virome (antoniewski ; watson et al. ) . a perl script, called paparazzi, enables reconstitution of viral genome using a viral sirna in a given sample (vodovar et al. ). host-pathogenic interactions play an important role in determining the pathogenicity of a pathogen or immune evasion mechanism of a host. to comprehend such interactions between viral and host cellular proteins, various databases and softwares are available. one such database is phever that enables to explore virusvirus and virus-host lateral gene transfers by providing evolutionary and phylogenetic information (palmeira et al. ). this distinct database catalogues homologous families between different viral sequences and between viral and host sequences. it compiles the extensive data from completely sequenced genomes ( nonredundant viral genomes, non-redundant prokaryotic genomes, eukaryotic genomes ranging from plants to vertebrates). thus, it enables compiling of various proteins into homologous families by selecting at least one viral sequence, related alignments and phylogenies for each of these families. with increasing availability of viral genome sequences, data mining, curation and genome annotation have become essential components to better comprehend the structure and function of genome components. this information can further be exploited to develop diagnostics, vaccines and therapeutics. there are a number of tools available capable of annotation and classification of viral sequences, such as ncbi genotyping tool (rozanov et al. ) , vigor (viral genome orf reader) (wang et al. ), viral genome organizer (vgo) (upton et al. ) , genome annotation transfer utility (gatu) (tcherepanov et al. ) , virus genotyping tools (alcantara et al. ), zcurve_v (guo and zhang ) and star (subtype analyser) (myers et al. ) . vgo is a web-based genome browser that allows viewing and predicting genes and orfs in one or more viral genomes. it also allows performing searches within viral genomes and acquiring information about a genome such as locating genes, orfs, start/stop codons, etc. within genome, the sequences can be searched for regular expression, fuzzy motif pattern, genes with highest at composition, etc. using vgo, comparative analyses can be made between different viral genomes. vgo uses the graphical user interface (gui) for constructing alignments and display orthologues in a set of genomes. it also allows searching the translated genome for matches to mass spec peptides. vigor is a gene prediction online tool that was developed by j. craig venter institute in . it started with gene prediction in small viral genomes such as coronavirus, influenza, rhinovirus and rotavirus. with the updated version in (https://www.ncbi.nlm.nih.gov/pmc/articles/pmc /), vigor is now capable of gene prediction in more viruses: measles virus, mumps virus, rubella virus, respiratory syncytial virus, alphavirus and venezuelan equine encephalitis virus, norovirus, metapneumovirus, yellow fever virus, japanese encephalitis virus, parainfluenza virus and sendai virus. with vigor, based on sequence similarity searches, users are able to predict protein coding regions, start and stop codons and other complex gene features such as rna editing, stop codon leakage and ribosomal shunting. further, various features such as frameshifts, overlapping genes, embedded genes, etc. can be predicted in the virus genome. additionally, a mature peptide can be predicted in a given polypeptide open reading frame. vigor is also capable of genotyping influenza virus and rotavirus. four output files -a gene prediction file, a complementary dna file, an alignment file, and a gene feature table file -are produced by vigor. genbank submission can be directly done using the gene feature table. genome annotation transfer utility (gatu) facilitates quick and efficient annotation of similar target genome using the reference genomes that have already been annotated. later, the users can manually curate the annotated genome. the newly annotated genomes can be saved as genbank, embl or xml file format. although it doesn't provide a complete annotation system, gatu serves as a very useful tool for the preliminary work in genome annotation. gatu utilizes tblastn and blastn algorithms to map genes onto the new target genome by using an annotated reference genome. as a result, majority of the new genome's genes are annotated in a single step. with gatu, users can also identify open reading frames present in the target genome and absent from the reference genome. these orfs can further be scrutinized by using other bioinformatics tools such as blast and vgo, which can determine if the orfs should be included in the annotation. multiple-exon genes and mature peptides can also be analysed using gatu. a primer design tool, primerhunter, allows to design highly sensitive and specific primers for virus subtyping by pcr (duitama et al. ). primerhunter allows predicting specific forward and reverse primers with respect to a given set of dna sequences. phylotype is a web-based as well as downloadable software that uses parsimony to reconstruct ancestral traits and to select phylotypes (chevenet et al. ) . rotac is an automated genotyping tool for group a rotaviruses (maes et al. ). it works by comparing a complete orf of interest to other complete orfs of cognate genes available in the genbank database by performing blast searches. viroligo is a database of virus-specific oligonucleotides. the viroligo database acts as a repository for virus-specific oligonucleotides for virus detection (onodera and melcher ) . the database comprises of oligo data and common data tables. the oligo data table enlists pcr primers and hybridization probes that are used for viral nucleic acid detection, while common data table contains pcr and hybridization experimental conditions used in their detection. each oligo data entry provides information on the name of the oligonucleotide, oligonucleotide sequence, target region, type of usage (pcr primer, pcr probe, hybridization or other), note and direction of the pcr oligonucleotide (forward or reverse). each oligonucleotide entry also contains direct links to pubmed, genbank, ncbi taxonomy databases and blast. on the updated version of viroligo as of september , the database contains complete listing of oligonucleotides specific to various animal viruses. the viruses are vaccinia virus; canine parvovirus; porcine parvovirus; rodent parvovirus; tobamovirus; potyvirus; borna virus; bovine herpesvirus types , , and ; bovine viral diarrhoea virus; bovine parainfluenza virus; bovine respiratory syncytial virus; bovine adenovirus; bovine rhinovirus; bovine coronavirus; bovine reovirus; bovine enterovirus; foot-and-mouth disease (fmd) virus; and alcelaphine herpesvirus. virus-ploc is a web server for prediction of subcellular localization of viral proteins within host and virus-infected cells (shen and chou ) . another web server developed a little later, iloc-virus, is a multi-label learning classifier that predicts the subcellular locations of viral proteins with single and multiple sites (xiao et al. ) . similarly, a most recent web server, ploc-mvirus (cheng et al. ) , is a new predictor that identifies subcellular localization of viral proteins with both single and multiple location sites. it works by extracting information from the gene ontology (go) database and is claimed to be more successful than the state-of-the-art method, iloc-virus, in predicting subcellular localization of viral proteins. avppred is an antiviral peptide prediction algorithm that contains the peptides with experimentally proven antiviral activity (thakur et al. ) . the prediction is based on peptide sequence features, peptide motifs, sequence alignment, amino acid composition and physicochemical properties. vips is a viral internal ribosomal entry site (ires) prediction system that can predict ires secondary structures (hong et al. ) . vips uses the rna fold program that predicts local rna secondary structures, rna align program that compares predicted structures and pknotsrg program (reeder et al. ) that calculates the pseudoknot structures. vazymolo, a database that deals with viral sequences at protein level, defines and classifies viral protein modularity (ferron et al. ) . it extracts information of complete genome sequences of various viruses from genbank and refseq and organizes the acquired information about modularity on viral orfs (fig. . f) . there are web-based tools available to predict and analyse structural aspects of viruses. the learncoil-vmf is a computational tool that allows to predict coiledcoil-like regions in viral membrane fusion proteins (singh et al. ) . the membrane fusion proteins are known to be diverse and share no sequence similarity between most pairs of viruses in the same or different families. the learncoil-vmf is also capable of characterizing the core structure of these membrane fusion proteins. viperdb (virus particle explorer database) is a web-based database that enables manual curation of icosahedral virus capsid structures (carrillo-tripp et al. ). this database serves as a comprehensive resource for specific needs of structural virology and comparatives of data derived from structural and computational analyses of capsids. with the updated version, viperdb ( ), capsid protein residues in the icosahedral asymmetric unit (iau) can be deduced using phi-psi (phi-psi) diagrams (azimuthal polar orthographic projections) (ref: https://www.ncbi.nlm.nih. gov/pubmed/ ). these diagrams can be depicted as dynamic interface and surface residues and interface and core residues and can be mapped to the database using a new application programming interface (api). this aids in identifying family-wide conserved residues at the interfaces. additionally, jmol and strap are built in the system to visualize an interactive model of viral molecular structures. vida is a database that organizes animal virus genome open reading frames from partial and complete genomic sequences (alba et al. ) . presently, vida includes a complete collection of homologous protein families from genbank for herpesviridae, papillomaviridae, poxviridae, coronaviridae and arteriviridae. the homologous proteins in vida include both orthologous and paralogous sequences. vida retrieves virus sequences from genbank and the files are parsed into subfields. the parsed fields contain all the information such as genbank accession number, genbank identifier (gi numbers), protein sequence source, sequence length, gene name and gene product. in order to eliminate % redundancy, the virus protein sequences thus retrieved are filtered and a list of synonymous gis is created for reference. the orfs from complete and partial virus genomes are further organized into homologous protein families, on the basis of sequence similarity. furthermore, the structure of known viral proteins or homologous to viral proteins is also mapped onto homologous protein families. vida also provides functional classification of virus proteins into broad functional classes based on typical virus processes such as dna and rna replication, virus structural proteins, nucleotide and nucleic acid metabolism, transcription, glycoproteins and others. this database also provides alignment of the conserved regions based on potential functional importance. apart from functional classification, vida also provides a taxonomical classification of the proteins and protein families. the protein families serve as a tool for functional and evolutionary studies, whereas alignments of conserved sequences provide crucial information on conserved amino acids or construction of sequence profiles. the viral bioinformatics resource center (vbrc) is one of eight nih-sponsored bioinformatics resource centers (http://www.oxfordjournals.org/nar/database/ summary/ ). it is an online platform that provides informational and analytical tools and resources to scientific community. the vbrc is oriented to conduct basic and applied research to better comprehend the viruses included on the nih/niaid list of priority pathogens. these viruses are selected based on their possibility of bioterrorism threats or as emerging or re-emerging infectious diseases. the vbrc focuses specifically on large dna viruses. it includes the viruses that belong to the arenaviridae, bunyaviridae, filoviridae, flaviviridae, paramyxoviridae, poxviridae and togaviridae families. it serves as a relational database and web application tool that allows data storage, annotation, analysis and information exchange of the data. the current version (v . ) consists of complete genomic sequences. using the vbrc, each of the viral gene and genome can be curated. as a result, a comprehensive and searchable summary is acquired that details about the genotype and phenotype of the genes. the role of the genes in host-pathogen relationships is also being emphasized in these curations. additionally, the vbrc also houses multiple analytical tools such as tools for genome annotation, comparative analysis, whole genome alignments and phylogenetic analysis. further, this database also looks forward to include high-throughput data derived from other studies such as microarray gene expression data, proteomic analyses and population genetics data. the poxvirus bioinformatics resource center (pbrc, now merged into vbrc) is an online platform that serves as an informational and analytical resource to better comprehend the poxviridae family of viruses. it allows data storage, annotation, analysis and information exchange of the data. influenza virus is one the major global concern. it gained attention after the emergence of pandemic influenza a virus (h n , swine flu) in . there are a total of web portals and tools that focus only on influenza virus. this includes the influenza virus database (ivdb), influenza research database (ird) and ncbi influenza virus resource (ncbi-ivr) (chang et al. ; bao et al. ; squires et al. ) . researchers can exploit all the three websites mentioned for sequence databases as well as various basic tools such as blast, multiple-sequence alignment, phylogenetic tree construction, etc. ivdb provides access to additional tools such as (i) the sequence distribution tool, which provides global geographical distribution of a given viral genotype as well as correlates its genomic data with epidemiological data, and (ii) the quality filter system, which according to their sequence content (coding sequence [cds], 'untranslated region [ 'utr] , and 'utr) and integrity (complete [c] or partial [p]) categorizes a given viral nucleotide sequence into either of the seven categories of c to c and p to p , respectively. ncbi-ivr is the most widely used and cited online resource. with ncbi-ivr, the given viral genomic sequences can be annotated using a genome annotation tool and flu annotation (flan) tool. additionally, large phylogenetic trees may be constructed and can be visualized in aggregated form with sub-scale details (bao et al. ; bao et al. ; zaslavsky et al. ) . ird provides tools for genomic and proteomic intervention, immune epitope prediction and surveillance data for viral nucleotide sequences (squires et al. ) . furthermore, this resource is also equipped with tools that provide insight into hostpathogen interactions, type of virulence, host range and a correlation of sequence variation and these processes. there are other repositories available: global initiative on sharing avian influenza data (gisaid) consortium that mediated the epiflu database and flugenome database that exclusively provides genotyping of influenza a virus and aids in detecting reassortments taking place in divergent lines (lu et al. ) . furthermore, reassortment events in influenza viruses exclusively can be identified by a program giraf (graph-incompatibility-based reassortment finder) that can be downloaded (nagarajan and kingsford ) . another distinct repository, influenza sequence and epitope database (ised), provides viral sequences and epitopes from asian countries; the information could be exploited to understand and study evolutionary divergence and migration of strains (yang et al. ). the web server ativs (analytical tool for influenza virus surveillance) provides an antigenic map for conducting surveillance and selection of vaccine strains by scrutinizing the serological data of haemagglutinin sequence data of influenza a/h n viruses and influenza subtypes (liao et al. ). there is another online repository openfludb (an isolate-centred inventory), where information of an isolate such as virus type, host, date of isolation, geographical distribution, predicted antiviral resistance, enhanced pathogenicity or human adaptation propensity may be obtained (liechti et al. ) . for influenza viruses, primers and probes can be designed using the influenza primer design resource (ipdr) (bose et al. ). further, prospective influenza seasonal epidemics or pandemics can be predicted using a stochastic model, flute (chao et al. ) (table . ). the ncbi virus variation resource (ncbi-vvr) is a web-based database of a set of viruses, viz. influenza virus, dengue virus, rotavirus, west nile virus, ebola virus, zika virus and mers coronavirus (resch et al. ). it enables the user to submit their viral sequences along with relevant metadata such as sample collection time, isolation source, geographic location, host, disease severity, etc. it further allows integrating and analysing the viral sequences using the generic tools such as multiple sequence alignment and phylogenetic tree construction. rotavirus a (rva) is the most frequent cause of severe diarrhoea in human and animal infants worldwide and remains as a major global threat for childhood morbidity and mortality (minakshi et al. ; basera et al. ) . in recent years, extensive research efforts have been done for the development of live, orally administered vaccines. in india, an orally administered vaccine rotavac was also introduced after successful clinical trials in which became available to clinicians in , although these vaccines will have to be scrutinized and have to be updated regularly to accommodate the emerging rotavirus genotype variations, following which molecular and genetic characterization of new circulating and emerging genotypes of rotavirus strains in humans and animals becomes necessary. recently, a classification system for rvas has been described by the rotavirus classification working group (rcwg) in which all the genomic rna segments are assigned a particular alphabet followed by the particular genotype number. the classification system will be helpful in explaining the importance of genetic reassortments among rvas, host range, transfer of gene segments among two different genotypes and adaptation to different hosts. to differentiate between different gene segments of rvas, an online web-based tool rotac was developed by the leading researchers from rega institute, ku leuven, belgium, in (table . ). it's an easy-to-use and reliable classification tool for rvas and works on the agreement with rcwg. it's a platform-independent tool which works on any web browser by simply going to its url (http://rotac.regatools.be/) and has been released without any restriction of use by academicians or anyone else. as claimed, the rotac web-based tool will be updated regularly to reflect the established as well as newly emerging genotypes announced by the rcwg from time to time. various researches in animal viral diseases are being conducted at the genomic level. often, handling an enormous data obtained from sequencing is daunting to researchers. the chapter categorically provides a list of bioinformatics approaches that are useful in data mining. there are tables that list all such bioinformatics programs as per the applications. the tables also list databases that organize information on human and animal viruses such as genomic data, orfs, oligonucleotides, etc. an illustration has also been provided in the chapter showing the application of the tool predictprotein, which is used for prediction of three-dimensional structures of viral proteins. the major goal of the chapter has been to provide a roadmap to bioinformatics approaches in the field of animal viral diseases. although the chapter elaborates on viruses-specific bioinformatics programs, most of these programs are designed for human viruses. nevertheless, there are bioinformatics tools that are animal-virus specific, but these are limited in number. henceforth, in many cases, researchers have to switch to either human virus-specific tools or other generic tools. application of such tools for studying animal viruses or animal diseases, in many situations, may not be as accurate as with specialized tools. the users should take precautions while using the settings of such tools. furthermore, the results, thus obtained, also need to be scrutinized. therefore, development of new bioinformatics programs/tools that are specifically designed for animal viruses/diseases should be taken up robustly. specialized tools will provide much accurate results and predictions, thereby accelerating the bioinformatics researches in the field of animal viral diseases. vida: a virus database system for the organization of animal virus genome open reading frames a standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences hmm: a hidden markov model for detecting recombination with microbial detection microarrays basic local alignment search tool phaccs, an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information visitor, an informatic pipeline for analysis of viral sirna sequencing datasets flan: a web server for influenza virus genome annotation the influenza virus resource at the national center for biotechnology information continuous and discontinuous protein antigenic determinants detection of rotavirus infection in bovine calves by rna-page and rt-pcr genemark: web software for gene finding in prokaryotes, eukaryotes and viruses mhcbn: a comprehensive database of mhc binding and non-binding peptides the influenza primer design resource: a new tool for translating influenza sequence data into effective diagnostics base-by-base: single nucleotidelevel analysis of whole viral genome alignments mhcpep, a database of mhc-binding peptides: update viperdb : an enhanced and web api enabled relational database for structural virology influenza virus database (ivdb): an integrated information resource and analysis platform for influenza virus research flute, a publicly available stochastic influenza epidemic simulation model virusseq: software to identify viruses and their integration sites using next-generation sequencing of human cancer tissue analysis of the complete genome sequence of the hz- virus suggests that it is related to members of the baculoviridae ploc-mvirus: predict subcellular localization of multi-location virus proteins via incorporating the optimal go information into general pseaac searching for virus phylotypes evidence for a novel gene associated with human influenza a viruses t-cell antigenic sites tend to be amphipathic structures viroblast: a stand-alone blast web server for flexible queries of multiple databases and user's datasets primerhunter: a primer design tool for pcr-based virus subtype identification alvira: comparative genomics of viral strains mathematics vs. evolution: mathematical evolutionary theory vazymolo: a tool to define and classify modularity in viral proteins zcurve_v: a new self-training system for recognizing protein-coding genes in viral and phage genomes identifying viral integration sites using seqmap . genome information broker for viruses (gib-v): database for comparative analysis of virus genomes viral ires prediction system -a web server for prediction of the ires secondary structure in silico vita: prediction of host micrornas targets on viruses covdb: a comprehensive database for comparative analysis of coronavirus genes and genomes viralzone: a knowledge resource to understand virus diversity vir-mir db: prediction of viral microrna candidate hairpins viralfusionseq: accurately discover viral integration events and reconstruct fusion transcripts at single-base resolution ativs: analytical tool for influenza virus surveillance openfludb, a database for human and animal influenza virus the viral meta genome annotation pipeline(vmgap):an automated tool for the functional annotation of viral metagenomic shotgun sequencing data flugenome: a web tool for genotyping influenza a virus rota c: a web-based tool for the complete genome classification of group a rotaviruses g and p genotyping of bovine group a rotaviruses in faecal samples of diarrheic calves by dig-labeled probes a statistical model for hiv- sequence classification using the subtype analyser (star) giraf: robust, computational identification of influenza reassortments via graph mining sivirus: web-based antiviral sirna design software for highly divergent viral sequences viroligo: a database of virus-specific oligonucleotides phever: a database for the global exploration of virus-host evolutionary relationships viralorfeome: an integrated database to generate a versatile collection of viral orfs virapops: a forward simulator dedicated to rapidly evolved viral populations syfpeithi: database for mhc ligands and peptide motifs epimhc: a curated database of mhcbinding peptides for customized computational vaccinology virus particle explorer (viper), a website for virus capsid structures and their computational analyses pknotsrg: rna pseudoknot folding including nearoptimal structures and sliding windows virus variation resources at the national center for biotechnology information: dengue virus orf-finder: a vector for high-throughput gene identification the predictprotein server discovery of functional genomic motifs in viruses with virema-a virus recombination mapper-for analysis of next-generation sequencing data metavir: a web server dedicated to virome analysis a web-based genotyping resource for viral sequences bcipep: a database of b-cell epitopes prevalence, diagnosis, management and control of important diseases of ruminants with special reference to indian scenario epitome: database of structure-inferred antigenic epitopes an update on the functional molecular immunology (fimm) database jphmm: improving the reliability of recombination prediction in hiv- virus-ploc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells sse: a nucleotide and amino acid sequence analysis platform learncoil-vmf: computational evidence for coiled-coil-like motifs in many viral membrane-fusion proteins advances in diagnosis, surveillance, and monitoring of zika virus: an update ebola virus -epidemiology, diagnosis and control: threat to humans, lessons learnt and preparedness plans-an update on its year's journey biohealthbase: informatics support in the elucidation of influenza virus host pathogen interactions and virulence influenza research database: an integrated bioinformatics resource for influenza research and surveillance selox--a locus of recombination site search tool for the detection and directed evolution of site-specific recombination systems genome annotation transfer utility (gatu): rapid annotation of viral genomes using a closely related reference genome virsirnadb: a curated database of experimentally validated viral sirna/shrna antijen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data hivsirdb: a database of hiv inhibiting sirnas viral genome organizer: a system for analyzing complete viral genomes the immune epitope database (iedb) . in silico reconstruction of viral genomes from small rnas improves virus-derived small interfering rna profiling vigor, an annotation program for small viral genomes virusfinder: software for efficient and accurate detection of viruses and their integration sites in host genomes through next generation sequencing data virome: an r package for the visualization and analysis of viral small rna sequence datasets virome: a standard operating procedure for analysis of viral metagenome sequences iloc-virus: a multi-label learning classifier for identifying the subcellular localization of virus proteins with both single and multiple sites influenza sequence and epitope database prism: a primer selection and matching tool for amplification and sequencing of viral genomes visualization of large influenza virus sequence datasets using adaptively aggregated trees with sampling-based subscale representation identification of novel viruses using virushunter--an automated data analysis pipeline acknowledgements all the authors of the manuscript thank and acknowledge their respective universities and institutes. there is no conflict of interest. key: cord- - ldxvbpz authors: pleschka, stephan; ludwig, stephan; wolff, thorsten; planz, oliver title: anti-viral approaches against influenza viruses date: journal: new concepts of antiviral therapy doi: . / - - - - _ sha: doc_id: cord_uid: ldxvbpz nan influenza is a highly contagious, acute respiratory disease with global significance that affects all age groups and can occur repeatedly in any individual. the etiological agent of the disease, influenza virus is responsible e. bogner and a. holzenburg (eds.) , new concepts of antiviral therapy, - . nordufer , d- berlin, germany; friedrich-loeffler-institute (fli) , germany; ä for an average between three and five mill. cases of severe influenza leading to about , - , mortalities annually in the industrialized world according to who estimations. compared to otherwise healthy persons, death rates in patients of risk groups (s. . ) are - fold higher in patients with cardiovascular or pulmonary disease as compared to healthy individuals. annual health cost, costs, e.g. due to work absenteeism (also related to parental care of infected children) or costs related to death, increased disabilities etc. can be higher than mil. € in european countries. furthermore. for a pandemic outbreak the centers for disease control (cdc) estimates that in the usa that % of all death will be caused by % of the population which are at high-risk. this will result in a financial burden of up to . billion us$ not including the commercial impact. the death rate would be up to , accompanied by up to , hospitalizations, - million outpatient visits and - million additional illnesses wilschut and mcelhaney, ) . this clearly would overrun the capacity of current supply and management of vaccines available. since waterfowl represents the natural reservoir for the virus (lamb and krug, ; webster, ; wilschut and mcelhaney, ; wright and webster, ) and many other animal species can be infected, the eradication of the virus is impossible and a constant reemergence of the disease will continue to occur. epidemics appear almost annually and are due to an antigenic change of the viral surface glycoproteins (fig. ) . furthermore, highly pathogenic strains of influenza-a-virus have emerged unpredictably but repeatedly in recent history as pandemics like the "spanish-flu" that caused the death of - millions people worldwide (taubenberger et al., ; webster, ) . since these pandemic virus strains usually possess different antigenic characteristics, current vaccines will be ineffective once such a virus emerges. regarding the vast possibilities for such a strain to "travel" around the world (hufnagel et al., ) it becomes evident that effective countermeasures are required for the fight against these foes. in recent outbreaks of avian viruses that infected humans ( , , / / ) hatta and kawaoka, ; li et al., ) from a total of confirmed cases people died ( / ) (world health organization, ) . fortunately, until now these particular viruses have not acquire the ability to spread in the human population. however, any novel virus strain emerging in the future may have such a capability (webby and webster, ) . here, we give an overview of current and new anti-influenza strategies, such as immunization methods and drugs against the virus. since every virus depends on its host cell, cellular functions essential for viral replication may also be suitable targets for anti-viral therapy. in this respect intra-cellular signaling cascades activated by the virus, in particular mapk pathways, have recently come into focus ludwig et al., ) . influenza viruses belong to the order of the orthomyxoviridae. they possess a segmented, single stranded rna-genome with negative orientation. they are divided into three types, a, b and c based on genetic and antigenic differences. among the three types influenza-a-viruses are clinically the most important pathogens since they have been responsible for severe epidemics in humans and domestic animals in the past. thus the focus of this chapter will be on type-a influenza viruses. a detailed description of the viral proteins and the replication cycle of influenza-a-viruses can be found elsewhere (lamb and krug, ; wright and webster, ) . therefore we will only give a brief overview on these topics without referring to individual references. the influenza-a-virus particle is composed of a lipid envelope derived from the host cell and of - structural virus proteins ( figure and table ). the components of the rna-dependent rna-polymerase complex (rdrp), pb , pb and pa are associated with the ribonucleoprotein complex (rnp) and are encoded by the vrna segments - . the pb segment of many, but not all, influenza-a-virus strains also contains a + -reading frame encoding the recently discovered pb -f protein (chen et al., ) . the viral surface glycoproteins hemagglutinin (ha) and neuraminidase (na) are expressed from vrna segments and , respectively. the nucleoprotein (np) is encoded by segment and associates with the vrna segments. it is the major component of the rnps. the two smallest vrna segments each code for two proteins. the matrix protein (m ) is colinear translated from the mrna of segment and forms an inner layer within the virion. a spliced version of the mrna gives rise to a third viral transmembrane component, the m protein, which functions as a ph-dependent ion channel. employing a similar coding strategy segment harbors the sequence information for the nonstructural ns protein and the nuclear export protein ns /nep. ns /nep is a minor component of the virion and is found associated with the m protein. figure . the influenza-a-virus particle schematic representation of the spherical influenza-a-virus particle that has a diameter of about nm. the eight viral rna segments were separated by urea-polyacrylamide gel electrophoresis and visualized by silver staining (left) . the corresponding gene products and their presumed location in the virus particle are indicated (right). ns is not a structural part of the mature virion. for details see text. table summarizes details of the genome segments, the encoded viral proteins and their according function. the viral replication cycle is initiated by binding of the ha to sialic-acid (neuraminic acid) containing cellular receptors and subsequent endocytosis of the virus (figure ) (for references: (lamb and krug, ; wright and webster, ) ). the active ha molecule consists of two subunits (ha / ha ) derived from the uncleaved precursor ha , which becomes proteolytically processed after release of the virion by extra-cellular proteases. this cleavage is absolutely essential for ha-function and cell infection. virus disassembly occurs in the acidic environment of late endosomal vesicles and involves two crucial events. first, the conformation of the ha is changed to a low-ph form, which results in exposure of a fusion active protein sequence within the ha . this fusion peptide is thought to contact the endosomal membrane and to initiate fusion with the viral envelope. second, the low ph in the endosomes activates the viral m ion channel protein resulting in a flow of protons into the interior of the virion. acidification facilitates dissociation of the rnps from the m protein. the rnps are subsequently released into the cytoplasm and rapidly imported into the nucleus through the nuclear pore complexes. the viral genomic segments are replicated and transcribed by the viral rdrp associated with the rnps in the nucleus of the infected cell. the vrna is directly transcribed to mrna and, in addition, serves as a template for a complementary copy (crna), which itself is the template for new vrna. in the late phase of infection newly synthesized viral rnps are exported to the cytoplasm. ns protein functions as a regulatory factor in the virus infected cell. the na, the m and the precursor ha (ha ) proteins follow the exocytotic transport pathway from the rer via the golgi complex and the trans golgi network. the mature ha and na glycoproteins and the nonglycosylated m are finally integrated into the plasma membrane as trimers (ha) or tetramers (na, m ), respectively. m assembles in patches at the cell membrane. it is thought to associate with the glycoproteins (ha and na) and to recruit the rnps to the plasma membrane in the late phase of the replication cycle. finally the viral rnps become enveloped by a cellular bilipid layer carrying the ha, na and m proteins resulting in budding of new virus particles from the apical cell surface. replicative viral proteins enter the nucleus to amplify the viral genome. in the late stage of the infection cycle newly synthesized rnps are exported from the nucleus and are assembled into progeny virions that bud from the cell surface. the polymerase complex of influenza viruses does not possess a proof reading activity, thus numerous mutations accumulate in the viral genome during ongoing replication (lamb and krug, ) leading to changes in all proteins. this includes conformational alteration of ha-and na-epitopes against which neutralizing antibodies are generated. influenza-a-viruses are categorized by antigenic differences of the ha-and na-proteins. the high mutation rate combined with the high replication rate results in a multitude of new variants produced in each replication cycle, thus allowing the virus to rapidly adapt to changes in the environment. this results in an escape of the existing immunity and in resistance to drugs acting directly against viral functions. gradual changes of the antigenic properties that make existing vaccines less or non effective are described as antigenic drift and demand for new compositions of the yearly vaccines. due to the nature of their segmented genome influenza virus can independently recombine segments upon the infection of a cell with two different viruses. this is described as genetic reassortment. today hasubtypes (h -h ) and na-subtypes (n -n ) are known, which can mix and lead to new antigenic properties. (lamb and krug, ; webster et al., ; wright and webster, ) . not all combination will ultimately be advantageous, but can lead to the generation of a virus that combines the ability to replicate in humans with novel antigenic properties (antigenic shift). this has happened at least three times in the last century resulting in the pandemics of ("spanish flu"), ("asian flu") and ("hong kong flu") that caused up to million death. therefore, the question is not "if" but "when" will such a pandemic occur again (horimoto and kawaoka, ; webby and webster, ; webster, b) . a vaccine against such "new" viruses can not be generated in advance and as vaccine production would need significantly more time than it takes for a pandemic virus to spread around the world (hufnagel et al., ) , alternative weapons in the fight against these enemies are urgently needed. besides pandemic variants that can occur when human and avian influenza virus reassort in porcine hosts (regarded as "mixing vessels") (webster, a; webster et al., ) , avian influenza virus strains have directly infected humans, as happened in hong kong in (claas et al., ; de jong et al., ; subbarao et al., subbarao et al., ) and recently ( subbarao et al., / koopmans et al., ) during vast outbreaks of avian influenza. these viruses show an extremely high virulence in humans with case fatality rates up to %. the virus that normally causes a respiratory disease (for references: (wilschut and mcelhaney, ) ) is transmitted by aerosol droplets and contaminated hands and can already be shed before onset of symptoms . therefore, high population density and dry air leading to reduced protection of respiratory epithelium by the mucus are conditions that promote transmission of the virus. the infection with influenza viruses is normally limited to the respiratory tract. here proteases released by clara cells in the epithelium are present that activate the ha to allow further infections (s. . ) (for review (ludwig et al., ) ). innate immunity as well as the adaptive immune system will normally restrict virus propagation. therefore population groups, that have a less protective immune system, such as young children up to two years and older persons over as well as immunocompromised or chronically diseased persons are especially of risk. the replication of the virus leads to the lysis of the epithelial cells and enhanced mucus production causing running nose and cough. furthermore, inflammation and oedema at the replication site are due to cytokines released. this can lead to fever and related symptoms. bacterial super-infections of the harmed tissue can further complicate the situation. normally onset of systemic (fever, myaglia, headaches, severe malaise) and respiratory (coughing, sore throat, rhinitis) symptoms occur after about two days incubation period and can last for about seven to ten days. coughing and overall weakness can persist for up to two weeks. if the virus spreads from the bronchiolar tract to the aveolars, viral pneumonia and interstitial pneumonitis with mononuclear and haemorrhage infiltration and finally lysis of the inter-aveolar space is possible (wilschut and mcelhaney, ) . this scenario is a likely picture in case of infection with a pandemic influenza strain, where the individual has not had a prior exposure to the virus and the innate immunity reaction can lead to a strong immunpathogenesis. high virus replication will induce secretion of large quantities of cytokines by the infected epithelia and will stimulate inflammatory processes. together with the destruction of the epithelia this results in an influx of fluid into the aveolars leading to hypoxia and acute respiratory distress syndrome, that may cause the death within a short period of time ( - days after onset). this scenario might also be caused by additional viral factors enhancing pathogenicity. such factors that are yet not well defined probably have contributed to the devastating outcome of the "spanish flu" (wilschut and mcelhaney, ) . accurate and rapid diagnosis of the disease is essential for an effective treatment, especially with anti-viral substances, as virus replication and therefore illness progresses rapidly. samples can be tested serologically, by cell culture or rt-pcr for strain typing and should be done within four days after onset of symptoms (wilschut and mcelhaney, ) . there are two main methods of influenza prophylaxis: the use of antiviral drugs and vaccines. several drugs are available for influenza prophylaxis functioning either as m -ion channel inhibitors (amantadine and rimantadine) or as inhibitors of the na (zanamivir and oseltamivir). despite these anti-viral drugs, which are a useful adjunct to influenza vaccines, vaccination itself remains the cornerstone of prophylaxis. vaccination induces a good degree of protection and is in general well tolerated by the recipient. nevertheless, while resistant virus variants can emerge after antiviral drug treatment the disadvantage of vaccination is that immunization needs to be refreshed almost every year, since the vaccine must reformulated to take account of the changing virus. in the immune response to influenza infection both the humoral and cell mediated immunity are involved. from the side of the humoral immune system, both the mucosal and the systemic immunity contribute to resistance to influenza infection. the cellular immune response is involved in recovery from influenza virus infection by eliminating virus-infected cells and by providing help for antibody production woodland et al., ) . consequently, the humoral immune response is the primary target of vaccination. after influenza virus infection antibodies directed against all major viral proteins can be detected in humans and the level of serum antibodies correlate with resistance to disease (couch, ; couch and kasel, ; coulter et al., ; nichol et al., ; potter and oxford, ) . only antibodies specific for the surface glycoproteins ha and na are associated with resistance to infection. in contrast, antibodies to the conserved internal antigens m and np are not protective (de jong et al., ; tamura and kurata, ) . the mucosal tissues of the respiratory system are the main portal entry of influenza virus and consequently the mucosal immune system functions as the first line of defense against infection apart from innate immunity (see paragraph ). antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection. secretory immunoglobulin a (siga) and to some extent igm are the major neutralizing antibodies directed against the entering virus. furthermore, these antibodies can function intra-cellular to inhibit influenza replication. iga and igm are involved in protection of the upper respiratory titre ≥ ) can be detected in approximately % of subjects after natural influenza virus infection and correlates with protection against the flu. plasma cells producing all three major ig classes are present in the peripheral blood in normal subjects (cox and subbarao, ; laforce et al., ) . tract while serum igg acts in protection of the lower respiratory tract ). an anti-ha antibody response (haemagglutination-inhibition (hi) the immune response induced by infection protects against reinfection with the same virus or an antigenically similar viral strain. cell mediated immunity plays a role in recovery from influenza virus infection and may also prevent flu-associated complications, but it does not seem to contribute significantly in preventing infection. influenza specific cellular t cells have been detected in the blood and the lower respiratory tract secretions of infected subjects . influenza virusspecific cytotoxic t-lymphocytes (ctl) regognize both external and internal proteins of virus on infected cells. in humans a major component of this response is directed toward the np-and m -protein. even though influenza virus specific ctl's are not able to protect against the infection, these cells are important for the clearance of the virus. futhermore, cytolysis of influenza virus-infected cells can be mediated by influenza virus-specific antibodies and complement mcmichael et al., ; mcmichael et al., ; townsend et al., ) . cd + t cells function as helper cells for antibody production. moreover, it is suggested, that cd cells might act as direct effectors in protection against influenza virusinfection (brown et al., ) . inactivated vaccines (iv) are availeble for about years. because of the antigenic drift observed in influenza ha-and na-glycoproteins these vaccines need to be matched with the randomly mutating molecular structure of the new occurring "drift" strain. besides these vaccines there are various new approaches for influenza vaccines in promising developmental stages. these new stratagies include vaccines with immunomodulators, virosomes and dna-vaccines. ivs vaccines are administered world wide each year with millions of doses. these vaccines have good safety and tolerance profiles, with very low number of adverse reactions reported. these reactions are tenderness and redness that arise locally at the injection site and are more frequent in healthy (< %) than in elderly recipients ( %) . ivs are produced by propagation of the virus in embryonated chicken eggs. the currently used bacterial endotoxin-free trivalent ivs (tiv) are formulated with µg ha each from a current influenza virus a/h n , a/h n , and a b-virus strain. the seed strain is prepared by co-infecting the allantoic sac of the chicken embryo with a laboratory-adapted high-growth phenotype of h n (a/pr/ / ) and the epidemic strain. this results in viral replication and genetic re-assortment leading to high growth reassortants. thereafter the new hybrid viruses are screened for the absence of genes encoding pr/ or pr/ -like surface glycoproteins. the selected seed strain containing ha-and na-components of the epidemic strain is mass propagated in chicken eggs to obtain sufficient quantities of vaccine virus. the allantoic fluid is harvested, and the virus is concentrated and highly purified by zonal centrifugation. as a next step the virus is inactivated. depending on the nature of inactivation the vaccine is used as whole inactivated vaccine after treatment with formalydehyde or β-propiolactone or as split vaccine (chemically disrupted by ethyl either or sds). furthermore, the vaccine is used as subunit vaccines (purified surface glycoproteins). even though influenza vaccines have excellent tolerant profiles, since propagation in chicken eggs may lead to contamination of the vaccine with trace amounts of residual egg proteins, they should not be administered to persons who have anaphylactic hypersensitivity to eggs. whole inactivated influenza vaccine is more immunogenic than split vaccine or subunit vaccine, but is also associated with more frequent side reactions. consequently, split or subunit are given to children younger than age and two half doses are recommended given at least month apart for naïve persons to develop protective immunity (bridges et al., ) . protection after vaccination against influenza virus infection is dependent on the antigenic match between the vaccine strains and circulating the influenza virus strain. moreover, protection is also dependent the age and the previous exposition to influenza of the vaccine recipient. if ivs have a good antigenic match they are - % effective in the prevention of morbidity and mortality among healthy adults (beyer et al., ) . in elderly people the effect of protection is reduced to - % because of decreased immune function. since the immune system is naïve in young children, they also show a reduction in protection against influenza vaccination (nichol et al., ) . immunosuppressed individuals, elderly people and subjects with underlying chronic diseases are at increased risk for influenza and related complications. for these people conventional influenza vaccines provide only limited protection. in order to enhance the immune reaction after influenza vaccination, several adjuvants (latin verb: adjuvare -to help) that function as immunopotentiators have been evaluated. the liposomal influenza vaccine (influsome-vac) consists of liposomes containing the viral surface proteins ha-and na-derived from various influenza strains and il- or granulocyte-macrophage colonystimulating factor (gm-csf), as an adjuvant (babai et al., ) . in clinical trails with either young adults or elderly vaccination of influsome-vac appeared to be both safe and more immunogenic than the currently used vaccine (ben-yehuda et al., a; ben-yehuda et al., b) . furthermore adjuvant emulsions combined with subunit influenza antigens are in use, such as the "oil in water"-emulsion containing squalene, mf , (fluad). this commercially available product was tested in clinical trials in comparison with non-adjuvanted conventional vaccines. again in elderly individuals the addition of the mf -adjuvant to subunit influenza vaccines enhances significantly the immune response without causing clinically important changes in the safety profile of the influenza vaccine (podda, ) . other adjuvants that increase immunoreactivity after influenza vaccination are immune stimulating complexes (iscoms). they are - nm cage-like structures, which consist of glycoside molecules of the adjuvant quil a, cholesterol and phospholipids in which the antigen can be integrated. . in animal models, even in the presence of pre-existing antibodies they function as a potent adjuvant system by inducing cellular and humoral immune responses. (coulter et al., ; rimmelzwaan et al., ; windon et al., ) . as mentioned, gm-csf has a potential role as a vaccine adjuvant. it may enhance the response to vaccination in immunosuppressed individuals. gm-csf stimulates maturation of hematopoietic progenitor cells, induces class ii major histocompatibility complex antigen expression on the surface of macrophages, and enhances dendritic cell migration and maturation (jones et al., ) . nevertheless, in various clinical trails with immunosuppressed individuals and cancer patents it was shown, that it is unlikely that gm-csf improves the immune response (ramanathan et al., ) . for production of influenza vaccines in large-scale cell culture systems several continuous cell lines have been tested for the production of influenza vaccines (kistner et al., ; pau et al., ; seo et al., ; youil et al., ) . production of influenza vaccine in mammalian cell lines has some advantages but also has disadvantages compared to production in chicken egg. (tree et al., ; youil et al., ) . process controllability, scalability and supply of substrates are much easier in cell culture systems. furthermore, cell culture production reduces the risk of microbial contamination. in contrast, the greatest disadvantage of cell culture based influenza vaccine is the relative low viral yield. on the other hand and a major disadvantage of production in chicken eggs is their supply and possible bacterial contaminations. additionally the lethality of h n influenza virus to chicken embryos (s. . . ). at the present ( ) two cell line derived vaccines have been licensed in europe . estimated time for production of such vaccines is about months. the power of this time gaining approach to generate a great variety of specific influenza-vaccines under controlled safety conditions is achieved by the direct use of field strains (kistner et al., ) as well as seed strains specifically designed by reverse genetics systems and the large scale cell culture system. nevertheless, the application of these techniques largely depends on meeting the needs of high viral yield, appropriate permissiveness, and ability to support replication of all influenza virus strains to immunopotentiating reconstituted influenza virosomes (irivs) possess several characteristics defining them as vaccine adjuvants. they are a liposomal carrier system. these reconstituted virus-like particles (vlp; diameter nm) contain a lipid bilayer of phosphatidylcholine and phosphatidylethanolamine. ha and na are intercalated into the lipid bilayer and give the irivs their fusogenic activity, but lack the viral genetic material. irivs are able to deliver proteins, rna/dna and peptides to immunocompetent cells. in addition, virosomes, as vaccine delivery systems, have been shown to be safe and not to engender any antibodies against the phospholipid components. therefore, their use in vaccination of children and elderly people is recommended. the system is already registered for human use and allows a specific targeting of antigens by a cellular or a humoral immune response. a virosome vaccine, inflexal-v, is used in switzerland and italy. (gluck et al., ; langley and faughnan, ; zurbriggen, ) . dna-vaccines are non-infectious and non-replicative plasmid constructs that encode either only the proteins of interest or the protein of interest in combination with immunomodulatory proteins. this kind of vaccination by direct intra-muscular injection of dna was first demonstrated in in a mouse model system et al. (wolff et al., ) . directed intra-muscular dna-vaccination is not very common. the creation of recombinant influenza vaccines based on dna-plasmids is more appropriate. with this technique rapid and flexible construction of dna-plasmid vectors can be achieved, which can address the problems of antigenic drift induced by the circulating influenza virus strain (ljungberg et al., ) . these above described techniques have a potential for the development of live and inactivated vaccines. the efficacy of the plasmid based dnavaccines expressing the immunogenic influenza virus genes alone or in combination with dna encoding various cytokines has also been demonstrated in several animal models (for detail see: bardiya and bae, ) . during dna-vaccination, the foreign genes are endogenously high titers in short time (reviewed in: bardiya and bae, ) . expressed in the host, the proteins subsequently processed, and recognized by the immune system of the host. dna-vaccines elicit a broad-based humoral and cellular immunity against influenza virus proteins (justewicz et al., ) . in addition, alterations in the vector, dose of the dna, inclusion of cpg-odn motifs, fusion with influenza virus-specific helper t cell or ctl-epitopes, and appropriate vaccine delivery mechanisms will further improve the efficacy of these vaccines (bowersock and martin, ; joseph et al., ) . an alternative to ivs are attenuated "live" vaccines such as cold-adapted vaccines (cav: caiv-t, flumist ® ) and ns -defective strains used as intranasal influenza vaccine, that may lead to long-lasting, broader immune response (humoral and cellular) that resembles more closely the natural immunity derived from viral infection. for example, ctls, which are important for the clearance of the virus are activated during an productive infection . additional cytokines produced by the infected cells during the innate immunity response enhance and support reaction of the humoral system. compared to ivs, that are strain-and subtype-specific the cavs (that also have to be adapted to circulating strains) can provide a broader immunity against circulating viruses (belshe et al., ; king et al., ; nichol, ; stepanova et al., ; treanor et al., ; wareing and tannock, ) . cavs that already have been used successfully in russia and are now licensed in the usa kendal, ) can be administered intra-nasally for example as aerosols (abramson, ) . this results in a limited viral replication in the upper and lower respiratory tract and circumvents the need for syringes. it also supports protective mucosal immunity, which is an important property of nasally applied live influenza virus vaccines. for the generation of a cav a donor and a wild type strain are reassorted in such a way, that the ha-and na-segments are wild type (wt) derived and the remaining six segments originate from the donor strain. for this purpose two master strains are currently used as donors in the usa. one to generate a-type and one b-type influenza cavs (mendelman et al., ; murphy and coelingh, ) . these strains are cold adapted ( °c) (kendal, ; maassab and bryant, ) and therefore temperature sensitive (ts) and attenuated, meaning that these viruses will not propagate efficiently at body temperatures. to prevent easy reversion of the genetic markers, that encode the ts-defect and allowing the virus to regain full virulence, all six donor-derived segments carry mutations. for the production of such cav strains embryonated eggs are infected with both viruses (wild type and donor strain) under the selection of antibodies directed against the ha and na of the donor strain. the attenuated donor strain by itself is unable to cause significant illness in humans, but is able to donate the ha-and na-proteins of the contemporary epidemic strain to produce live attenuated vaccine by the traditional egg-based process (belshe, ; clements and murphy, ; jin et al., ) . the live attenuated vaccines were shown to be safe and effective in the general population kendal, ; langley and faughnan, ) . cav are trivalent like the ivs and are composed according to the who recommendations (mendelman et al., ) . new possibilities of reverse genetic techniques will certainly improve production of vaccine strains in time and quality (s. . . ). even though one should consider the possibility of reassortment with another human strain in the vaccinated person, which could produce an aggressive virus, cavs have been successfully used in russia without reports of severe side effects and seem to be safe. they show a comparable effectiveness to trivalent iv's (tivs) and both vaccines can also be used in combination belshe et al., ; boyce and poland, ; edwards et al., ; glezen, ; jackson et al., ; mendelman et al., ; swierkosz et al., ; treanor and betts, ; treanor et al., ) . in addition to the traditional live attenuated vaccines, production by reverse genetics (s. . . ) of replication-incompetent influenza virus-like particles (vlps) by deletion of either the entire ns gene (encoding both the ns and ns protein) or only the ns gene has also been reported. these vlps were entirely produced from cdnas (watanabe et al., b) . although, these technologies are in the very early stages of development and so far only tested in animal models, the vlp incapable of replication and spread to other cells due to deletion of a major protion of the ns or m , are expected to be good novel influenza vaccine candidates (galarza et al., ; watanabe et al., a) . a variation of the theme is presented by influenza virus strains (generated by reverse genetics (s. . . )) that express a modified ns (palese and garcia-sastre, ; palese et al., ; talon et al., ) . this non-structural protein is the major viral interferon (ifn)antagonist (s. tab. ). even though ns is a multifunctional viral protein that supports viral replication it seems to be an accessory protein as a virus without the ns -gene can replicate in ifn-deficient systems (garcia-sastre et al., a; garcia-sastre et al., b; ludwig et al., ) . ifnα/β are two important cytokines expressed in primary infected epithelia cells, that induce innate immunity. ifnα/β-induction severely reduces viral replication even in the presence of ns . therefore recombinant viruses expressing altered ns with reduced capacity to suppress cellular ifn-induction could raise protective immunity and might represent interesting attenuated live vaccine candidates (talon et al., ) . such viruses have been generated by reverse genetic techniques (s. . . ) and have been successfully tested in experimental settings . after initial experiments that implied the in vivo reconstitution of rnps from plasmid-expressed rdrp, np and vrna it became possible to generate recombinant influenza virus de novo totally from plasmid dna (fodor et al., ; , allowing complete genetic manipulation. this manipulation can either concern the combination/mixture of the genomic rna-segments and/or the genesequences themselves. the technique involves the transfection of four plasmids expressing the viral rdrp and the np together with eight plasmids (for all eight genomic rnas) that generate a vrna-like transcript. this again results in the in vivo reconstitution of active rnp-complexes, which will replicated and transcribe the vrnas. thereby all viral rnas and proteins are generated and the viral replication cycle is established resulting ; palese et al., ) . reverse genetics technique can be used to produce influenza vaccines based on recombinant virus (for detail see: bardiya and bae, ) . these methods do not require selection procedures and eliminate the need for multiple passages in eggs, thereby reducing the time required for vaccine production. it is known that interference among the vaccine viruses of type-a and -b can occur that affect the efficacy of the live attenuated vaccines by restricting their replication. to overcome that problem a chimeric virus (a/b) possessing chimeric (a/b) ha, and full-length b-type na in the background of a type-a vaccine virus was created (horimoto et al., ) . this study provided a novel method for creating live attenuated vaccine from a single donor strain. for different reasons the technique of reverse genetics has become highly relevant for anti-viral vaccine approaches. (i) for the production of regular ivs against wild type strains, that either grow poorly or are too pathogenic in eggs (s. later) one can generate strains carrying the ha and na needed in the background of an egg adapted virus. this is normally done by reassortment of the wild type with the egg-adapted strain and can not be well controlled. this problem can be circumvented by plasmid based reverse genetics that allow the controlled design of the reassortant. (ii) as mentioned the cav are composed of ha-and na-genes from the wild type in the production of infectious influenza viruses (for review: garcia-sastre, strain and a mixture of the other six segments from wild type and donor virus. by choice of the according plasmids one can compose a cav-strain that carries all six segments from the donor strain each with an adaptive mutation. this way it is less likely that a revertant virus will arise by mutation in one of the donor strain segments (s. . . ) (maassab and bryant, ; schickli et al., ) . (iii) it is possible to specifically design viruses with altered ns -genes that could be used as highly attenuated life vaccines (s. . . ), additionally modification of other viral genes (murphy et al., ; parkin et al., ) or of the replications efficiency of the genesegment (muster et al., ) can be applied to further attanuate the virus. (iv) viruses could be produced that lack an essential gene (e.g. nep) (watanabe et al., b) . the missing gene-product can be transcomplemented from an expression-plasmid in the transfected cell during virus generation. the recombinant viruses would be still infectious and lead to expression and presentation of viral proteins, but could not themselves establish a productive propagation as they are lacking the according gene. currently used ivs are prepared from egg-grown viruses (wilschut and mcelhaney, ) . this method is not without limitations but has proven to be efficient. as mentioned earlyer ( . . ), one particular problem that could arise would be production of a vaccine strain against an highly pathogenic avain influenza virus like the types that have recently infected humans. besides bio-safety questions they pose further problems. the ha of these viruses is activated within the infected cell by ubiquitous proteases allowing the virus to spread through out the organism. due to the special hacharacteristics these viruses themselves are highly pathogenic birds and eggs as well as a vaccine strain that would carry the according ha. therefore efficient virus production in embryonated eggs will be problematic (lipatov et al., ) . by plasmid based reverse genetic techniques recombinant viruses can be produced that have lost the pathogenic character of the ha and can replicate well in eggs li et al., ; liu et al., ; subbarao et al., ) . this could additionally be combined with virus production in cell culture systems (s. p. ) romanova et al., ) thereby overcoming the limitation posed by the number of embryonated eggs available at a given time (stephenson et al., ) . it should also be mentioned that not only type-a influenza viruses but also type-b influenza viruses can be generated and manipulated by reverse genetic systems and can therefore also be engineered to fit the circulating wild type strains (dauber et al., ; hatta et al., ; jackson et al., ; maassab and bryant, ) . inhibitors of viral functions (treatment and anti-viral chemoprophylaxis of influenza) anti-viral treatment is generally considered a supporting measure to prevent and control outbreaks of epidemic influenza in addition to immunoprophylaxis. however, chemotherapy is the only option to combat the disease when there is no type-specific vaccine available as for instance upon the emergence of a pandemic shift variant. two classes of substances are currently licensed in many countries for the treatment and/or prophylaxis of influenza, which includes the adamantane compounds amantadine and rimantadine, and the na-inhibitors oseltamivir and zanamivir. other small inhibitory compounds that target the viral polymerase complex are also introduced in this section, although none of them has been converted into a pharmaceutical product so far. amantadine ( -amino adamantane hydrochloride) and its derivative rimantadine (α-methyl- -adamantane methylamine hydrochloride) have potent anti-viral activity against most influenza-a-viruses, because they block the viral m ion channel protein during the early stage of viral uncoating (pinto et al., ) . specifically, the adamantane compounds inhibit the acidification of the virion inside the endosome, which prevents the intra-cellular release of the viral rnps. the % inhibitory concentration (ic ) of most natural influenza-a-virus strains against adamantane compounds is in the range of . to . µg/ml as determined by plaque reduction assay (appleyard et al., ; hayden et al., ; scholtissek and faulkner, ) . amantadine and rimantadine have proven effectiveness in the treatment of uncomplicated influenza-a-virus infection. they can reduce the duration of fever and system symptoms by approximately one day when given within two days after onset of disease signs (demicheli et al., ; tominack and hayden, ) . furthermore, both substances also have prophylactic effectiveness in reducing influenza-associated morbidity and clinical symptoms. a survey of studies undertaken with healthy adults demonstrated average effectiveness of % for amantadine and % for rimantadine in preventing laboratory confirmed influenza (demicheli et al., ) . during long-term prophylaxis amantadine was found to cause mild reversible adverse effects in a small proportion of the recipients, which involved central nervous system (cns) and minor gastrointestinal complaints. no increase in side effects was observed during treatment with rimantadine compared to placebo (n.n., ) . an early recognized limitation for widespread clinical use of m -blockers is the rapid emergence of drug-resistant viruses in tissue culture, in animal models and in patients (appleyard et al., ; hayden et al., ; oxford et al., ) . one study found that a total of % of children with laboratory-confirmed influenza shed resistant viruses after seven days of treatment with rimantadine (hall et al., ) . unfortunately, such selected drug-resistant viruses are virulent, as they can transmit to family members and cause disease even when the contact persons were treated prophylactically with rimantadine (hayden et al., ) . viruses that become insensitive to amantadine show complete cross-resistance to rimantadine and vice versa. thus, the clinical usage of adamantane amine compounds has been limited by the reported adverse effects, the induction of viral drug resistance and the inactivity towards influenza-b-viruses. nevertheless, these drugs are still recommended as a cost-effective choice particularly in influenza chemoprophylaxis (harper et al., ) . it is noteworthy, that amantadine resistance has also been detected in the highlypathogenic h n viruses currently circulating in south east asia (puthavathana et al., ) . thus, adamantane compounds are not an option to treat such infections. amantadine and rimantadine are approved for treatment of adults and children older than years at two daily mg doses. the substances should carefully be used in individuals above years in age and patients with impaired renal functions and halving of the daily doses is recommended. only amantadine is licensed for treatment of children between and years and should be dosed with mg/kg per day. in order to avoid emergence and transmission of drug-resistent viruses, treatment should be kept to a minimal time of to days until disease symptoms disappear. chemoprophylaxis can be considered for protection among high-risk groups including children and adults with chronic pulmonary or cardiac disease, immunocompromised persons with a reduced response to vaccines or in the case of a poor match between an epidemic virus strain and the current vaccine. since the adamantane compounds do not interfere with the development of neutralizing antibodies (tominack and hayden, ) , they can also be used for the protection of persons at high risk to bridge the time gap between vaccination and the establishment of an efficient immune status. for adults and children older than years two mg doses of amantadine or rimantadine per day are recommended. children between and years should receive a maximum of mg per day in two divided doses. two anti-viral drugs that inhibit both influenza-a and b-viruses, zanamivir (relenza™, glaxosmithcline) and oseltamivir (tamiflu™, roche pharmaceuticals) have recently been approved for general use in the usa, australia, europe and japan. the current knowledge suggests that nainhibitors (ni) will have a better clinical utility than the m -blockers, because these substances are broadly effective against type-a and -b influenza viruses including highly virulent avian virus strains. further, they appear to have a very low frequency of adverse effects and are less prone to induce drug resistance. zanamivir and oseltamivir function as slow binding, substrate competitive inhibitors that strongly reduce viral na-activity by interacting with five sub-sites close to the enzymatic pocket of the na. the ic values of these inhibitors were found to be in the range of . - . nm depending on the virus types and subtypes (mckimm-breschkin et al., ) . targeting of the viral na does not require the delivery of an inhibitor into the cell interior, because the enzyme is a surface glycoprotein. influenza viruses attach to the host cell through binding of the viral ha to sialic acid moieties that are conjugated to cellular glycoproteins. by the time of progeny virus budding these receptor determinants need to be removed to allow efficient release from the host cell. this is accomplished by the viral na (acylneuraminyl hydrolase, ec . . . ) that hydrolyzes glycosidic linkages adjacent to n-acetyl-neuraminic acid (neu ac, sialic acid). thus, blockade of na-activity by antibodies, temperature-sensitive mutation or inhibitory substances results in the aggregation of budding virions at the cell membrane and, hence, reduction of virus release (compans et al., ; palese and compans, ; palese et al., ) . in infected animals or humans, na probably also enhances penetration of the virion through the viscous mucus on respiratory epithelia, which contains sialic acids (matrosovich et al., ) . thus, inhibition of viral na-activity was the rationale behind several efforts to identify substances that would reduce influenza virus spread and replication. the development of the current nis was based on early characterizations of the sialic acid transition state analogue -deoxy- , dehydro nacetylneuraminc acid (neu ac en) (meindl et al., ) and the determination of the three-dimensional structure of the na by x-ray crystallography varghese et al., ; varghese et al., ) . neu ac en had been shown to inhibit viral na-acitvity but was not protective in a mouse model of influenza (palese and schulman, ) . based upon computer-assisted drug design, von itzstein et al. demonstrated that the introduction of positively charged amino-or guanidino moieties at position of neu ac en increased na inhibition by two to four orders of magnitude (von itzstein et al., ) . importantly, the inhibition of naactivity by guanidino-neu ac en that is now also termed zanamivir translated into efficient reduction of viral replication of type-a and binfluenza viruses in the nanomolar range in vitro and dose-dependent decrease of viral titers in infected animals (von itzstein et al., ; woods et al., ) . zanamivir has low oral bioavailability, but shows high antiviral activity in humans or animals when administered topically by inhalation of dry-powder aerosol (cass et al., ) . the second currently approved na inhibitor compound oseltamivir ( r, r, s- acetamido- -amino- -( -ethylpropoxyl)- -cyclohexene- carboxylic acid¸ also termed gs /ro - ) has similarly potent activities against type a and b influenza viruses . oseltamivir emerged from an independent na structure-based study and is based on a cyclohexen ring structure in which the polar glycerol side chain of the sialic acid analogues is replaced by a lipophilic -pentyloxy moiety (kim et al., ) . importantly, oseltamivir has high oral anti-viral activity when administered as its methylester pro-drug, gs /oseltamivir phosphate, that is converted to the active drug by hepatic enzymes (hayden et al., b; li et al., ; mendel et al., ) . zanamivir and oseltamivir have potent anti-viral effectiveness against community-acquired influenza and are in general safe to use in healthy adults (abramson, ; boivin et al., ; hayden et al., ; makela et al., ; monto et al., ; n.n., ) . in clinical trials the nis significantly shortened disease duration and reduced symptoms and viral loads when treatment was initiated within hours post infection (hayden et al., ; hayden et al., b) . even, when inhalation of zanamivir was begun within hours after onset of symptoms the time to alleviation of major disease signs (cough, myalgias, fever, headache) was shortened by one to two days and patients were able to resume normal activities earlier (hayden et al., ; monto et al., ) . initiation of therapy later than hours after disease onset still reduced viral loads but was less beneficial for symptom recovery. two mg daily doses of oseltamivir for five days were shown to reduce shedding of virus and the severity and duration of influenza symptoms by one to two days when therapy was begun within hours after onset of disease signs (nicholson et al., ; treanor et al., ) . some side effects that included diarrhea, nausea and nasal symptoms were observed during clinical testings of zanamivir but were similar in placebo groups (glaxowellcome, ) . the ni substances are also highly effective to prevent spread of the disease. a post-exposure protection study with zanamivir demonstrated % efficacy in preventing transmission of influenza to family members, when the index case was treated with zanamivir . oseltamivir had a comparably high efficacy in preventing laboratory-confirmed influenza by % and influenza with fever by % (hayden et al., a) . within households, one mg dose oseltamivir per day was % protective against clinical influenza even when the index cases were not treated (welliver et al., ) . thus, to prevent the spread of the flu within household contacts the nis appear to be preferable compared to the m -blockers that can induce the emergence and transmission of virulent drug-resistant viruses. during the development of nis for clinical use it was recognized that viruses with a reduced drug sensitivity could be selected in tissue culture (summarized in (mckimm-breschkin, ; tisdale, ) ). resistance can be characterized by various methods including ic -determination of the viral na, by plaque reduction assays (number and size) and yield reduction assay in tissue culture (matrosovich et al., ; tisdale, ) . under laboratory conditions several passages are usually required to select such variants, which is different to amantadine-resistant viruses that can emerge in a single cycle experiment. drug-resistant viruses were also isolated from diseased persons treated with nis (gubareva et al., ; gubareva et al., ; kiso et al., ; zambon and hayden, ) . however, the available data on the pathogenicity of these mutant viruses in animal models suggest that they have reduced replication capability in vivo and may therefore be clinically less relevant in humans. resistance to nis was found to be complex, because it can be associated with mutations in the na, the ha or synergistically in both genes. namutations that confer reduced drug sensitivity were identified at amino acid residues , , , and (based on n -na numbering) (gubareva, ; zambon and hayden, ) . these amino acids are part of or cluster around the conserved catalytic pocket and their mutation can decrease the enzymatic activity to below % and some also destabilize the enzyme (varghese et al., ) . the various ni-molecules slightly differ in their interactions with the enzyme. thus, a given na-mutant enzyme may show a range of sensitivity against different inhibitors (gubareva et al., ) . interestingly, some viruses with a reduced sensitivity to nis were found to carry mutations in the ha, which affected the receptor binding site in the globular head region, the stalk region and the ha -subunit (mckimm-breschkin, ) . apparently, the ha-mutations reduce drug sensitivity by decreasing the affinity for cellular sialic acid receptor molecules and thereby easing the release of budding viruses from the plasma membrane. these findings corroborate the concept that efficient viral replication requires a carefully balanced interplay between the strength of ha/receptor binding and the activity of the na that removes these receptor determinants . the use of zanamivir (relenza™) and oseltamivir (tamiflu™) is recommended for the treatment of uncomplicated influenza caused by type-a and b-viruses (harper et al., ) . therapy with either drug should be initiated within hours after the onset of disease signs and should be continued for five days (glaxowellcome, ; roche, ) . it is important to consider that bacterial superinfections may occur that would not be affected by these anti-virals. neither substance has been shown to prevent serious complications of influenza like pneumonia. zanamivir is approved for treatment of influenza in persons aged years and older. the recommended dosage is two inhalations of mg doses twice a day using the inhalation device provided by the manufacturer. zanamivir is not recommended for persons with underlying respiratory conditions like asthma or chronic obstructive pulmonary disease, because of the risk of precipitating bronchospasm in such patients (glaxowellcome, ) . oseltamivir can be used for treatment of patients of year or older. depending on the age, the recommended doses for children above years and adults are two mg capsules a day. two daily doses of - mg is recommended for children under kg, x mg for children between - kg and x mg for persons weighing > - kg. currently, tamiflu™ but not relenza™ is licensed for chemoprophylaxis in children older than years and in adults. for persons with creatinine clearance of - ml/min, halving of the usual dosage for therapy or prophylaxis is recommended. two approaches are possible, a seasonal prophylaxis that provides a % reduction of confirmed influenza infection in a vaccinated population of frail elderly persons (mcclellan and perry, ) , and a short-term prophylaxis for controlling institutional outbreaks by breaking the virus circulation. several further compounds that inhibit the influenza virus na were identified in independent efforts and have been evaluated as anti-influenza agents. thus, the cyclopentane derivatives bcx- (rwj- ), bcx- , bcx- and bcx- as well as the pyrrolidine-based a (from abbott laboratories) showed strong potent anti-viral activies at least in vitro (kati et al., ; smee et al., ) . thus, although development of bcx- has been halted after showing a lack of activity in a phase iii clinical trial (chand et al., ) , additional nis may emerge as anti-influenza drugs in the future. two unique properties of the trimeric rna-dependent rna-polymerase of influenza viruses, which are not shared by cellular enzymes, provide attractive opportunities for anti-viral interference with possibly little disturbances of the host cell. first, the polymerase exhibits an endonuclease activity that cleaves the first - nucleotides including the '-cap structure from nascent host rna-polymerase ii cap transcripts and use them to prime viral mrnas (lamb and krug, ) . second, the viral polymerase replicates the negative-sense viral rna-segments via unprimed synthesis of a complementary positive-strand rna-intermediate. for both of these activities, inhibitory small molecule compounds have been identified, some of which were also shown to reduce viral propagation in tissue culture and/or in infected mice. however, further clinical development has not been reported for any of those substances so far. the viral endonuclease activity is associated with the pb -subunit and depends on binding of the polymerase to the terminal ends of the vrnatemplate and the cap structures of nascent mrna-transcripts (li et al., ) . the endonuclease most likely utilizes a two metal ion mechanism for cleavage of the cellular nucleic acid (klumpp, a) . it has been shown that derivatives of the fungal metabolite flutimide as well as a class of substituted , -dioxobutanoic acids specifically inhibited the cap-dependent endonuclease, presumably by interaction with the active catalytic site of the enzyme (hastings et al., ; parkes et al., ; tomassini et al., ; tomassini, ) . the most potent compounds of these two classes had ic values in the range of . - µm when tested in virus yield assays in tissue culture experiments. further, intranasal instillation of the l- , compound was reported to inhibit viral titers in nasal washes of mice infected with influenza virus a/pr/ / virus, but the effects on disease progression were not studied (hastings et al., ) . another screening effort has identified t- ( -fluoro- -hydroxy- pyrazinecarboxamide) to have potent and selective anti-influenza activity. t- showed ic values of less than . µg/ml in virus yield assays in mdck cells against all three influenza virus types (a, b, c) with no signs of cytotoxicity (furuta et al., ) . importantly, t- was also orally active in a mouse model and shown to significantly reduce viral lung titers and enhance survival rates from % to % after infection with influenza virus a/pr/ / virus at a dose of mg/kg per day (furuta et al., ; takahashi et al., ) . although the basis for its anti-viral activity was unclear at that time, t- was found to inhibit replication of an oseltamivirresistant mutant virus in vitro suggesting that this inhibitor targets a different viral function (takahashi et al., ) . indeed, recent analyses showed that the compound is metabolized inside the cell into t- -ribofuranosyl- 'triphosphate (t- -rtp), which is a potent and selective inhibitor of apgprimed viral rna-polymerase activity (furuta et al., ) . these findings show that t- may have the potential to become a novel oral antiinfluenza drug that targets a viral function not blocked by one of the currently licensed nis or m -blockers. influenza viruses only have a limited coding capacity. thus, these viruses employ functions of their host-cell for efficient replication. these dependencies create opportunities to design novel anti-viral strategies by targeting specific host cell functions. cell fate decisions in response to extra-cellular agents, including pathogenic invaders are commonly mediated by intra-cellular signaling cascades that transduce signals into stimulus specific actions, e.g. changes in gene expression patterns, alterations in the metabolic state of the cell or induction of programmed cell death (apoptosis). thus, these signaling molecules are at the bottleneck of the control of cellular responses. in this section we will review the recent advances in the analysis of influenza virus induced signaling pathways and first attempts to use signaling mediators as targets for anti-viral approaches. mitogen activated protein kinase (mapk)-cascades have gained much attention as being critical transducers to convert a variety of extra-cellular signals into a multitude of responses (english et al., ; hazzalin and mahadevan, ; widmann et al., ) thereby, these pathways regulate numerous cellular decision processes, such as proliferation and differentiation, but also cell activation and immune responses (dong et al., ) . four different members of the mapk-family that are organized in separate cascades have been identified so far: erk (extra-cellular signal regulated kinase), jnk (jun-n-terminal kinase), p and erk /bmk- (big map kinase) (garrington and johnson, ; widmann et al., ) . these mapks are activated by a dual phosphorylation event on threonine and tyrosine mediated by mapk-kinases (mapkk also termed meks or mkks). the mapk "erk" is activated by the dual-specific mapkk mek and - that are controlled by the upstream serine threonine mapkkkinase raf. raf, mek and erk form the prototype module of a mapk-pathway and are also known as the classical mitogenic cascade. the mapk p and jnk are activated by mkk / and mkk / , respectively, and are predominantly activated by pro-inflammatory cytokines and certain environmental stress conditions. the mek /erk module is both activated by mitogens and certain stress inducers. there is evidence that all these different mapk-cascades are activated upon infection with rna-viruses, including influenza viruses. thus, these signaling cascades may serve different functions in viral replication and host cell response. another important signaling pathway, which is commonly activated upon virus infection is the iκb-kinase (ikk)/nfκb-signaling module (hiscott et al., ) . the nfκb/iκb family of transcription factors promote the expression of well over different genes, such as cytokine or chemokine genes, or genes encoding for adhesion molecules or anti-and pro-apoptotic protein (pahl, ) . the canonical mechanism of nfκb activation includes activation of iκb-kinase (ikk) that phosphorylates the inhibitor of nfκb (iκb) and targets the protein for subsequent degradation (delhase and karin, ; karin, b) . this leads to the release and migration of the transcriptionally active nfκb factors to the nucleus (ghosh, ; karin and ben-neriah, ) . the ikk-complex consists of at least three isozymes of ikk: (i) ikk /ikkα, (ii) ikk /ikkβ and (iii) nemo/ikkγ. the most important isozyme for nfκb-activation via the degradation of iκb is ikk (karin, a) . nemo acts as a scaffolding protein for the large ikk complex (courtois et al., ) that contains still other kinases such as mekk (mapkk-kinase ) (lee et al., ) , nik (nfκb inducing kinase) (nemoto et al., ; woronicz et al., ) and the dsrnaactivated protein-kinase (pkr) (gil et al., ; zamanian-daryoush et al., ) . both nfκb and the jnk mapk-pathway regulate one of the most important anti-viral gene expression events, the transcriptional induction of interferon beta (ifnβ) . ifnβ is one of the first antiviral cytokines to be expressed upon virus infection, initiating an autoamplification loop to cause an efficient and strong type-i ifn response. the ifnβ enhanceosome, which mediates the inducible expression of ifnβ, carries binding sites for transcription factors of three families, namely the ap- family members and jnk targets c-jun and atf- , the nfκb factors p and p , and the interferon-regulatory factors (irfs) (hiscott et al., ; thanos and maniatis, ) . in the initial phase of a virus infection this promoter element specifically binds the constitutively expressed and specifically activated irf -dimer (taniguchi and takaoka, ) . ap- and nfκb-transcription factors are activated by a variety of stimuli. however, a strong irf -activation is selectively induced upon infection with several rna-viruses, in particular by the dsrna, which accumulates during replication yoneyama et al., ) . thus, irf is the major determinant of a strong virus-and dsrna-induced ifnβ-response. interestingly all four so far defined mapk-family members are activated upon an influenza virus infection (kujime et al., ; ludwig et al., ; has helped to get a clearer picture of the importance of the erk-signaling pathway for influenza virus replication. the activation of the map-kinase erk upon productive influenza virus infection (kujime et al., ) appears to serve a mechanism that is beneficial for the virus . strikingly, blockade of the pathway by specific inhibitors of the upstream kinase mek and dominantnegative mutants of erk or the mek-activator raf resulted in a strongly impaired growth of both, influenza a-and b-type viruses . conversely, virus titers are enhanced in cells expressing active mutants of raf or mek olschlager et al., ) . this has not only been demonstrated in cell culture but also in vivo in infected mice expressing a constitutively active form of the raf-kinase in the alveolar epithelial cells of the lung (olschlager et al., ) . while in the wt-situation influenza viruses primarily infect bronchiolar epithelial cells, there is efficient replication in the alveolar layer most exclusively in the cells carrying the transgene. as a consequence this results in an earlier death of the transgenic animals (olschlager et al., ) . this indicates that activation of the raf/mek/erk pathway is required for efficient virus growth. noticeably, inhibition of the pathway did not significantly affect viral rna-or protein-synthesis . the pathway rather appears to control the active nuclear export of the viral rnp-complexes that are readily retained in the nucleus upon blockade of the signaling pathway. most likely this is due to an impaired activity of the viral nuclear export protein nep . this indicates that active rnp-export is an induced rather than a constitutive event, a hypothesis supported by a late activation of erk in the viral life cycle. so far the detailed mechanism of how erk regulates export of the rnps is unsolved. there are two likely scenarios: either it does occur directly via phosphorylation of a viral protein involved in rnp-transport or by control of a cellular export factor. although in the initial studies no alteration of the overall phosphorlyation status of the np, m and nep proteins was observed there are now first indications that certain phosphorylation sites of the np indeed are affected by mek-inhibition (s.p., unpublished data). it remains to be shown, whether this is of functional relevance for the rnp-export process. it is striking that mek-inhibitors are not toxic for the cell, while more general blockers of the active transport machinery, such as leptomycin-b exert a high toxicity even in quite low concentrations. this may indicate that mekinhibitors are no general export blockers but only block a distinct nuclear export pathway. indeed there are first evidences that the classical mitogenic cascade specifically regulates nuclear export of certain cellular rna-protein complexes. in lps-treated mouse macrophages mek-inhibition results in a specific retention of the tnf-mrna in the nucleus (dumitru et al., ) . this is also observed in cells deficient for tpl- , an activator of mek and erk. in these cells the failure to activate mek and erk by lps again correlated with tnf mrna retention while other cytokines are normally expressed (dumitru et al., ) . thus the erk-pathway may regulate a specific cellular export process but leaves other export mechanisms unaffected. it is likely that such a specific export pathway is employed by influenza-a and b-viruses. the finding of an anti-viral action of mek-inhibitors prompted further research showing that replication of other viruses, such as borna disease virus , visna virus (barber et al., ) or coxsackie b virus (luo et al., ) is also impaired upon mek-inhibition. requirement of raf/mek/erk-activation for efficient influenza virus replication may suggest that this pathway may be a cellular target for antiviral approaches. besides the anti-viral action against both, a-and b-type viruses , mek-inhibitors meet two further criteria which are a prerequisite for a potential clinical use. although targeting an important signaling pathway in the cell the inhibitors showed a surprisingly little toxicity (a) in cell culture planz et al., ; pleschka et al., ) (b) in an in vivo mouse model (sebolt-leopold et al., ) and (c) in clinical trials for the use as anti-cancer agent (cohen, ) . in the light of these findings it was hypothesized that the mitogenic pathway may only be of major importance during early development of an organism and may be dispensable in adult tissues (cohen, ) . another very important feature of mek-inhibitors is that they showed no tendency to induce formation of resistant virus variants . although targeting of a cellular factor may still raise the concern about side effects of a drug, it appears likely that local administration of an agent such as a mek-inhibitor to the primary site of influenza virus infection, the lung, is well tolerated. here the drug primarily affects differentiated lung epithelial cells for which a proliferative signaling cascade like the raf/mek/erk-cascade may be dispensable. following this approach it was recently demonstrated that the mek inhibitor u is effective in reducing virus titers in the lung of infected mice after local administration (o.p., s.p. and s.l., unpublished). activation of the classical mitogenic raf/mek/erk-cascade is initiated by yet other phosphorylation events. the kinase raf is known to be regulated by phosphorylation of different upstream kinases including members of the protein kinase c (pkc)-family (cai et al., ; kolch et al., ) . the pkc-superfamily consists of at least different pkc-isoforms that carry out diverse regulatory roles in cellular processes by linking into several downstream signaling pathways (toker, ) . beside a regulation of the raf/mek/erk-cascade and other downstream pathways, pkcs may have additional functions during viral replication. a role of pkcs in the process of entry of several enveloped viruses has been proposed based on the action of protein kinase inhibitors h and staurosporine (constantinescu et al., ) as well as by the calcium-channel blocker verapamil (nugent and shanley, ) . influenza virus infection or treatment of cells with purified viral ha results in rapid activation of pkcs upon binding to host-cell surface receptors (arora and gasse, ; kunzelmann et al., ; rott et al., ) . in a recent study it was shown that the pan pkc-inhibitor bisindolylmaleimide-i prevented influenza virus entry and subsequent infection in a dose dependent and reversible manner (root et al., ) . using a dominant-negative mutant approach this function was assigned to the pkcßii-isoform. overexpression of a phosphorylation-deficient mutant of pkcßii revealed that the kinase is a regulator of late endosomal sorting. accordingly, expression of the pkcßii-mutant resulted in a block of virus entry at the level of late endosomes (sieczkarski et al., ; sieczkarski and whittaker, ) . thus, a specific inhibition of pkcßii may be a suitable approach to blunt virus replication at a very early time point in the replication cycle. activation of the transcription factor nfκb is a hallmark of most infections by viral pathogens (hiscott et al., (hiscott et al., ) including influenza et al., . influenza viral nfκb-induction involves activation of iκbkinase (ikk) and is also achieved with isolated influenza virus components. this includes dsrna (chu et al., ) or over-expression of the viral ha, np or m proteins (flory et al., ) . since gene expression of many pro-inflammatory or anti-viral cytokines, such as ifnβ or tnfα, is controlled by nfκb the concept emerged that ikk and nfκb are essential components in the innate immune response to virus infections (chu et al., ) . accordingly, influenza virus-induced ifnβpromoter activity is impaired in cells expressing transdominant negative mutants of ikk or iκbα (wang et al., ; wurzer et al., ) . nevertheless, ikk and nfκb might not only have anti-viral functions as two recent studies demonstrate that influenza viruses replicate much better in cells where nfκb is pre-activated (nimmerjahn et al., ; wurzer et al., ) . conversely, influenza virus titers from different host cells in which nfκb-signaling was impaired by means of specific inhibitors or dominantnegative mutants, a dramatic reduction could be observed (nimmerjahn et al., ; wurzer et al., ) . thus, in the context of an influenza virus infection a function of nfκb to support virus replication appears to be dominant over the function as a transcription factor in the anti-viral response. on a molecular basis this was shown to be due to the nfκbdependent expression of pro-apoptotic factors, such as tnf-related apoptosis inducing ligand (trail) or fasl . inhibition of virus induced expression of these factors results in strongly impaired viral growth. this links the pro-viral action of nfκb to the induction of apoptosis, a process that will be discussed in the next section. finally, viral need for nfκb-activity suggests that this pathway may be suitable as a target for anti-viral intervention. to this end we have shown recently that several pharmacological inhibitors of nfκb act anti-viral in vivo, without toxic side effects or the tendency to induce resistant virus variants (i. mazur, w. wurzer, c. erhardt, t. silberzahn, t.w., o.p., s.p. and s.l, unpublished). another cellular signaling response commonly observed upon virus infections, including influenza virus is the induction of the apoptotic cascade. apoptosis is a morphological and biochemical defined form of cell death (kerr et al., ) and has been demonstrated to play a role in a variety of diseases, including virus infections (razvi and welsh, ) . apoptosis is mainly regarded to be a host cell defense against virus viruses (reviewed in: julkunen et al., ; ludwig et al., ; infections since many viruses express anti-apoptotic proteins to prevent this cellular response. the central component of the apoptotic machinery is a proteolytic system consisting of a family of cysteinyl proteases, termed two groups of caspases can be distinguished: upstream initiator caspases such as caspase- or caspase- , which cleave and activate other caspases and downstream effector caspases, including caspase- , - and - , that cleave a variety of cellular substrates, thereby disassembling cellular structures or inactivating enzymes (thornberry and lazebnik, ) . caspase- is the most intensively studied effector caspase. work on caspase- deficient mcf- breast carcinoma cells has revealed a caspase- driven feedback loop, that is crucial to mediate the apoptotic process (janicke et al., ; slee et al., ) . thus, caspase- is a central player in apoptosis regulation and the level of pro-caspase- in the cell determines the impact of a given apoptotic stimulus. although it is now well established that influenza virus infection induces caspses and subsequent apoptosis, the consequence for virus replication or host cell defense is still under a heavy debate (reviewed in (lowy, ; ludwig et al., ; schultz-cherry et al., ) . with the identification of pb -f , a new influenza virus protein expressed from a + reading frame of the pb polymerase gene segment, a pro-apoptotic influenza virus protein has been discovered (chen et al., it is long known that influenza virus infection with a-and b-type viruses results in the induction of apoptosis both in permissive and un-permissive cultured cells as well as in vivo (fesq et al., ; hinshaw et al., ; ito et al., ; mori et al., ; takizawa et al., ) . interestingly, viral activation of mapks or upstream kinases has been linked to the onset of apoptosis. in a mouse model for a neurovirulent influenza infection, jnkactivity correlated with apoptosis induction in the infected brain (mori et al., ) . in embryonic fibroblasts deficient for the mapkk-kinase ask- the virus-induced jnk-activation was blunted concomitant with an inhibition of caspase- activation and virus-induced apoptosis (maruoka et al., ) . as an extrinsic mechanism of viral apoptosis induction it has been noted quite early on that the fas receptor/fasl-apoptosis inducing system (fujimoto et al., ; takizawa et al., ; takizawa et al., ; wada et al., ) is expressed in a pkr-dependent manner in infected cells (takizawa et al., ) . this most likely contributes to virus-induced cell death via a receptor mediated fadd/caspase- -dependent pathway (balachandran et al., ) . another mode of viral apoptosis induction might occur via activation of tgf-β that is converted from its latent form by the viral na (schultz-cherry and . within the influenza virus infected cell the apoptotic program is mediated by activation of caspases (lin et al., ; takizawa et al., ; zhirnov et al., ) with a most crucial role of caspase- (wurzer et al., ) . although it is now well established that influenza virus infection induces although it is now well established that influenza virus infection induces caspases (for review see: cohen, ; thornberry and lazebnik, ) . ). pb -f induces apoptosis via the mitochondrial pathway if added to cells and infection with recombinant viruses lacking the protein results in reduced apoptotic rates of lymphocytes (chen et al., ) . however, most of the avian virus strains are lacking the pb -f reading frame and pb -f deficient viruses do not affect apoptosis in a variety of other host cells (chen et al., ) . these results have let to the assumption that apoptosis induction by pb -f may be required for the specific depletion of lymphocytes during an influenza virus infection, a process which is observed in infected animals (tumpey et al., ; van campen et al., a; van campen et al., b) . a recent study adds a new aspect to the open discussion by the surprising observation that influenza virus propagation was strongly impaired in the presence of caspase inhibitors (wurzer et al., ) . this dependency on caspase activity was most obvious in cells where caspase- was partially knocked-down by sirna (wurzer et al., ) . consistent with these findings, poor replication efficiencies of influenza-a-viruses in cells deficient for caspase- could be boosted -fold by ectopic expression of the protein. mechanistically, the block in virus propagation appeared to be due to the retention of viral rnp-complexes in the nucleus preventing formation of progeny virus particles (wurzer et al., ) . interestingly the findings are consistent with a much earlier report showing that upon infection of cells over expressing the anti-apoptotic protein bcl- the viral rnp-complexes were retained in the nucleus (hinshaw et al., ) resulting in repressed virus titers (olsen et al., ) . furthermore the recently identified proapoptotic pb -f (chen et al., ) is only expressed in later phases of replication consistent with a later step in the virus life cycle that requires caspase activity (wurzer et al., ) . the observation of a caspase requirement for rnp-nuclear export was quite puzzling since this export process was shown before to be mediated by the active cellular export machinery involving the viral nuclear export protein (ns /nep) (neumann et al., ; o'neill et al., ) and the anti-apoptotic raf/mek/erkcascade . caspase activation does not support, but rather inhibit the active nuclear export machinery by cleavage of transport proteins. this suggests an alternate strategy by which caspases may regulate rnp-export, e.g. by directly or indirectly increase the diffusion limit of nuclear pores (faleiro and lazebnik, ) to allow passive diffusion of larger proteins. such a scenario is supported by the finding that isolated nps or rnp-complexes, which are nuclear if ectopically expressed, can partially translocate to the cytoplasm upon stimulation with an apoptosis inducer in a caspase- -dependent manner (wurzer et al., ) . these findings can be merged into a model in which the rnps are transported via an active export mechanism in intermediate steps of the virus life cycle. once caspase activity increases in the cells, proteins of the transport machinery get destroyed, however, widening of nuclear pores may allow the viral rnps to use a second mode of exit from the nucleus (faleiro and lazebnik, ) . that would be a likely mechanism to further enhance rnp-migration to the cytoplasm in late phase of the viral life cycle and thereby support virus replication. such a complementary use of both "active" (raf/mek/erkdependent) and "passive" (caspase-dependent) transport mechanisms is supported by the observation, that at concentrations of mek-and caspaseinhibitors, which only poorly block influenza virus replication alone, efficiently impaired virus propagation if used in combination (wurzer et al., ) . thus, while both pathways do not interfere with each other (wurzer et al., ) they appear to synergize to mediate rnp-export via different routes. therefore one may conclude that influenza virus has acquired the capability to take advantage of supposedly anti-viral host cell responses to support viral propagation. this includes early induction of caspase activity but not necessarily execution of the full apoptotic process that most likely is an anti-viral response. this dual role of "early" versus "late" apoptotic events during virus replication may exclude the use of caspase-inhibitors as anti-flu agents, although in cell culture these inhibitors may have a beneficial outcome for the host cell. besides mediators of signaling and apoptosis a variety of other cellular enzymes are required for efficient virus growth. there are also some initial attempts to use these components as target for an anti-viral intervention. another important requirement for a cellular enzyme is the proteolytic cleavage of the ha by proteases. the infectivity and pathogenicity of influenza virus is based on the proteolytic cleavage of the precursor ha into ha and ha chains by an arginine-specific, trypsin-like host protease. the viral glycoproteins are glycosylated in the endoplasmic reticulum (er) and the er-α-glycosidase-i is responsible for the removal of terminal α- , glucose residues from precursor oligosaccharides in the er. a variety of viruses such as hiv, hsv and dengue-virus have been shown to be highly sensitive to inhibitors of these enzymes (mehta et al., ; mehta et al., ) . one of these inhibitors, castanopermine, has been demonstrated to inhibit replication of influenza virus a/hongkong/ / in mdck cells with an ic value of < µm (klumpp, b) . the inhibitor also acted antiviral in vivo in a mouse model and reduced lung titers of a/pr / infected mice by tenfold when administered intranasal. the compound has reached phase ii clinical trials for the treatment of hiv and has been licensed for a potential treatment of hepatitis-c-virus infections (reviewed in (klumpp, b) ). several exogenous protease inhibitors were investigated with respect to their anti-influenza activity: camostat, a serine protease inhibitor; was shown to exhibit strong anti-influenza effects in vitro and in vivo in mice and in chicken embryos. the compound also showed strong anti-influenza effects in amantadine-resistant type-a and -b virus infection in vitro (lee et al., ) . other protease inhibitors nafamostat mesilate, camostat mesilate, gabexate mesilate and aprotinin also inhibited virus replication in vitro protease inhibitors gordox, contrycal and epsilonaminocapronic acid were tested in both animal and clinical experiments. inhalation of aminocapronic acid-containing aerosols exerted the most effective therapeutic effect, reducing the duration of viral antigen in the nasopharyngeal epithelium / to fold (zhirnov et al., ) 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factor complex containing irf- and cbp/p comparative study of influenza virus replication in vero and mdck cell lines nf-kappab activation by double-stranded-rna-activated protein kinase (pkr) is mediated through nf-kappab-inducing kinase and ikappab kinase position statement: global neuraminidase inhibitor susceptibility network caspase-dependent nterminal cleavage of influenza virus nucleocapsid protein in infected cells immunostimulating reconstituted influenza virosomes we would like to thank all the colleagues who have helped in many invaluable ways in the production of this chapter, in particular, dr. c. erhardt, dr. w. wurzer, h. marjuki, b. daubner, v. oehlschlaeger, j. lampe and k. oesterle. this work is dedicated to prof. dr. c. scholtissek's th birthday. key: cord- -e fofgxq authors: ryhal, bruce title: viral disease, air pollutants, nanoparticles, and asthma date: - - journal: bronchial asthma doi: . / - - - - _ sha: doc_id: cord_uid: e fofgxq health care providers who treat patients with respiratory disease are often asked by their patients, “what caused my asthma? and what causes my asthma suddenly to become worse?” these questions have always been difficult to answer, and moving directly to a discussion of the management of asthma is a much easier road to take. in recent years, though, enough information has accumulated about the causes of asthma that one can weave a story containing useful advice that may help patients participate in the management of their disease. and there are also recent studies that can provide answers to the questions posed by physicians who have watched in puzzlement as their previously well-controlled asthma patients have spiraled rapidly out of control. this story has been growing increasingly complex, with an ever-expanding cast of players that sometimes creates a tangled web of interactions. viral respiratory tract infections are the most common triggers of significant asthma • exacerbations. "upper respiratory tract infections" (uris) do not just involve the upper respiratory • tract. human rhinovirus (hrv), which causes the common cold, is the virus most likely to • trigger an asthma exacerbation. in contrast to the usual spring and summer temperate zone pollen season, viral infec-• tions begin to peak in the fall. the number, species, and typical course of viral respiratory tract infections that trigger • asthma vary with a person's age. both acute sinusitis and asthma exacerbations are associated with viral respiratory tract • infection and therefore antibiotics are rarely needed in uncomplicated cases. sulfur dioxide, nitrogen dioxide, ozone, and particulate matter in air pollution may • exacerbate asthma, and patients should be cautioned to stay indoors when levels of these irritants are high. indoor air pollution, especially from tobacco smoke, can be reduced with benefit to the • asthma patient. introduction health care providers who treat patients with respiratory disease are often asked by their patients, "what caused my asthma? and what causes my asthma suddenly to become worse?" these questions have always been difficult to answer, and moving directly to a discussion of the management of asthma is a much easier road to take. in recent years, though, enough information has accumulated about the causes of asthma that one can weave a story containing useful advice that may help patients participate in the management of their disease. and there are also recent studies that can provide answers to the questions posed by physicians who have watched in puzzlement as their previously well-controlled asthma patients have spiraled rapidly out of control. this story has been growing increasingly complex, with an ever-expanding cast of players that sometimes creates a tangled web of interactions. this chapter will look at how viral infections, air pollution, and possibly nanoparticles may act as causal agents of asthma. the concept of causal agent, though, has a variety of different interpretations. in general, agents may act on the respiratory tract to initiate asthma or to exacerbate it. initiation (or inception or development) of asthma refers to the start of asthma in a patient who was previously entirely free of this problem. an exacerbation (or trigger or precipitating event) means the significant and often sudden worsening of an established chronic asthmatic condition. avoidance of a proven initiating factor, if possible, could permit the primary prevention of asthma. in contrast, avoidance of triggering events will not halt the disease but only decrease the number of exacerbations in someone who already has chronic illness. in studying and treating asthma, identification of a specific trigger is usually much easier than trying to prove an initiating cause. viruses that affect asthma are acting on a complex and varied phenotype, and therefore the outcome of each infection can be quite varied. a simple linear cause-and-effect relationship between a viral infection and an asthmatic episode usually does not exist. koch's modified postulates for infection-caused disease are: the microorganism must be present in every case of the disease. • the microorganism must be isolated from a diseased organism and grown in pure • culture. the cultured microorganism should cause disease when introduced into a healthy • organism. the microorganism must be recovered from an inoculated, diseased experimental host. • this linear way of looking at viral-induced disease is not comprehensive enough to allow sufficient insight into the relationship between viral illness and asthma. no one viral infection consistently causes asthma in all or even most individuals. systems biology, though, can provide a conceptual framework for better understanding of the virus-asthma interaction. systems biology looks at the web of factors in the initial state of the individual patient and then examines how one or more external or internal influences perturb this state ( ). in fig. , the path taken by system a illustrates how one factor, for example a simple rhinovirus infection, may have very little long-term effect on mucosal inflammation in an individual with no atopic stressors and no genetic propensity toward asthma. this individual will return quickly to equilibrium and a low inflammatory state. the path of system b illustrates how multiple stressors, including genetic factors and atopic immune development, may interact with a viral infection to cause a long lasting or perhaps permanent change in the level of mucosal inflammation. some details of risk factors will be outlined and discussed in this chapter, but systems biology or systems medicine cannot yet specify each feature of the set of interactions in a way that leads to firm predictions about asthma. out of the complexity of the systems approach, though, some simple and compact principles do emerge, so that every precondition does not have to be known to predict the outcome of intervention or treatment. some general factors that appear to be important in the asthmatic response to viral infection include: though two-dimensional paper does not allow multidimensional maps, we can walk down a branching path in a narrative fashion to show the interaction of factors important in viral-caused asthma. in most of the twentieth century, the office or hospital diagnosis of viral respiratory infection was most often a good guess, a probability statement. common and more affordable viral molecular diagnostics, especially reverse transcriptase pcr (rt-pcr), and viral culture can now improve the accuracy of the guess when precision is needed. viruses may be detected in symptomatic or in asymptomatic patients. two thirds or more of acute respiratory tract infections (rtis) occurring in the community can be identified as viral. traditionally, these have been divided into upper and lower rti, but the difference between upper and lower infection seems to be more indistinct than previously believed. human rhinovirus (hrv), for example, replicates initially in the upper respiratory tract yet may cause extensive lower respiratory tract illness. the frequently used term viral upper rti (urti) is somewhat of a misnomer. the most commonly occurring respiratory virus is hrv, which accounts for nearly half of cases of viral respiratory illness, followed by influenza virus and coronavirus, with lesser contributions from parainfluenza virus, respiratory syncytial virus (rsv), adenovirus, metapneumovirus, and other miscellaneous viral species ( ) (see table ). the three main types of viruses that are known to affect asthma are hrv, rsv, and influenza. the peak periods of viral infection tend to vary from year to year, but generally in north america rhinovirus peaks in the fall and early spring, influenza in the early winter, and rsv in midwinter (fig. ) . many communities can monitor the progress of these annual epidemics with viral culture and molecular diagnostics, thereby giving physicians a higher probability of knowing in advance what virus a patient may have. a molecular diagnostic panel is commercially available for identifying acute viral respiratory infection, though the cost-effectiveness of this type of testing for routine clinical use is yet to be determined. more details of the immunobiology of the major asthmogenic viral infection, hrv, have been revealed in the past several years ( ) . the intercellular adhesion molecule icam- found on nasal epithelial cells is the attachment point for the majority of serotypes of hrv( ). hrv is divided into clades or strains hrv-a, hrv-b, and hrv-c. hrv-c has proven extremely difficult to culture. there are over different serotypes ( ). viral species influence asthma in the various age groups in different ways. age is a marker for the development and maturation of the immune system, which diversifies greatly over time. as the human body ages, the immune system molds itself to the environment to become a mirror image of specific, usually protein, molecules in the external local surroundings. age also has an important effect on the physics of scaling in the respiratory system. airway resistance is inversely proportional to the fourth power of diameter, which enlarges with age until young adulthood and then slowly declines. small increases in airway diameter therefore lead to huge reductions in airway resistance and give more "breathing room." about % of significant, prolonged wheezing episodes in children are triggered by respiratory viruses and hrv is most often involved ( ). the common rhinovirus cold accounts in large part for the fall seasonal peak of asthma in school-age children. epidemiologic evidence combined with viral molecular diagnosis has suggested that this peak is a consequence of children returning to the classroom with the subsequent spread of respiratory viruses, mainly rhinoviruses ( ) . viral exacerbations of asthma tend to be prolonged and severe. triggers such as a gust of pollen-laden breeze may be ameliorated by moving the young patient indoors, and exercise triggers can be removed by stopping the exercise, but a viral trigger is usually steady and persistent and replicates within the body. a study of children aged - years with asthma concluded that an asthma exacerbation was of a greater severity if a viral infection was present as opposed to a nonviral illness ( ) . airway hyperreactivity and a corresponding cough and wheeze may be noted for well over weeks after a rhinovirus infection in the asthmatic child. atopy confers additional risk on asthmatic children who become ill with respiratory virus infection ( ) . school-aged children with atopic asthma, as opposed to those with nonatopic asthma, have been noted in a number of studies to experience more frequent symptomatic colds, more episodes of viral-triggered asthma, and more prolonged airway hyperreactivity after the colds ( ) ( ) ( ) . the tendency to have higher numbers of symptomatic rtis and a longer duration of illness was also noted for allergic children in general, with and without asthma ( ) . parents of children with atopy and asthma tend to be frustrated by the prolonged recovery time compared with their nonatopic siblings, and school absences are more problematic. inhaled corticosteroids and leukotriene receptor antagonists (ltras) are well known to control the number of wheezing exacerbations in school-age children with chronic persistent asthma, an effect that appears to encompass those episodes caused by viral illness. a survey of school children in ontario found that children presenting in september to the emergency department for asthma exacerbations, presumably mostly viral triggered, were less likely to have used preventive anti-inflammatory medications than their counterparts who did not have such severe exacerbations ( ) . a retrospective study suggested that inhaled fluticasone and salmeterol administered prior to and during the fall could reduce the morbidity of the fall viral season in patients with asthma ( ) . a trial of a montelukast added to usual asthma therapy was able to attenuate the fall asthma peak in one study ( ) though this effect did not reach statistical significance in a later trial ( ) . inhaled corticosteroids might be expected to prevent viral-induced wheezing in children with minimal chronic disease as well. a preventive effect, though, has not been consistently shown in clinical trials. a study conducted in school-aged children without persistent disease but with a history of viral-triggered wheezing demon strated that inhaled beclomethasone diproponate was not superior to placebo in reducing future episodes. the inhaled steroid failed to reduce days with symptoms, or the frequency, severity, or duration of episodes of upper or lower respiratory illness ( ) . preventive medication should therefore be targeted especially to those patients with persistent chronic asthma. for acute treatment of a viral-provoked asthma exacerbation, oral systemic corticosteroids continue to be the most effective choice ( ) and are part of the current therapy protocols ( ). use of high-dose acute corticosteroid inhalers continues to be studied with varying success. whether vaccination can prevent asthma exacerbations is unclear. the expert panel report concluded that influenza vaccine does not reduce the frequency or severity of asthma exacerbations during the influenza season ( ). many patients in the community with asthma experience severe complications from an influenza infection, so all reasonable means of prevention should still be taken, including vaccination. the influenza virus appears to be a less potent trigger of asthma than hrv, and influenza peaks are not as well correlated with childhood asthma peaks as in the case of hrv. an oral influenza antiviral (oseltamivir) improved pulmonary function and reduced exacerbation frequency in one randomized, placebo-controlled trial in school-age asthmatic children who had influenza ( ) . unfortunately, increasing resistance of the influenza virus to antiviral agents limits their use as a long-term strategy to reduce illness in asthmatic children. the concept of using antivirals to reduce asthma morbidity in children seems theoretically promising. the preschool years can lay the groundwork for the later asthma issues of the type that have been discussed. diagnosing viral-triggered asthma in infants and preschool children, though, must be done with caution. asthma is defined as a chronic disease, and several, or even many, self-limited acute wheezing illnesses do not necessarily add up to a chronic illness. often children in this age group will experience wheezing in association with a variety of viral infections. parents are naturally anxious about treatment and prognosis in these children. preschool children who experience rsv-and hrv-induced wheezing are more likely to develop asthma in later years. the childhood origins of asthma study (coast) showed that viral wheezing illnesses in infancy and early childhood caused by hrv were the most significant predictors of the subsequent development of asthma at age ( ) . a bidirectional causation has been proposed with rsv: severe rsv was associated with a short-term increase in bronchial hyperresponsiveness, and, in turn, the presence of asthma was associated with long-term increased susceptibility for severe rsv disease ( ) . whether early childhood viral infection initiates a series of events that lead to asthma has been an area of much interest and study. one analysis showed that infants reaching months of age at the winter virus peak had a % increased risk of developing later asthma compared with those reaching age year at the winter peak ( ) . if viruses do initiate asthma in some patients, then prevention of rsv or hrv or a similar illness in a critical time period might prevent or reduce the frequency of asthma in later years. nonatopic infants who had received palivizumab (a humanized mab against rsv) for prevention of rsv infection showed an % reduction in risk of recurrent wheezing from ages to ( ) , though no effect was noted in atopic children. the hypothesis that early viral infections lead to asthma is made less convincing by epidemiologic studies showing that frequent exposure to viral rtis throughout early childhood may actually decrease the risk of later asthma. studies in the united states and in the united kingdom have shown that day care attendance and other factors that increase the frequency of viral rtis reduce the risk of later (after - years) frequent wheezing ( , ) . one interesting medical editorial on this topic was subtitled with tongue-in-cheek, "please sneeze on my child" ( ) . that strategy may not be practical, but clinicians should be able to reassure worried parents that day care exposure does not seem to result in a long-term risk of asthma. while the factors that contribute to the development of asthma are still unclear, there is little doubt that viral infections act as potent triggers of asthma in preschool children. as noted, hrv is the most potent of triggers, though all hrvs do not seem to be alike. pathogenicity of hrv appears to vary among groups a, b, and c. hrv-c was found in a study of hospitalized preschool children to be associated with asthma more often than hrv-a ( ), and hrv-c was noted to be the most frequent type found in patients presenting to the emergency department ( ) . in contrast, experimental infection with a type of hrv-a resulted in no worse illness in allergic than in nonallergic subjects ( ) . knowledge of a circulating virulent hrv strain in the community could put clinicians on the alert for more serious symptoms in their asthmatic patients with colds. there are several competing classification systems for the wheezing preschool child that aim to help with prognosis and treatment ( table ) . as a conceptual model, one can create two opposing poles. at one pole is the small child who experiences rare mild wheezing with acute viral illness, has no wheezing or cough between episodes, and has no atopy or parental asthma. these children appear to benefit very little or not at all from acute or chronic corticosteroid therapy for viral-triggered wheezing illness ( ) . at the other pole are children who wheeze daily or weekly, have an atopic history, have a parental history of asthma, and may be on chronic controller therapy. a viral infection in these children appears to be a trigger that requires a step up in asthma therapy, perhaps to a burst of oral corticosteroids. between these poles of severity are many children whose therapy must be individualized. the criteria from the national asthma education and prevention program help select preschool children who may benefit from acute and/ or chronic corticosteroids. these guidelines use the asthma predictive index ( ) to specify which wheezy small children have or likely will have chronic asthma and could benefit from various forms of inhaled and oral corticosteroid therapy. owing to concerns about oral corticosteroids, other forms of treatment for viral wheezing have been examined in preschool children. a study in -to -year-old children showed a benefit of episodic high-dose inhaled steroids with viral rti and wheezing ( ) , though some adverse effects on growth were noted. the effectiveness of a ltra, montelukast, was examined in a study of - year olds with a history of intermittent asthma. this study showed a reduction of typically viral-induced asthma exacerbations in children given the ltra as a daily controller ( ) . both inhaled corticosteroids and ltras are options to control chronic asthmatic wheezing in this age group ( ). prolonged or chronic cough after viral rti may be a problem. preschool children, whether asthmatic or not, spend a considerable percentage of the year with viral rti symptoms that are distressing to patient and parent. the years from teen through young adulthood tend to be the healthiest years of an individual's life. an expanded antiviral immunologic repertoire helps in reducing the number of annual viral rtis. while childhood is the time of most frequent viral rtis, young adults who are exposed to their own small children may have a secondary peak near their s. acute sinusitis is a common problem in this age group. sinusitis has been known to precede a worsening of asthma, and episodes of acute sinusitis have often been the occasion for a course of antibiotics. the entity of viral rhinosinusitis, though, is far more common than previously believed. a viral rti can produce a week or more of purulent discharge and radiographic abnormalities of the sinus cavities on ct scans ( ) . most acute sinusitis is not predominantly initiated by bacteria nor, at least in the first week or so, antibiotic-responsive ( , ) . the mechanism by which acute viral sinusitis becomes linked with worsening asthma is generally through the association of both diseases with viral infection (fig. ) . the adult group of patients with asthma diverges into several different phenotypes, likely representing various diseases. asthma is often said to be a syndrome rather than one disease. different phenotypes may have varying responses to viral infection. a cluster analysis divided asthma patients into five different groups. one group, "benign asthma" seemed to have well-controlled symptoms regardless of triggers, viral or otherwise. another group that was female preponderant, "obese nonesosinophilic," had minimal atopy or eosinophilic inflammation yet a high level of symptoms in response to typical triggers ( ) . chronic adult diseases of previously unknown cause have occasionally been found, in whole or in part, to have an infectious etiology. these include peptic ulcer disease (helicobacter pylori), polyarteritis nodosa (hepatitis b and c), reactive arthritis (shigella and chlamydia), and lyme arthritis (borrelia burgdorferi). a survey of asthma patients, of mean age , suggested that % of initial attacks started after an illness suggestive of a respiratory infection ( ) . this subset tended to be nonatopic and may represent a distinct phenotype. viral and nonviral initiating infectious agents have been proposed for adult "infectious asthma," including mycoplasma, chlamydia, adenovirus, and adult rsv, but reasonable proof of an infectious mechanism is still pending. regardless of initiating cause, asthma is exacerbated in adults, as in children, by viral respiratory infections. respiratory virus is found at least % of the time in adults with asthma exacerbations, but not as frequently as in childhood acute significant wheezing episodes ( ). older and elderly adults experience some degree of immune senescence but also have expanded specific antiviral immunity. the types of viral illness that exacerbate asthma are slightly different than in younger years. the peak of ed visits and asthma admissions for adults over tends not to be in the fall but rather from december to january, suggesting a broader range of viral triggers than in the fall rhinovirus peak ( ) . the contribution of influenza to excess morbidity in older adults is well known, but less generally appreciated is the contribution of rhinovirus to serious illness. concomitant heart disease, chronic obstruction pulmonary disease, and hypertension can make viralexacerbated asthma a more complicated and serious illness in older adults. a rhinovirus outbreak in a nursing home for elderly patients resulted in two thirds of the affected patients having lower respiratory tract symptoms, nearly one-third requiring corticosteroid or bronchodilator therapy, and three individuals having serious morbidity including one death ( ) . a peak of rhinovirus rti may be seen in grandparents who care for small children. consistently effective treatments for viral-caused respiratory disease have been frustratingly slow to arrive in the modern pharmacopoeia. despite these obstacles, however, a proactive approach, including vaccination and respiratory hygiene, can improve the care of the patient at risk for viral illness and bronchospasm and avert complications. since the time of albert einstein, scientists have known to be wary of "spooky action at distance." particles that affect the respiratory tract must first be dispersed into the air and enter and contact the respiratory tissue to have an effect. these particles vary in size from molecules in the angstrom range ( × − m), to so-called nanoparticles ( - × − m), to large pollen grains ( × − m), on up to the largest dust particles that can remain suspended in air (about × − m). air particles are divided into several common ranges for study purposes: • pm -particulates of an aerodynamic diameter of less than mm or , nm • fine particles of diameters below . mm or , nm • ultrafine particles or nanoparticles of diameters below . mm or nm study of the real-world, clinical effects of the individual components of air pollution is challenging since most or all components tend to be released into the air at about the same time. unwanted and/or unhealthy gases and particles make up the components of air pollution. outdoor air quality issues vary to great extent by specific location and depend on weather and climate, the level of vehicle traffic, and the type of fuel used for energy and manufacture. in the united states, the office of air quality planning and standards (oaqps) has established the national ambient air quality standard (naaqs) for each of the several pollutants. carbon monoxide, lead, nitrogen dioxide (no ), ozone, sulfur dioxide (so ), and particulate matter in the air have maximum exposure standards (fig. ) . studies of the effect of air pollution on health attempt to use statistical analysis to separate the individual contribution of each component of pollution. additionally, provocation/exposure testing can be performed in the laboratory. many of the same questions that can be asked about viral disease can be asked about air pollution -does it initiate asthma and does it trigger asthma? clearly not everyone breathing air pollution gets asthma or wheezing, but exposure does seem to increase the risk. a population study in the netherlands found that children with higher exposure to traffic-related pollutants (no , particulate matter) were more likely to develop asthma ( ) . data from the taiwan children health study showed an increased prevalence of bronchitic symptoms among children with long-term exposure to outdoor air pollutants ( ) . in addition to irritant properties, air pollution may contain immunologically active particles. nanoparticles, including particles of diesel exhaust, which are suspended in air are especially interesting to immunologists studying the development of asthma. they have been proposed to act as adjuvants and immunomodulators ( ) . most diesel particulates have sizes of less than mm and represent a mixture of fine particles and nanoparticles. acute wheezing may be triggered by exposure to high levels of pollutant gases including nitrogen dioxide, sulfur dioxide, and carbon monoxide. burning of fossil fuels is the main source of these pollutants in most locations. nitrogen dioxide and sulfur dioxide gases diffuse rapidly and impact upon the wet respiratory tract to produce highly irritating acids. sulfur dioxide can cause respiratory constriction in asthmatic patients at concentrations of . ppm when exercising ( ) . healthy adults begin experiencing increased airway resistance at ppm, and even nonasthmatic adults will develop bronchospasm at ppm, though these levels would be highly unusual in outdoor air pollution. nitrogen oxides, and especially no , are also irritating to the upper and lower respiratory tracts at low levels, and patients with asthma are more susceptible to these adverse effects. higher concentrations of outdoor no were associated with more asthma symptoms in a study of inner city children ( ) . though the mechanism of action is uncertain, exposure to carbon monoxide in city air was found in one european study to worsen lung function in adult patients with asthma ( ) . ozone, while of critical importance to global health in the upper atmosphere, is an especially noxious chemical when generated at or near ground level. ozone (o ) is not produced directly by traffic or by hydrocarbon burning. instead, the combination of no and hydrocarbons with air and sunlight form the secondary pollutant ozone. high average ozone and airborne particulate matter were associated with more frequent asthma symptoms and ed visits and hospital stays in a study of asthma sufferers in the san joaquin valley in california ( ) . ozone from air pollution has been shown to exacerbate asthma in children and adults, though this effect may be greater in children ( ) . a study of over , emergency department visits in atlanta for pediatric asthma showed a relationship to ozone and primary pollutant concentrations from traffic sources. these pollutants increased ed visits even at relatively low concentrations ( ) . the study of particulate matter in the air is quite complex, since the exact composition varies geographically. in general, high levels of particulate matter have long been associated in epidemiological studies with increased levels of respiratory disease. ongoing research is examining the importance of particle size, fine versus more coarse, in asthma and chronic respiratory illness. one study in turkey showed an % rise in asthma admissions when air contained high levels of coarse particles ( ) . in contrast, a us study did not find increased hospitalizations for respiratory disease during those periods with high coarse particle levels ( ) . the evidence for a negative effect on health from suspended fine particles is stronger ( ) . genetic and phenotype differences may be important in the sensitivity of the asthma patient to air pollution. the risk of childhood asthma in mexico city was modulated in some children by genes controlling the response to oxidative stress, such as might occur while breathing ozone ( ) . advice on how to avoid high concentrations of air pollutants is important for asthma patients. air pollution, like pollens and viruses, follows a seasonal pattern, and knowledge of the local pattern can help the primary physician with diagnosis and treatment. in the united states, a daily air quality index (aqi) is computed and distributed for most large population areas. the aqi, which is determined on the basis of the highest pollutant of the day, may be considered safe for patients with chronic respiratory disease if less than (green zone). on days with poor air quality, asthma sufferers should come inside where pollutant levels are typically much lower. indoor ozone levels vary from to % of outdoor concentrations, depending on the size of outdoor air flows into a building ( ) . although asthma patients should continue their controller therapy during periods of high air pollution, pretreatment with controller medications may not always be successful. budesonide treatment in one study was not successful in preventing ozone-triggered functional airways impairment in test subjects with mild persistent asthma ( ). while outdoor air pollution rightly receives a great deal of media and government attention, indoor air pollution can make living inside hazardous as well. fortunately, indoor air problems are usually amenable to personal control and behavioral advice. air quality issues may occur in both home and work environments. the field of occupational medicine examines workplace concerns and is reviewed in another chapter. home air quality is typically not regulated, though pollution may result from several indoor sources. hydrocarbon fuels are, of course, burned inside as well as outdoors. indoor gas cooking and heating stoves may produce no , high levels of which have been associated with increased asthma symptoms in children ( ) . good ventilation is essential if natural gas is to be burned indoors. indoor nitrogen oxides are also produced by wood-burning stoves, especially if not well vented. the most serious and prevalent type of home air pollution is secondhand or environmental tobacco smoke (ets). the risk from this indoor pollutant begins in utero. maternal smoking during pregnancy is associated with increased infant wheezing ( ) . this risk of respiratory morbidity continues to increase with postnatal parental smoking ( ) . laws regulating indoor tobacco smoking in one european country were followed by improved quality-of-life scores in a group of asthmatic indoor workers ( ) and also by a reduction in the overall rate of hospitalizations for childhood asthma ( ) . as noted previously, indoor ozone is usually much less than outdoor levels. in recent years, though, indoor ozone generators have been marketed to the public for odor control and purported heath benefits. a us epa review has warned the public about the potential hazards of adding an additional amount of a measured air pollutant to the indoor air. this chapter has examined some of the most significant initiating and exacerbating causes of asthma. viral respiratory infections, and to a lesser extent air pollution, are common triggers of exacerbations and may interact with individuals to affect the development of some forms of asthma. these causal factors do not exist in isolation but rather interact with the personal attributes of each patient, including his or her genetic and environmental background. the role of viruses and pollutants in asthma is important knowledge that has consequences for prevention, treatment, and avoidance of illness. helpful education may be given to patients and appropriate treatments selected, and health care providers can avoid the considerable human effort and resources wasted on interventions that are useless or harmful. viral and pollutant triggers demonstrate that the highly complex inflammatory asthmatic response is called forth on many more occasions than simply by the contact of pollen grains and other allergens with the respiratory tract. since so much of immune inflammation seems to have arisen from the need to combat infection, the interaction between asthmatic inflammation and viral infection is a natural topic for further investigation. some of the most significant advances in medical care have come through the treatment and prevention of viral illnesses, and furthering the understanding of respiratory viruses is a worthy priority for the future study of asthma prevention and treatment. in addition to natural harmful infectious particles, humans in recent decades have added many substances of their own creation, including the molecules and particles that constitute indoor and outdoor air pollution. control of this problem is very important for overall respiratory health. important action and advice is available for each asthma patient. by understanding and anticipating respiratory viral infections and air pollution as important causes of asthma, the health care provider can provide superior care for those who suffer from this chronic disease. thanks to albin leong md and denise ryhal bsn for helpful comments and for reviewing portions of this manuscript. the clinical applications of a systems approach a case-control study of acute respiratory tract infection in general practice patients in the netherlands the role of rhinovirus in asthma exacerbations expresssion of intercellular adhesion molecule- (icam- ) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection? common cold, uncommon variation understanding the september asthma epidemic weekly monitoring of children with asthma for infections and illness during common cold seasons duration of postviral airway hyperresponsiveness in 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prednisolone for preschool children with acute virus-induced wheezing the asthma predictive index: a very useful tool for predicting asthma in young children preemptive use of high-dose fluticasone for virus induced wheezing in young children montelukast reduces asthma exacerbations in -to -year-old children with intermittent asthma computed tomographic study of the common cold effect of amoxicillin-clavulanate in clinically diagnosed acute rhinosinusitis antibiotics for acute maxillary sinusitis cluster analysis and clinical asthma phenotypes infectious asthma: a reemerging clinical entity? epidemiology of asthma exacerbations a rhinovirus outbreak among residents of a long-term care facility traffic-related air pollution and the development of asthma and allergies during the first years of life air pollution and prevalence of bronchitic symptoms among children in taiwan the immune effects of naturally occurring and synthetic nanoparticles carbon monoxide pollution is associated with decreased lung function in asthmatic adults medical management guidelines for sulfur dioxide (so ): agency for toxic substances and disease registry acute respiratory health effects of air pollution on children with asthma in us inner cities outdoor air pollution and uncontrolled asthma in the relationship between visits to emergency departments for asthma and ozone exposure in greater short-term associations between ambient air pollutants and pediatric emergency department visits particulate matter (pm( . ), pm( - . ), and pm( ))and children's hospital admissions for asthma and respiratory diseases: a bidirectional casecrossover study coarse particulate matter air pollution and hospital admissions for cardiovascular and respiratory diseases among medicare patients epidemiological evidence of effects of coarse airborne particles on health nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase and glutathione s-transferase m polymorphisms and childhood asthma indoor ozone: north carolina public health epidemiology budesonide reduces neutrophilic but not functional airway response to ozone in mild asthmatics health effects of indoor nitrogen dioxide and passive smoking on urban asthmatic children international study of wheezing in infants:risk factors in affluent and non-affluent countries during the first year of life interactive effect of family history and environmental factors on respiratory tract-related morbidity in infancy respiratory symptoms, pulmonary function, and markers of inflammation among bar workers before and after a legislative ban on smoking in public places smoke-free legislation and hospitalizations for childhood asthma asthma and wheezing in the first years of life definition, assessment, and treatment of wheezing disorders in preschool children: an evidence-based approach key: cord- - ce authors: meskill, sarah d.; o’bryant, shelease c. title: respiratory virus co-infection in acute respiratory infections in children date: - - journal: curr infect dis rep doi: . /s - - - sha: doc_id: cord_uid: ce purpose of review: this investigation aims to understand the role and burden of viral co-infections for acute respiratory illnesses in children. co-infection can be either viral-viral or viral-bacterial and with new technology there is more information on the role they play on the health of children. recent findings: with the proliferation of multiplex pcr for rapid diagnosis of multiple viruses as well as innovations on identification of bacterial infections, research has been attempting to discover how these co-infections affect each other and the host. studies are aiming to discern if the epidemiology of viruses seen at a population level is related to the interaction between different viruses on a host level. studies are also attempting to discover the burden of morbidity and mortality of these viral-viral co-infections on the pediatric population. it is also becoming important to understand the interplay of certain viruses with specific bacteria and understanding the impact of viral-bacterial co-infections. summary: rsv continues to contribute to a large burden of disease for pediatric patients with acute respiratory illnesses. however, recent literature suggests that viral-viral co-infections do not add to this burden and might, in some cases, be protective of severe disease. viral-bacterial co-infections, on the other hand, are most likely adding to the burden of morbidity in pediatric patients because of the synergistic way they can infect the nasopharyngeal space. future research needs to focus on confirming these conclusions as it could affect hospital cohorting, role of molecular testing, and therapeutic interventions. acute respiratory illnesses are the most common cause of under- -year-old mortality worldwide [ ] . in particular, pneumonia is responsible for approximately . - . million fatal cases in children under age five globally [ ] . beyond the mortality burden, there is significant morbidity with symptomatic viral infections estimated at more than five episodes per year in children under age three [ ] . while single viral infections are relatively straightforward, there is interest in understanding the role of dual respiratory infections in pediatric patients. dual infections could either be viral-viral or viral-bacterial as the human respiratory tract is a reservoir for many organisms. molecular diagnostics have increased the ability to diagnose causative agents in patients with respiratory illnesses. real-time polymerase chain reaction (rt-pcr) allows for the detection of multiple viruses at once and is found to be more reliable and expedient than viral culture [ ] . furthermore, newer methods, such as multiplex pcr and next-generation sequencing are producing more accurate and quicker results towards identifying these organisms [ ] . given this ability, studies have attempted to discern if the positive test for pathogen is actually causative or if it is asymptomatic shedding leading to a positive test result. one study evaluated adult visitors to a tourist attraction taking clinical history of symptoms as well as testing pcr for common respiratory illnesses [ ] . of those participants who tested negative for respiratory viral infections, . - . % reported to be asymptomatic depending on the definition applied. only . % of patients tested positive for a respiratory virus. of these positive results, up to . % were considered symptomatic. another study focused on respiratory viral infection positivity specifically in the pediatric population [ ] . testing children biweekly whether symptomatic or not, % of symptomatic episodes had detection of a viral pathogen compared to % of asymptomatic episodes. the younger the patient, the less likely a pathogen positive episode was asymptomatic (p = . ); in addition, multiple pathogens were found in significantly less asymptomatic episodes (p = . ). further studies attempted to discern which viruses were more likely to cause symptoms. one study in children under age years old found that respiratory syncytial virus (rsv), human metapneumovirus (hmpv), and parainfluenza viruses (piv) were more likely to be causative of disease [ ] . another study still found high rates of asymptomatic shedding of rsv and piv with more than half of positive tests associated with an asymptomatic patient [ ] . however, within these data, younger patients were associated with higher rates of positive test results correlating with a symptomatic event demonstrating the importance of age in clinical manifestation of viral infections. rsv is responsible for an extremely high burden of disease in children. infection with rsv is one of the leading causes of death in children under year of age worldwide, second only to malaria [ ] . in a surveillance of acute respiratory infections in children under age , rsv was responsible for % of annual hospitalizations, % of emergency department visits, and % of office visits [ ] . in an evaluation of children under years old admitted to the hospital with a diagnosis of pneumonia, the most common cause of infection, whether bacterial or viral, was rsv quickly followed by rhinovirus [ ] . rhinovirus is the most common cause of respiratory viral illness during all seasons except winter when rsv is predominant [ ] . rhinovirus is second only to rsv in causing bronchiolitis in hospitalized patients [ ] . unfortunately, the true burden of rhinovirus is under-reported as many studies do not include this virus in there molecular diagnostic testing as the rates of asymptomatic shedding are quite high and it is difficult to differentiate shedding from actual cause of disease [ ] . while some studies do have rsv and rhinovirus as the leading cause of pneumonia in children, another important common viral contributor is influenza [ ] . influenza and its complications are the leading cause of morbidity and mortality [ ] . despite antiviral medications and vaccines, it is estimated by the world health organization (who) that the annual influenza epidemics cause to million severe infections and , - , deaths each year in developed countries and , hospitalizations and , deaths in the usa [ , ] . influenza has recently been recognized as having a higher burden of disease than previously thought because of poor recognition and diagnosis even with molecular testing [ ] . although vaccination against influenza decreases the risk of infection, now with less vaccination we are witnessing less herd immunity and those more vulnerable are becoming severely ill [ ] . in my practice, i've witnessed a -month-old infant-too young for the influenza vaccine and caregivers opted out of the influenza vaccination for themselves-develop respiratory failure requiring endotracheal intubation due to influenza a epiglottitis [ ] . human metapneumovirus (hmpv) also causes acute respiratory infections globally. hmpv has been shown to cause high hospitalization rates of per in children years old or younger, with an estimated , hospitalizations annually which is similar to the rates seen with influenza [ ] . outpatient visits were estimated at one million clinic visits and , emergency department visits annually in the same population [ ] . admitted pediatric patients with hmpv were more likely than those without the infection to require supplemental oxygen and had longer intensive care stays [ ] . at the host level, the outcome of dual infection is commonly viral interference, such as when one virus competitively inhibits the replication of another virus, but it can also enhance replication in some cases [ ] . it is postulated that the sequence of infections, the time interval between viral exposure, and the route of infection affect the pathogenicity of the co-infection [ ] . one example of this used mice models. when the mice cell models that were infected with rhinovirus were then infected with influenza a virus days later, there was an attenuated response to the influenza infection with less severe manifestation of disease [ ] . this demonstrates that the preceding infection altered the host response and the timing and order of infection of the host are crucial in outward disease manifestation. co-infections can also alter the epidemiology of viral infections. for example, rhinovirus is a rapidly replicating virus and can interfere with the replication of other viruses while piv is an extremely slow replicator and its replication can be interrupted by the presence of other viruses [ ] . viral loads can be compared among patients with one or multiple infections. one study found that viral loads were consistently high regardless of co-infection status (such as rsv, influenza a, and hmpv) but others had viral load vary based on coinfection status (such as piv and adenovirus) [ ] . of these viruses, influenza a is least likely to be identified in coinfected patients. in mathematical modeling, the idea of resource competition can explain viral loads as the fasterreplicating viruses overcome slower replicating viruses by leaving no resources [ ] . in this model, influenza a replicates faster than rsv and therefore keeps the rsv viral load below detection level. the pathophysiology behind dual viral infections can explain some of the epidemiology of viral-viral co-infections seen at the population level. given the high rates of rsv and rhinovirus infections, it makes sense that they are the most commonly identified viruses in co-infected patients across multiple studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, one study did conclude that the odds of rhinovirus detection were lower when rsv was present and the odds of rhinovirus were significantly higher in those patients who received rsv immunoprophylaxis [ ] . this indicates that despite the high prevalence of co-infection with these viruses, there is still viral interference occurring. there is also evidence of viral competition when it comes to rsv and influenza. in terms of viral competition, when rsv infection rates are high, influenza rates of infection are low and the converse is true [ ] . in addition, when both viruses are circulating in a small population the rates of co-infection of rsv and influenza are over times less than expected [ ] . on review of influenza interactions with other viral pathogens, there is evidence of competitive interference with rsv, rhinovirus, other influenza strains, and hmpv virus by evaluating population incidence and co-infection detection [ ] . multiple studies have attempted to evaluate the clinical importance of viral co-infections. it would seem intuitive to think that having more than one virus causing disease in a person would lead to more severe symptoms and sequelae. one study did support this conclusion. in it the authors looked at all pediatric patients who had a respiratory viral panel sent, they found an unadjusted increase in the risk of moderate-severe illness, non-invasive ventilation, ecmo, and death in coinfected patients however in the adjusted analysis only the risk moderate-severe disease continued to be increased [ ] . some studies found certain clinical outcomes to be at increased risk but not a persistence of high risk in all outcomes. one study evaluated patients under months in france and found that viral co-infections were . times more likely to be in the picu however once in the picu there was no difference in length of stay, duration of mechanical ventilation, duration of supplemental oxygen [ ] . another study supported this finding by evaluating children in the picu retrospectively comparing one virus vs co-infected viral status and found that the co-infected patients had longer average length of stay in the picu and longer time of intubation [ ] . however, there was no difference in rates of needing highflow nasal cannula, mechanical ventilation, or having cardiovascular dysfunction. it is possible that the prolonged intubation times could be because patients who were co-infected with viruses had higher odds of bacterial co-infection. there are some studies that also found increased risk of clinical outcomes but these actually were based on specific viral combinations. one study found that specifically influenza viral co-infection with another influenza strain significantly increased the risk of icu admission or death but this did not carry through to any other co-infected status [ ] . another study did a multicenter evaluation of bronchiolitis over years and found longer length of stay in those with rsv/rv coinfection but no proof that these patients were sicker (as in no difference in icu admission or support with cpap or mechanical ventilation) [ ] . another study also found an increase in length of stay and oxygen use in those patients with specifically rsv/rv co-infected status [ ] . there are a few studies that found no difference in clinical outcomes on co-infected patients. the study looking at tourists did not find any association between viral co-infections and the likelihood of being symptomatic or presenting with more severe symptoms [ ] . another one looking at hospitalized pediatric patients found no increased risk of icu admissions in those with multiple viruses identified [ ] . beyond no difference in clinical outcomes, quite a few found less severity in those patients with multiple viral infections. one study found age to be important in understanding the risk of severe disease. in this study, they evaluated children with bronchiolitis in the netherlands and found those patients with dual infection had no difference in severity under months of age but significantly less severe disease in patients over months [ ] . another study in the netherlands looking at pediatric patients had shorter mean length of stay and no difference in oxygen supply needs or intensive care (icu) admission [ ] . another found that viral co-infected patients were significantly less likely on the inpatient ward (or . ), icu stay (or . ), require oxygen supplementation (or . ), or have length of stay greater than days (or . ) [ ] . one study was actually able to see a linear response to length of stay with a shorter length of stay the more viruses detected when evaluating all pediatric patients with hospitalized respiratory infections [ ] . the best evaluation of the literature on viral co-infections and clinical severity is going to be in systematic reviews. there are three looking at viral co-infections that warrant discussion. scotta et al. [ • ] did a large systematic review of respiratory viral coinfections with illness severity in children with over , patients in the combined evaluation. in this review, viral co-infections did not influence risks of all outcomes assessed: mean length of stay, length of supplemental oxygen, need for hospitalization, need for intensive care admission, mechanical ventilation, or death. they also looked at sub-analyses of specific viral combinations and did not see any influence on outcomes. lim et al. [ ] did a systemic review for children under age years with respiratory illness and found insufficient evidence to suggest a difference in any clinical outcome based on co-infected status. in a very small subset of patients, they found a suggestion that children without co-morbidities actually did worse when only a single virus was identified. goka et al. [ ] did a systemic review of patients of all ages with respiratory illnesses and found that studies that recruited young children were more likely to report high rates of co-infection and that there were inconclusive results on risk of hospitalization or icu admission. to cause respiratory illnesses, bacterial pathogens first need to colonize the nasopharyngeal space [ ] . organisms achieve this colonization via positive and/or negative associations: positive association exists through mutualism, symbiosis or helping to evade the host's immune system; negative association exists through ammensalism, predation, or the host immune system disproportionally affecting one organism over the other [ ] . overall, there are multiple mechanisms, for viruses and bacteria, to aid in the success of invasion and colonization of the human body. ) viral pre-disposition to bacterial adherence: alteration of the host's respiratory epithelium causes viruses to increase the susceptibility of bacterial colonization during a simultaneous infection and after full recovery of a viral illness [ , ] . examples include: influenza and streptococcus pneumoniae and adenovirus and streptococcus pneumoniae [ , ] . ) disruption of the epithelium barrier: viruses can intracellularly disarrange cellular processes or destroy infected cells through metabolic exhaustion or lysis [ ] . the destruction of cells leads to the denuding of the epithelial layer, exposing the basement membrane; therefore, causing introduction of bacterial organisms [ , ] . examples of this mechanism are s. pneumoniae binding to fibronectin after the denudation of the epithelium layer, and staphylococcus aeurus and morexella catarrhalis binding to extracellular matrix protein after destruction to the epithelium [ ] [ ] [ ] . loss of the epithelium integrity and promotion of bacterial translocation are also seen in rhinovirus-induced paracellular migration of haemophilus influenzae [ ] . ) upregulation of adhesion proteins: viral infected cells may decrease the innate immune response by altering the expression of antimicrobial peptides (defensins), which are secreted in the respiratory mucosa [ ] . during viral infections, there are cascades of proinflammatory responses leading to the upregulation of adhesion proteins found on epithelial cells, which leads to the cellular invasion of pathogenic organisms [ ] . for example, rsvand parainfluenza viruses upregulate intracellular and outer membranes proteins such as intracellular adhesion molecule (icam- ), p -homologous fimbriae (p fimbriae), carcinoembryonic adhesion molecule- (ceacam- ), and platelet-activating factor receptor (pafr) [ , ] . with the expression of these proteins, several bacterial organisms, s. pneumoniae and h. influenzae, are able to adhere to these molecules leading to invasion of the host's cells [ , ] . ) production of viral factors: production of viral components such as neuramindase (na), a glycoprotein produced by influenza and parainfluenza, and protein-gexpressed on rsv cells-destroy the integrity of infected cells. this destruction exposes bacterial receptors and aids in bacterial co-infections [ ] [ ] [ ] [ ] . ) dysfunction of immune system components: respiratory viruses may affect the immune system by impairing neutrophil function, decreasing oxidative burst, and enhancing neutrophil apoptosis, thus increasing the susceptibility to bacterial superinfection [ , ] . also, viruses can predispose to bacteria superinfection by rendering natural killer (nk) cells recruitment and activation ineffective, which is seen with influenza and s. pneumoniae [ ] . viruses also alter monocyte function, decrease production and activity of cytokines, and prevent appropriate immune response routing, leading to enhanced bacterial colonization and increasing risk for mortality [ ] [ ] [ ] [ ] . because of the interaction between viruses and bacteria, there are many known specific virus-bacteria relationships ( table ) . for example, influenza interacts with both streptococcus pneumoniae and staphylococcus aureus. the most common bacteria found in viral, secondary bacterial infections is s. pneumoniae [ ] . s. pneumoniae has over distinct serotypes and is a common cause of acute otitis media (aom), pneumonia, sepsis, and bacterial meningitis [ ] [ ] [ ] . one of the most common complications of influenza infection is aom from either s. pneumonia or s. aureus [ , ] . since influenza is one of the common viral contributors to pneumonia, it also increases the rates of bacterial pneumonia from s. pneumoniae and s. aureus. this is because influenza with both of these bacteria has synergistic relationships using the mechanisms listed above [ , ] . in addition, the bacteria increase influenza's infectivity by activating hemagglutinin on the membrane, thus neutralizing antibodies and allowing more virions to be replicated inside the host cell [ , ] . table reviews the most common viral-bacterial co-infections and their associated complications. there are also reports of preceding bacterial infections leading to increase viral susceptibilities [ ] . for example, s. pneumoniae and hmpv have a unidirectional synergistic relationship where s. pneumoniae predisposes children less than years old to hmpv infections [ ] . similarly, h. influenza stimulates adhesion proteins on human epithelial cells, creating an entry point for rhinovirus [ ] . rhinovirus also has a bi-directional relationship with s. aureus: rhinovirus aids in the bacterial adhesion and engulfment into the epithelium cell and s. aureus promotes the replication of rhinovirus [ ] . rhinovirus can actually increase the nasal load of s. aureus by % over baseline [ ] . the evaluation of viral-bacterial co-infection on disease severity is an advancing field however no study reports a decrease in severity with viral-bacterial co-infection. one study [ ] evaluated children presenting with bronchiolitis using nasal swabs to identify a plethora of viruses in addition to s. pneumoniae, m. catarrhalis, and h. influenzae. in this study, rsv and s. pneumoniae co-detection was significantly associated with severe disease in the regression analysis. in a cohort study of children admitted with acute respiratory disease, bacterial superinfections were significantly associated with higher illness severity scores (or = . ), more severe respiratory distress (or = . ), required more respiratory support (or . ), and have longer hospital length of stay [ ] . it is encouraging to note that the children who received the pneumococcal vaccine had lower illness severity, less respiratory distress, required less respiratory support, and had less admissions to the picu [ ] . in a review of the clinical significance of viral-bacterial coinfections in pediatric patients [ •] , studies were found to evaluate clinical severity in this co-infected group. only four of these studies did not observe increased clinical severity, such as more frequent and longer picu admissions or longer ventilation requirements. it is also important to place this discussion in a historical context and discuss the influenza a pandemic of . this pandemic had a mortality rate of - million people worldwide [ ] . it is now known that secondary bacterial pneumonia caused the majority of the deaths during this time [ ] . this further underscores that the viral infection alone was not as detrimental as the viral-bacterial co-infection. parainfluenza s. pneumoniae aom, acute rhinosinuisitis [ ] a invasive pneumococcal disease: pneumococcus is isolated from a sterile site, i.e., sepsis, meningitis [ ] cap community acquired pneumonia, aom acute otitis media, copd chronic obstructive pulmonary disease, lrti lower respiratory tract infection there is still a lot to learn about pediatric respiratory illness co-infections, as the interplay is intricate and not fully understood. looking at viral-viral co-infections, it seems that ultimately rsv seems to be the major decider of severity of infection whether or not the child has one or multiple viruses identified [ ] . this information could influence the role of molecular testing in routine hospitalized patients. viralbacterial co-infections, on the other hand, usually lead to more severe diseases. overall, being able to better understand coinfection relationships can aid in the development of therapeutic methods as well as potentially affect the role of molecular testing. conflict of interest all authors declare no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance global, regional, and national causes of under- mortality in - : an updated systematic analysis with implications for the sustainable development goals viral-bacterial co-infections in the respiratory 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description of the pathogen, disease epidemiology, treatment, and prevention effect of clonal and serotype-specific properties on the invasive capacity of streptococcus pneumoniae use of a whole genome approach to identify vaccine molecules affording protection against streptococcus pneumoniae infection complications and associated bacterial coinfections among children hospitalized with seasonal or pandemic influenza synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro hmpv infection influenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam- and tlr expression streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children does viral co-infection influence the severity of acute respiratory infection in children? review of the literature on the role of viral-bacterial co-infections in pediatric respiratory illnesses predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness respiratory syncytial virus coinfections with rhinovirus and human bocavirus in hospitalized children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - b ycv authors: gavora, js title: resistance of livestock to viruses: mechanisms and strategies for genetic engineering date: - - journal: genet sel evol doi: . / - - - - sha: doc_id: cord_uid: b ycv nan maximum survival of livestock, with good health and well being are conditions for efficient animal production. many of the current livestock disease problems that prevent the realization of this optimal production goal are caused by viruses, described by peter medawar as &dquo;pieces of bad news wrapped in protein coat&dquo;. this review deals with possible new, genetic engineering strategies for the improvement of resistance to viruses in livestock. since work on genetic engineering of disease resistance is more advanced in plants than in livestock, information on research in plants is also reviewed. the use of livestock for food, fibre and draft over hundreds of years has led to a significant influence by humans on the evolution of domesticated animal species. some of the changes induced by artificial selection parallel in their significance speciation. a modern meat-type chicken can be viewed as a species different from a modern egg-type chicken. similar differences exist between breeds of dairy and beef cattle. this 'genetic engineering' of livestock was achieved through the long-term use of conventional genetic improvement methods. it can be argued that gene transfer represents just another phase in the development of genetic engineering of livestock and that it would be foolish not to take advantage of the new technologies. thus introduction of new mechanisms of disease resistance in livestock by gene transfer may be viewed as a logical continuation of the creative influence of humans on the evolution of farm animals and birds that could benefit mankind by improvements in food safety and production efficiency. increased disease resistance will also improve the welfare of livestock. the latter consequence may make this type of genetic engineering more acceptable to the general public than other types of gene transfer. if there is one attribute that is common to viruses, it is the lack of uniformity in all aspects of their existence. nevertheless, this review attempts to find general elements and common patterns in the subject discussed. as background for the discussion of the subject, the article deals briefly with coevolution of hosts and parasites and principal elements of virus-host interactions, and reviews past improvement of disease resistance in plants and livestock by conventional breeding and genetic engineering, as well as the potential 'biological cost' of genetic manipulation. it includes prerequisites for and principles of the design of new resistance mechanisms, and proposes possible strategies for the introduction of disease resistance mechanisms by gene transfer. the main goal of this review is to inform readers from both research and industry about this area of long-term interest to animal agriculture and outline the potential use of the concept of new resistance mechanisms for the benefit of mankind and improvement of animal welfare. basic understanding of the parallel evolution of viruses and their hosts provides a useful starting point for the consideration of strategies for genetic engineering of new mechanisms of resistance. therefore, principal elements of the coevolution of viruses and hosts are briefly reviewed. viruses are obligatory, intracellular parasistes with limited genome sizes that code for functions the virus cannot adopt from host cells (strauss et al, ) . viruses have their own evolutionary histories, independent of those of their hosts. it is not clear whether viruses had a single or multiple origin. the origin of a virus is defined as that time when its replication and evolution became independent of the macromolecules from which it was derived (strauss et al, ) . viruses may have arisen ( ) by selection from an organelle; ( ) from cellular dna or rna components that donate macromolecules which gain the ability to replicate and evolve independently; or ( ) from self-replicating molecules. polymers of ribonucleotides can contain both the information required and the functional capacity to form a self-replicating system (watson et al, ) . the main mechanisms of viral evolution are mutation, recombination, and gene duplication. viruses have a very short generation interval and high mutation rate. for example, the mutation rate of a chicken retrovirus is - nucleotide/replication cycles -approximately eight orders higher than that of the host cell genome (dougherty and temin, ) . nevertheless, the virus always retains its origin of replication. recombination has also a large role in viral evolution because it allowed viruses to 'try out new gene combinations'. an example of an unusual acquisition of genes by a virus are three trna genes in bacteriophage t a type of gene only observed in eukaryotes (gott et al, ) . although it is possible that the genes evolved within t , the phage may also have acquired the genes from an eukaryotic host (michel and dujon, ) . similarly some retroviruses such as rous sarcoma virus acquired oncogenes for their genome. in general, dna viruses are more stable than rna viruses and do not cause rapidly moving pandemics as is the rule for rna viruses; in contrast, dna viruses tend to establish persistent or latent infections which may lead to malignant transformations (strauss et al, ) . exceptions to the general rule include the herpesvirus of marek's disease, a dna virus that can cause rapidly moving disease outbreaks in chickens, and the avian leukosis viruses, rna viruses that exhibit a period of latency and seldom cause high mortality. a disease of the host is not an evolutionary goal of the parasite. compatibility is preferable to incompatibility. subclinical infections are common; they are the rule -diseases the exception. there is no selective advantage to the virus in making the host ill, unless the disease aids in the transmission of the virus to new hosts, such as in the case of diarrhea. in some instances, disease may also result from an overzealous immune system. hence the interplay between microbes and hosts should not necessarily be seen as an ongoing battle but as a coevolution of species (pincus et al, ) . general considerations susceptibility (in the narrow sense) is the capacity of cells to become infected. for a virus to survive and reproduce, essential viral genes have to ensure: ( ) replication of viral genomes in which the involvement of viral genes varies from assisting host enzymes, to actually replicating the viral genome, although even the most selfdependent viruses use some host cell function in the process; ( ) packaging of the genome into virus particle -viral proteins do the packaging, although host proteins may complex with viral ones in the process; and ( ) alteration of the structure or function of the infected cell -the effects may range from cell destruction to subtle, but significant changes in function and antigenic specificity of infected cells. in general, once it enters, no virus leaves a cell unchanged. during their replication, viruses exploit host cell molecules at the expense of the cells. there are three types of viral infection (knipe, ) . ( ) ( ) productive infection is when the cell produces the virus but, as a consequence, dies and lyses. ( ) productive infection is when the cell survives and continues to produce the virus. the levels of injury to the cells resulting from viral infection range from no visible effects to cell death and include inclusion body or syncytium formation and cell lysis. in most instances cell injury is a consequence of processes necessary for virus replication but at least in one known instance, the penton protein of the adenovirus, which has no known purpose in the viral cycle, causes cytopathic effects in monolayer cells (valentine and pereira, ) . genetic engineering strategies that prevent entry of viruses into host cells would be effective against all three types of viral infection. other strategies discussed below can deal with various stages of viral life cycles and would accordingly affect the outcome of viral infection. to provide a basis for the examination of the opportunities to devise and genetically engineer new resistance mechanisms, the viral life cycle that consists of three fundamental steps, attachment, penetration, and replication (roizman, ) will be examined in sequence. attachment of virus to the host cell attachment of the virus to the host cell is, in most instances, through a specific binding of a virion protein, the antireceptor, to a constituent of the cell surface, the receptor. complex viruses, such as vaccinia, may have more than one species of antireceptor or antireceptors may have several domains, each reacting with a different receptor. mutations of receptors may cause a loss of the capacity of a receptor and antireceptor to interact and thus lead to resistance to viral infection. it seems likely that mutations in antireceptors preventing viral attachment will be automatically eliminated from viral evolution, unless they are able to interact with a substitute host. the number of receptors for which information is accumulating is rapidly increasing. examples in table i show that receptors are mostly glycoproteins. not all cells in a susceptible organism express viral receptors, a phenomenon that may limit susceptibility. even though our understanding of receptors is still at an early stage, it is obvious that viral receptors are molecules that have a normal physiological function in the host. while there is a great deal of variability in the types of molecule in viral receptors, some cell surface molecules are used by multiple, often unrelated viruses (table i) . when viewed across host species, for example, histocompatibility molecules are receptors for both semliki-forest togavirus and human coronavirus; sialic acid residues serve as receptors for both the influenza myxovirus and reoviruses, although there are rotaviruses that do not require their presence (mendez et al, ) and low density lipoproteins (ldl) are receptors for both the human minor cold picorna virus and avian leukosis viruses. viruses compete with molecules that require receptors for a physiological function of the host. for example, ldl and the human minor rhinovirus compete for ldl receptors (table i) , and cells with down-regulated ldl receptor expression yield much less virus than up-regulated cells (hofer et al, ) . viruses tend to use abundant molecules as receptors, so that reduction in availability of the molecules for the physiological function is not lethal, or molecules whose function can be substituted by other molecules. there are alternative viral strategies to deal with the receptor problem. the part of the sodium-independent transporter of cationic amino acids, used as the receptor for ecotropic bovine leukemia virus (table i), is different from the part of the protein directly involved in the amino-acid transport function. thus the physiological function of the receptor can continue, despite binding of virus to the receptor (wang et al, ) . another example confirming this possibility is the sodium-dependent transporter of inorganic phosphate that serves as the receptor for the gibbon ape leukemia virus (table i). productive infection of cells expressing this receptor results in complete blockage of the uptake of inorganic phosphate mediated by the receptor. nevertheless, the infection is not cytotoxic. hence, there is likely more than one phosphate transport mechanism in these cells (olah et al, ) . this aspect of viral strategies may open up possibilities to block the receptor sites, thus preventing entry of a virus without serious impairment of physiological function of the receptor. the receptor for herpes simplex virus exemplifies a situation of special interest from the point of view of future engineering of disease resistance. the viral receptor heparan sulfate is present on cell surfaces but body fluids also contain heparin and heparin-binding proteins, either of which can prevent binding of herpes simplex virus to cells (spear et al, ) . hence spread of the virus is likely influenced by both immune response and the probability that the virus will be entrapped and inhibited from binding to cells by extracellular forms of the receptor (heparin or heparan sulfate). similarly, soluble molecules of the cd receptor for human immunodeficiency virus, as well as fragments of the critical cd domains can inhibit infection . it has been suggested that a secreted receptor for avian leukosis virus might similarly be able to neutralize the virus (bates et al, ) . penetration of a virus into the cells is usually an energy-dependent process that occurs almost instantly after attachment. as summarized by roizman ( ) , penetration can occur as ( ) translocation of the entire virus particle across the cell membrane; ( ) endocytosis resulting in accumulation of virus particles in-side cytoplasmatic vacuoles; or ( ) fusion of the cell membrane with the virion envelope. non-enveloped viruses penetrate host cells by the first two processes. uncoating of the virus particle takes place after penetration. for some viruses, such as orthomyxoviruses and picorna viruses, divestiture of the protective envelope or capsid takes place upon their entry into cells. for others, such as herpes viruses, the capsid is transported along the cytoplasmic cytoskeleton into nuclear pores. with reoviruses, only a portion of the capsid is removed and the viral genome expresses all its functions even though it is never fully released from the capsid. while several genetic engineering strategies to prevent attachment of viruses to host cells can be devised and are proposed below, strategies to prevent penetration of viruses attached to cells are much less obvious. viruses use many strategies for replication leading to ( ) encoding and organization of viral genomes, ( ) expression of viral genes, ( ) replication of viral genes, and ( ) assembly and maturation of viral progeny. the key event in these processes is the synthesis of viral proteins. regardless of its size, organization, or composition, a virus must present to the cell's protein synthesizing mechanisms an mrna that the cell recognizes and translates. the interaction between the viral cell attachment protein and host-cell receptors is the principal determinant of tropism, but there are other factors involved. for retroviruses and papovaviruses, cis-acting elements of the viral genome, gene enhancers, which are usually - bp in size and often repeated in tandem, stimulate transcription (serfling et al, ) . they may serve as an entry point for rna polymerase ii. enhancers may be both cell-type-specific and cell-differentiationspecific, in that they function mainly in certain cell types (tyler and fields, ) . for avian retroviruses, enhancer regions within the long terminal repeat (ltr) are an element of the viral genome that determines cell tropism of disease expression ). the cell imposes three constraints on the virus at the point of virus multiplication. ( ) the cell may lack enzymes to synthesize mrna off the viral rna genome, or may lack enzymes to transcribe viral dna. ( ) eukaryotic host cell protein-synthesis machinery translates only monocistronic messages and does not recognize internal initiation sites within mrna. as a consequence the virus must synthesize either a separate mrna for each gene or an mrna encompassing a 'polyprotein' to be later cleaved. ( ) the expression of viral proteins is in competition with cellular genes. viruses evolved strategies that either confer competitive advantage to viral mrna or abolish translation of cellular mrnas. the host range of a virus defines both the kinds of tissue or cells and animal species in which a virus can enter and multiply (roizman, ) . receptors may be species specific. for example, the poliovirus receptor is only found on primate mammalian cells (mclaren et al, ) . a tissue-specific receptor is exemplified by the cd receptor for the hiv virus, which is present only on t-lymphocytes (table i) . species-specifity of receptors is one of the components of non-host resistance that will be discussed in more detail below. infection with some viruses leads to inhibition of transcription of cellular proteincoding genes by host polymerase ii, possibly through competition for transcription between cellular and viral genes. herpes simplex virions contain a transcriptional activator complex (post et al, ) , while adenovirus provides a trans-acting eia gene product responsible for increased polymerase activity after adenovirus infection (nevins, ) . viruses can also induce or express new dna-binding proteins. thus a retrovirus encodes a homolog to cellular transcription factor ap- (bohmann et al, ) . splicing of viral mrna precursors is accomplished by cellular enzymes. influenza and retroviruses can regulate the extent of the splicing, adenovirus inhibits maturation of cellular mrna, and influenza virus transcription complexes intervene in the host mrna maturation (knipe, ) . many viral mrnas are capped, in that they contain a single major initiation site near their ' end, and their translation is similar to that of host mrna. however, inhibition of host mrna translation provides the virus with increased availability of ribosomal units. thus herpes simplex and poxvirus degrade cellular mrna to decrease its translation (inglis, ; fenwick and mcmenamin, ) . other mechanisms include competition for the host translational apparatus by production of large amounts of viral mrna, or viral mrna with higher affinity to ribosomes than cellular mrna (knipe, ) and changes in the specificity of host translational apparatus; for example, extracts from poliovirus-infected cells translate poliovirus but not host mrna (rose et al, ) . both rna and dna viruses cause inhibition of host-cell dna synthesis (knipe, ) . eukaryotic cell proteins contain signals that target them to a specific cell compartment or organelle. viral proteins may also contain similar signals for their localization within the cell. viral proteins make use of cellular chaperone proteins to secure their proper folding. similarly, many post-translational modifications of viral proteins are performed by cellular enzymes. for example, tissue-specific proteases cleave specific proteins on the virion surface thus facilitating virion infectivity (scheid and choppin, ) . maintenance of viral dna in the host cell and release of progeny virus there are two types of mechanism for maintaining viral dna in the host cell: ( ) virus dna is integrated into the cellular genome, eg, in retroviruses; or ( ) viral dna is maintained as extrachromosomal circular molecule in the infected cell, eg, epstein-barr virus, or bovine papilloma virus. viruses that persist in the body may cause damage, and prevention of persistence may be the next best defence if prevention of virus entry is impossible. persistence is usually in differentiated cells that remain morphologically unchanged but may lose their differentiated or 'luxury' function, as well as their homeostasis. persistent viruses can negatively influence host cells in two ways: ( ) virus presence and replication causes damage resulting in a selective disadvantage; and ( ) in such a way that the virus will gain an evolutionary advantage for which there will be selection pressure to maintain. alternatively, some viruses undergo a latency stage in their life cycle that seems to cause little damage. enveloped viruses move from infected cells either by budding through the plasma membrane or by secretion vesicles containing virus particles within the plasma membrane (knipe, ) . non-enveloped viruses are mostly released by lysis of the cells but they can also leave without cell lysis as in simian virus (norkin and ouelette, ). to facilitate their survival and spread throughout the body, some viruses have evolved strategies to modulate the immune response of their host to their favor, a phenomenon recently reviewed by fujinami ( ) . virus infection can lead to development of immune responses against the host's own tissues and viruses can also code for proteins, homologous to cellular proteins, that modify the host's immune response. for example, epstein-barr virus produces a bcrf protein similar to the interleukin il- protein (a cytokine-inhibiting factor) that inhibits the production of il- and il- , tumor necrosis factor, gamma interferon, and macrophage-granulocyte colony-stimulating factor. the herpes simplex virus- (hsv- ) but not hsv- can interfere with the complement system by producing a protein that acts as a receptor for the component of the complement cascade. virus infections can also interfere directly with the major histocompatibility system (mhc). cytomegalovirus encodes an mhc class i heavy-chain homolog that limits expression of the cellular class i molecules on cell surfaces and this may reduce killing of infected cells by host defences. most animal and plant species are resistant to the great majority of viruses. nonhost resistance is the rule, susceptibility the exception. however, the nature of nonhost resistance is not sufficiently understood to fully explore the incompatibility between viruses and non-hosts (wilson, ) . nevertheless, it is certain that we, as well as all animals, are &dquo;continuously bathed in a sea of microbes, yet harmed by a relatively few&dquo; (oldstone, ) . to coexist, viruses and their hosts have established, to a greater or lesser degree, an equilibrium. in general, normal coevolution of parasites and their hosts is from disoperation, through exploitation, to toleration and from facultative to obligatory mutualism, but genetic changes may also bring reversals to this process (dobzhansky, ) . none of the strategies for the creation of new, genetically engineered viral resistance mechanisms proposed in this article are derived from non-host resistance. nevertheless, a brief discussion of the subject is included to stimulate further exploration of this widespread phenomenon as the possible basis for protection of livestock against viruses. some knowledge of non-host resistance mechanisms is emerging from experimentation with plant viruses that infect permissible but normally resistant cells by bypassing the resistance barrier (dawson and hilf, ) . viral host range is determined by interactions between existing viral gene products and corresponding host components. because of the obligately parasitic nature of viruses, viral host range is not determined by a particular gene product that enables the virus to overcome host defences but by a 'fit' between viral gene product and certain gene products of the host. there are two general prerequisites for successful infection: ( ) presence of all conditions necessary for viral infection. absence of the conditions results in 'passive resistance mechanisms' in plants, that tend to be recessive or incompletely dominant. ( ) absence of successful host defences. adaptation mechanisms of viruses that enable them to infect potential hosts protected by non-host mechanisms may include an ability to overcome a host block by a mutation or recombination with another virus, or acquisition by the virus of capabilities formerly provided by the hosts that are not available in resistant plants. a virus can capture such genetic information from the host. there are many mechanisms of resistance to viral diseases. for our purposes, emphasis will be placed on non-immune mechanisms. of particular interest in this review are those mechanisms that prevent the entry of viruses into host cells. viral receptors can be variable so that some alleles of the receptor may make the potential host resistant to viral infection. however, it is only rarely that resistance to infection is observed in otherwise susceptible host species. this indicates that during virushost coevolution, viruses tend to utilize evolutionarily stable molecules as receptors. resistance to infection by parvovirus b in some humans is due to lack of a specific virus receptor. people who do not have the erythrocyte p antigen parvovirus receptor (brown et al, ) are naturally resistant to the virus (brown et al, ) . another example is resistance to coronaviruses in mice. a monomeric protein has been identified as a receptor for mouse hepatitis virus on intestinal and liver cells. the presence of this receptor appears to be the principal determinant of susceptibility to infection (boyle et al, ) . similar variation in viral receptors is observed in genetic resistance to avian leukosis virus (alv) infection in chickens (payne, ) . the alv receptors, which belong to the family of receptors for ldl (bates et al, ) , include recessive alleles that do not allow viral entry into potential host cells and render some chickens resistant to the virus. the receptor for subgroup a alv was shown to map to tva * s known as the dominant gene for susceptibility to subgroup a virus (bates et al, ) . susceptibility of cells to infection needs to be distinguished from permissiveness, which can be defined as the ability of a cell to support viral replication. for example, chick cells are not susceptible to poliovirus but are permissive to its replication following their transfection with poliovirus rna (roizman, ) . such cells are potential hosts for a virus, providing a mutation provides means for the virus to enter the cells. in laboratory mice, alleles at the fv- locus determine susceptibility to infection with ecotropic murine leukemia viruses and the resistance is dominant in heterozygous mice (ikeda and odaka, ) . a viral protein gp normally interacts with the viral receptors on cells. however, in resistant mice, the specific receptor on cell membranes seems already bound by the gp whose production is controlled by the mouse fv- '' resistant allele. this system is similar to that in chickens, where the endogenous retroviral gene ev- , expressing the subgroup e endogenous viral envelope also controls resistance to infection by subgroup e virus (robinson et al, ) . resistance of mice to certain strains of influenza virus is a dominant trait associated with the allele mx on chromosome (staehli et al, ). the resistance is mediated by action of alpha-and beta-interferons that induce mx protein expression which inhibits synthesis of viral mrna (krug et al, ) . a recent review of natural, 'preimmune' resistance loci in mice (malo and skamene, ) includes genes controlling resistance to influenza virus, cytomegalovirus, ecromelia, friend leukemia virus, mink cell focus-forming virus, moloney leukemia, radiation leukemia, and rous sarcoma virus. the resistance genes represent a variety of mechanisms that do not involve viral receptors. for example, the cmvl gene, associated with resistance to cytomegalovirus, appears to control host responses mediated by natural killer and inflammatory response cells. similarly, the resistance loci in friend leukemia control the susceptibility of target cells to viral replication. it is not the purpose of this review to provide a detailed account of immune mechanisms that protect against virus infection. the brief text below will give only a general outline of immune responses and examples of how the system may be influenced by viruses. acquired immune responses involve phagocytic, humoral and cell-mediated systems. only the cell-mediated immune response that is especially effective against cells containing actively replicating virus and, as a rule, is the most important defence against viral infections will be discussed briefly. the cellular immune system becomes sensitized to viral infection only after viral proteins are degraded to short linear peptide epitopes that become complexed with class i or ii major histocompatibility complex proteins. the resulting complexes are transported to cell surface, where they are presented as 'non-self' entities to t-lymphocytes. if the viral antigen has not previously encountered the t-cell repertoire of the host, the initial antigen-specific activation event requires appearance of mhc-peptide complexes on antigen-presenting cells. but if activated t-cells, previously sensitized to the viral epitopes are available, then a broader class of antigen-presenting cells can be targeted for clearance by cytotoxic t cells. in both events, the ability to discriminate self molecules from the viral epitopes depends on the presentation of the non-self peptide to t-cells in specific peptide-binding grooves of the mhc molecules on antigen-presenting cells. mcfadden and kane ( ) summarized how dna viruses perturb the mhc and alter immune recognition. a number of gene products of dna viruses have been identified as directly affecting mhc expression or antigen presentation, whereas rna viruses interact with mhc by indirect mechanisms. most dna viruses are able to modulate cellular immunity. it seems that many viral gene products remain to be identified among the open reading frames of as yet unknown function that exists in these viruses. besides a trivial strategy of hiding dna molecules in cells, such as neurons that lack mhc surface molecules, viruses can modify mhc expression directly within cells or indirectly at the level of cytokine regulation. there is now evidence that viruses can combat antiviral effector t cells directly by blocking their antiviral activity (bertoletti et al, ) . in humans infected with hiv- and hepatitis b viruses, naturally occurring variants of epitopes recognized by cytotoxic t lymphocytes may act as antagonists in vivo because the corresponding peptides prevent a cytotoxic t cell response. although exactly how the antagonists function is not known, it is evident that the presence of these antagonists prevents the t cell from performing its function. endogenous viruses represent a separate phenomenon with regards to the immune system. as a rule, the host is completely immunologically tolerant to endogenous viruses. however, antibodies against endogenous retroviruses were found in mice (miyazawa et al, ) . how the immune system makes antibodies against endogenous retroviral gene products is unknown but this ability may relate to the expression of such genes after the establishment of immunological tolerance to endogenous retroviral antigens expressed earlier in life (miyazawa and fujisawa, ) . a similar delay in expression of the endogenous viral gene ev- has been described in chickens (crittenden, ) and may serve as a model for construction of similar 'self-vaccinating' transgenes in the future. given the potential benefits that can be derived from the use by the host of parts of a pathogen's genome to induce resistance, the paucity of pathogen-mediated resistance mechanisms in nature is surprising. the situation begs the question whether evolution exhausted all such possibilities in the development of host defences. why did certain mechanisms develop and others not? a reason for the absence or rare occurrence of pathogen-mediated defence mechanisms may be that they encompass some disadvantage for the host. one example in which a viral genome has become an integral part of the host are endogenous proviruses found in germ cells of all vertebrates. for example, in the laboratory mouse endogenous proviruses occupy more than . % of the cellular dna (pincus et al, ) . in the genomes of chickens, there are several families of retrovirus-related permanent insertions. in the most thoroughly studied family of endogenous viral genes, there are more than endogenous proviruses in various parts of the genome (crittenden, ) . the presence of some of these proviruses may interfere in the spread of the generally non-pathogenic endogenous virus produced by other such proviruses. however, the endogenous proviruses do not protect the host against infection with similar but more harmful, pathogenic exogenous viruses. on the contrary, the antigenic similarity between the products of the endogenous proviruses and the exogenous viral antigens reduces the ability of birds with certain types of these proviruses to mount an immune response against the exogenous virus (crittenden et al, ; gavora et al, b) . a possible reason why other endogenous proviral sequences did not evolve as resistance mechanisms is that their expression may adversely affect important physiological processes of the host (gavora et al, a,b) and reduce the ability of the host to resist the exogenous analogues of the proviruses. genetic variation is a prime prerequisite for genetic change by selection. as a general rule, genetic variation exists in the ability of livestock to tolerate infectious diseases. and it was this variation that allowed populations of domestic animals and birds to survive under continuous exposure to rapidly evolving disease agents. before domestication, disease resistance of today's livestock species was influenced by natural selection and the current status of variable resistance to multiple disease agents can be considered to be the result of a response to the selection pressure of multiple pathogens. as a consequence of domestication, a significant new element that entered this evolutionary system was artificial selection for characters that benefit humans as users of livestock. simultaneously, housing conditions evolved towards increased concentration of animals and birds and thus provided opportunities for spread of pathogens. improved disease prevention and control measures now provide some compensation for the larger population sizes used in current production systems. selection for disease resistance plays a relatively minor but increasingly important role in livestock improvement. the choice of selection criteria and the emphasis they receive in the context of total selection pressure available to a practical breeder are decided by market demands and economic considerations. disease resistance traits receive attention from the breeders mainly when a specific disease is a major cause of economic loss. although in most instances existing genetic variation provides an adequate basis for resistance selection, selection may not always be practised. such selection is expensive because the expression of resistance traits requires exposure of selection candidates or their relatives to the disease agent. this is why industries prefer to look for indirect selection techniques that do not require pathogen challenge. recent developments in gene mapping provide good prospects for progress in this direction. indirect selection for resistance to the herpesvirus of marek's disease in chickens, by increasing the frequency of the 'resistant' major histocompatibility haplotypes, is one example of such a technique. it has been practised by most of the world's poultry breeding companies over the past two decades (gavora, ). conventional procedures for direct and indirect selection for disease resistance will in the foreseeable future be the main route for genetic improvement of disease resistance. one disadvantage of their application is the general absence, with rare exceptions mentioned above, of genetic variation in resistance to infection. thus genetic improvements in disease resistance by conventional means lead mostly to better resistance of livestock to disease developmenta situation where the organism becomes infected but tolerates the pathogen and reduces its ill effects. hence development of new genetic mechanisms that prevent entry of a pathogen into the host, or otherwise substantially improve the position of the host in the pathogen-host interaction is justified. while conventional selection leads to quantitative improvement of resistance, the new mechanisms would represent a qualitative change that, at least in some instances, will justify the large effort and cost. the expenses will be further justified if the new, engineered mechanism proves to be stable and remains effective despite evolution of the pathogen and functions without harmful effects on the animal's production capacity. improvement in the welfare of the modified livestock will be an automatic, additional benefit. in crops despite large differences between animals and plants, sufficient similarities exist in their resistance mechanisms to justify examination of the situation in plants with regards to genetic engineering of viral resistance. for example, normal virus replication requires a subtle balance of virus and host coded proteins, present in critical relative concentrations at specific times and locations. therefore, wilson ( ) suggests that any unregulated superimposition of protein or nucleic acid species interacting with the virus can result in plants in an apparently virusresistant phenotype. the results from experimentation with animal cells into which a viral gene was inserted indicate that a similar situation may also exist in animals (gavora et al, ) . the idea that viral components contained in plants might interfere with virus infection was first proposed well before gene transfer techniques became available (hamilton, ) and the concept of pathogen-derived resistance was first put forward in a formal statement by sanford and johnston ( ) . there are several approaches to the introduction of disease resistance by gene transfer in plants (fitchen and beachy, ) . they include transfers of segments of viral genome encoding capsid or coat proteins, viral sequences encoding proteins that may be subunits of viral replicase, sequences incapable of encoding proteins, entire genomes of defective, interfering viruses, and complete genomes of mild virus strains. the transgenes may act on initiation of infection, replication of virus, spread of infection throughout the plant, and symptom development. the level of protection derived from the transgene ranges from low to high and its breadth of host range from broad to narrow. the available data are not sufficient to firmly establish the molecular mechanisms of the protection. in general, although a viral sequence may confer resistance in one virus-host system, an analogous sequence from a different virus in another virus-host system may not be effective. the conceptual simplicity of the approach and availability of virus coat gene sequences facilitated broad implementation of this strategy. fichten and beachy ( ) list published examples of this approach. it is unlikely that a single mechanism accounts for the observed resistance of the transgenic plants but regardless of the mode of the transgene action, resistance results from a block in an early event in the infection process (fichten and beachy, ) . in resistance to some viruses other than tobacco mosaic, it seems that accumulation of the coat protein transgene rna, rather than the virus coat protein itself is responsible for resistance. resistance has been observed even in plants that transcribed a translation-incompetent coat protein mrna (kawchuk et al, ; de haan et al, ) . it seems that even in the absence of understanding of its mechanism, the strategy can be extended to other plant species and viruses. protection by sequences encoding replicase-related proteins replicase-mediated resistance was first demonstrated against tobacco mosaic virus (golemboski et al, ). the number of initially infected cells in transgenic and non-transgenic plants was the same but virus replication was markedly reduced in cells of the transgenic plants. replication of the virus was severely impeded and little or no systemic spread of the virus occurred (carr and zaitlin, ) . protection by the accumulation of rna plants were protected by rna-mediated resistance to a degree comparable to protein-mediated resistance. transgenic tobacco plants, carrying a translationally defective tomato spotted wilt virus nucleocapsid gene exhibited resistance similar to that in experiments with translationally competent gene constructs (de haan et al, ) . other examples include potato plants with constructs producing sense and antisense transcripts of potato leafroll virus (kawchuk et al, ) and tobacco plants and similar transcripts of tobacco mosaic virus (powell et al, ) . protection by transgene copies of mild strains, satellites and satellite rnas, and defective interfering viruses transgenic tobacco plants carrying cdna of a mild strain of tobacco mosaic virus developed only mild symptoms when challenged with severe strains of the virus (yamaya et al, (gerlach et al, ) . nevertheless, this approach does not seem desirable because the transgenes may produce active pathogens by recombination or a pathogenic mixture. also, transgene components may recombine with another virus, thus extending its host range or virulence (fitchen and beachy, ) . the identification of a variety of disease resistance (r) genes is expected to facilitate identification and introgression of new resistance from wild species into new plant varieties. it is well known that a new resistant plant variety developed over a long time and with great effort is often overcome by a new pathogenic race an immensely wasteful situation. such breakdown of resistance is much less likely in varietal mixtures that carry an array of different r genes. once different r genes are cloned, varieties can be produced that consist of mixtures of lines differing only in the r gene allele they carry (staskawitz et al, ) . for genetically engineered resistance, pathogen-inducible promoters, such as the prpl-i promoter in potato (martini et al, ) may be the most advantageous as they induce the 'resistance' peptide(s) only in cells that are being challenged by a compatible pathogen (de wit, ) . the extent of the research effort to genetically engineer new resistance mechanisms in animals is much smaller than that in plants and available data on the subject are reviewed below. the first successful introduction of pathogen-mediated resistance to disease in animals was reported by salter and crittenden ( ) . they produced several lines of chickens, each with an insert of a recombinant avian leukosis retroviral genome at a different locus within the host genome. the transgenic birds that expressed only the viral envelope coding region of the recombinant genome were shown to be resistant to the corresponding subgroup of the avian leukosis virus (salter and crittenden, ; gavora et al, a) , due to a blockage of virus receptors by the viral envelope proteins. another introduction of a new virus resistance mechanism into a livestock host was attempted by clements et al ( (owens et al, ) . expression under the control of metallothionein of a single glycoprotein d gene from herpes simplex virus (hsv- ) rendered cells resistant to infection by hsv but not by other viruses (johnson and spear, ) . the mechanism of this resistance is not known but it seems likely that d interacts with a cell surface component required for viral penetration. in an attempt to introduce resistance to bovine rotavirus that causes calf diarrhea and results in large economic losses, two genes that code for rotavirus capsid proteins, implicated in early virus-host cell interactions, were transferred into the genomes of susceptible cells in culture and, one of the genes, also into genomes of laboratory mice (gavora et al, ) . the transgenes produced mrna of the relevant viral genes but no corresponding protein was detected either in the cells or in the mice. nevertheless, several of the transformed cell lines showed significantly increased resistance to bovine rotavirus (gavora et al, ) , while no increase in the resistance of four similarly transformed lines of mice was detected following challenge of pups shortly after birth with the virus (js gavora, unpublished results). antisense rna although not yet tested in vivo, the use of antisense rna to combat viruses has received attention by researchers and presents another possible avenue for the construction of new resistance mechanisms. the possibilities of inhibiting retroviral replication by antisense molecules before its integration into a host chromosome has been demonstrated (to and neiman, ) . to block viral integration, antisense sequences can be designed to target regions essential in the synthesis of viral dna intermediates or viral integration. replication of a recombinant avian retrovirus, carrying a neomycin resistance gene neo' in the antisense orientation was blocked when cells expressed high levels of neo' rna molecules in the sense orientation, suggesting that antisense rna inhibition may be a useful strategy for inhibition of retroviral infections . it was hypothesized that when sequences immediately upstream of the polypurine tract are hybridized to antisense molecules, rnase h failed to process the rna sequences in the polypurine tract into a functional primer for the synthesis of plus-strand dna (to and neiman, ) . they suggested that an antisense segment in that region can be defined for use in a large number of pathogenic retroviruses. these experiments also showed that constructs expressing the antisense rnas can be delivered by replication-competent retroviral vectors to host cells in culture, thereby immunizing the host cells against superinfection with different retroviruses. the advantage of the antisense rna approach may be that only about basepairs are needed to bind the antisense rna with absolute precision to a unique mrna and intensive research is now under way to develop antisense therapeutics (bradley et al, ) . even though the mechanism will not prevent viral entry into host cells, it may prevent integration of the viral genome in the host chromosome. catalytic rnas, known as ribozymes, are not rare in nature and it is possible to engineer an intron that can repeatedly perform the first chemical step in the splicing process (parker et al, ) . ribozymes have been shown to cleave target rna and to inhibit mrna transcript activity (edington and nelson, ) . the principal advantage of ribozymes is their ability to cleave and thus inactivate multiple targets. even though ribozyme-mediated gene inhibition involves a mechanism (target cleavage) different from that of bacterial antisense rnas, many of the essential steps of the two mechanisms are identical. ribozymes were shown to successfully inhibit gene expression in xenop!s oocytes in tissue culture (cotten and birnstiel, ) and may be another possible approach to the engineering of new disease resistance mechanisms for livestock. transfer of resistance genes from another species as was mentioned above, the murine mxl is a protein with activity against influenza virus. garber et al ( ) inserted cdna encoding this protein into chicken embryo fibroblasts through the use of a replication-competent avian retroviral vector. cells infected with the vector were resistant to infection with avian, as well as human influenza viruses but susceptible to enveloped rna viruses. biological costs of and risks associated with genetic engineering conventional methods of genetic improvement are rather forgiving in the sense that they induce gradual changes and provide time for the breeder to correct disturbances in biological equilibria that might be harmful to the animals. gene transfer, on the other hand, may induce dramatic, undesirable changes that will disturb development or physiological functions that are difficult to correct. however, new technological developments, such as homologous recombination and use of embryonic stem cells for gene transfer will likely reduce the risks. given the extent of work on transfer of disease resistance-inducing genes in both plants and animals, surprisingly little research has been done on the possible physiological consequences of adding such new genes to cells. consequences of transgenes have been demonstrated in plants by hilder and gatehouse ( ) . they studied lines of transgenic tobacco containing a cowpea trypsin inhibitor gene construct which expressed the transgene at various levels and plants that possessed, but did not express, the gene. small, but in some instances, significant differences between the transgenic and non-transformed plants were found in various parameters but there was no additional difference between plants that expressed the transgene and those that did not. they concluded that although the transformation may have some small effects on non-targeted phenotypic characteristics, the expression of the transgene at high levels imposed no additional yield penalty on the plants. negative genetic correlations between disease resistance and production traits have been reported (eg, gavora, ) but their basis as to linkage or pleiotropy is not clearly established. design of genetically engineered resistance mechanisms may have to take possibility of such negative correlations with production traits into consideration. as mentioned above, a transgene that successfully induced resistance of chickens to avian leukosis retrovirus subgroup a in chickens (salter and crittenden, ) was shown to result in a sizeable reduction of egg production rate (gavora et al, a) . it was suggested that the reduced ovulation rate was due to interference of the viral envelope protein produced by the transgene with the attachment of the virus to host cells and also with transport of lipids into the developing egg yolk, since the virus uses an ldl receptor for entry into host cells (bates et al, ) . on the other hand, a transgene containing a gene for a capsid protein of bovine rotavirus in laboratory mice (gavora et al, ) was not associated with any significant effects on their growth and reproductive performance (j nagai and js gavora, unpublished results). hence, significant 'biological costs' may not always accompany insertion of transgenes but they need to be considered in strategies for genetic engineering of new resistance mechanisms. reports on work on assessment of risks involved in the production of varieties with new, genetically engineered resistance are only available for plants. transgenic plants expressing viral pathogen-derived dna sequences have been considered sites for hyperevolution of viruses through recombination of a mild or defective viral genome with the transgene (de zoetten, ) . however, there is no experimental evidence to confirm this supposition. on the contrary, evidence against this type of event exists through one to up to eight viral passages, even though heteroincapsidation of viral rna by transgenically expressed viral coat proteins has been observed (wilson, ) . the danger that transgenic crops may generate new viruses and diseases has been assessed by falk and bruening ( ) . they provide evidence that genomic recombination was observed when transgenic tobacco plants expressing a segment of cowpea chlorotic mottle virus genomic rna were inoculated with a mutant of the same virus that contained a deletion (greene and allison, ) . the important question is whether such recombination can produce dangerous new viruses. rna-rna recombination has indeed been demonstrated for four groups of rna plant viruses. the recombination occurs between closely related rna molecules, possibly at sites of similar rna structure. under usual crop production circumstances, opportunities exist for genetic interaction between plant viruses in mixed virus infections. since both crop plants and weeds may be present in a field, recombinations between a virus that cannot infect a plant and one that can, do not have a zero probability. nevertheless, mixed infections rarely result in new plant pathogenic viruses. instead, new viral diseases are usually due to minor variants of already known viruses. generally, however, existing viruses are stable, having to fit hosts that evolve only slowly. falk and bruening ( ) believe it is unlikely that recombinations between transgene rna and viral genomic rna will occur at greater frequencies than the recombinations already occurring between virus genomic rnas in natural infections. in the past, development of resistant plants by traditional breeding fostered the emergence of virulent virus strains (dawson and hilf, ) but the cost of this phenomenon is much less than the cost of abandoning plant breeding. similarly, the benefits of engineered plant resistance genes far outweigh the vanishingly small risk of creating harmful new viruses in significant excess over those being created by natural processes (falk and bruening, ) . in mice, endogenous proviruses are known to recombine with exogenous viral sequences to give rise to novel viruses with unique properties (pincus et al, ) . similar recombinants between exogenous and endogenous avian retroviruses had been produced in vitro and used as transgenes to induce resistance to the exogenous retrovirus in chickens (salter and crittenden, ) . endogenous viral genes may be regarded as prototypes of transgenes in animals. early evidence that rous sarcoma virus recombined with envelope protein of endogenous avian virus was provided by hanafusa et al ( ) . recently, an env gene related to endogenous viral gene was found on the exogenous avian leukosis virus subgroup j (bai et al, ) . there is also evidence that the alv transgene that expresses the avian leukosis virus subgroup a envelope can recombine with endogenous virus from gene ev to produce subgroup a infectious virus (lb crittenden, personal communication). until more results become available in animals, we could assume that a situation similar to that described above for plants will also exist in livestock. however, it is imperative to keep the possible risks in mind in designing strategies for induction of resistance by genetic engineering and to experimentally assess the recombinations, if any, between transgenes and existing viruses in farm animals and birds. an example of an increase in the virulence of an animal virus that may be associated with improved resistance of the host by vaccination and genetic means is the emergence of highly virulent marek's disease herpesviruses in chickens (witter, ) . the viruses may have emerged as a consequence of vaccination and conventional selection for resistance that included efforts to increase the frequency of major histocompatibility haplotypes associated with such resistance. genetically engineered resistance may provide a more stable solution to the marek's disease problem. conventional breeding and vaccination improved survival of chickens infected by marek's disease virus. however, the virus continues to be present in vaccinated birds so there are ample opportunities for its mutations towards higher virulence. a genetically engineered mechanism that would prevent the entry of the virus into the host cells would reduce the size of the viral population and thus reduce the possibility of such viral evolution. unfortunately emergence of viral mutations to overcome the genetically engineered barrier to virus entry would be difficult to eliminate. it seems that the arguments used by plant breeders in favor of continuing research toward new, engineered resistance genes should also be valid for livestock. a necessary prerequisite for this development has to be an adequate system of controls and thorough testing of the engineered livestock. as mentioned above, any introduction of new genetic material into a cell carries with it a risk of disrupting cell functions. this risk has to be kept in mind in the design of new resistance mechanisms. it may be possible to minimize such risks on the basis of a thorough understanding of the physiology of virus-infected animals and interactions between the virus and the host. another, no less important aspect of the design of new resistance mechanisms is their long-term stability. the new mechanism may become ineffective through evolution of the virus which will overcome the resistance provided by the transgene. evolution of pathogen virulence genes that overcame resistance induced by conventional breeding is well known and documented in plants (flor, ; wilson, ) , and a possible instance of a similar phenomenon observed with marek's disease herpesvirus in chickens was mentioned above. the design of new mechanisms and strategies of disease resistance to be introduced into livestock by genetic engineering techniques is a search for mechanisms that did not, for whatever reason, develop by evolution. unlike most of the mechanisms of defence of the hosts against viruses that resulted in virus tolerance by the host, the ideal goal of the new, engineered mechanisms should be prevention of viral entry into host cells. it may be easier to develop new resistance strategies for viruses which depend for most of their functions on the host cell than for those that provide for the functions in their genome. new techniques of molecular and cell biology allow transfers of genes between species, taxonomic genera and even kingdoms so that we are no longer limited by the constraints of sexual compatibility. recent progress in the development of techniques of homologous recombination, together with the use of embryonic stem cells for gene transfer provide good prospects for progress in this area of research (first et al, ) . while the use of both of these techniques is now routine in laboratory mice, their application in animal agriculture is hampered by the unavailability of a reliable technique for the production of embryonic stem cells in any of the livestock species. nevertheless, given the high level of interest and scientific activity in this area in several countries, it is likely only a matter of time before embryonic stem cells will become available for introduction of new genetic information into the genomes of farm animals and birds. homologous recombination and use of embryonic stem cells will allow insertion of a transgene in a predetermined location in the genome. in the case of gene constructs designed to induce new resistance mechanisms, the insertion will likely be targeted into a 'neutral' region of the genome, to minimize the potential disruption of important genomic functions. after successful insertion, it will be possible to test the transformed embryonic stem cells in culture for the expression of the transgene, its stability and, as much as possible, its undesirable effects on the cells. preliminary testing in cell culture for resistance to the pathogen in question will be also possible. only the embryonic cell lines that will meet criteria of acceptability in the above tests will be used for the introduction into developing embryos with the goal of producing disease resistant transgenic individuals. it is anticipated that the protocol will make the introduction of new disease resistance mechanisms into livestock less expensive. the approach will also be less risky as the dangers of disruption of important genetic mechanisms by the transgene insertion will be reduced by gene targeting. moreover, the reduction of such risks will make the research more acceptable for both livestock producers and the general public. unfortunately, the use of advanced techniques of gene transfer will likely be limited to developed countries. because of their relative simplicity and small size, the genomes of viruses are generally better understood than those of host cells. many viral genomes have been sequenced and it is generally easy to obtain the necessary sequence information for viral genes that are candidates for inclusion into potential resistance-inducing transgene constructs. the general principles for the design of new resistance mechanisms and the new defence strategies can be summarized as follows. the most useful would be mechanisms based on an element common to the life cycle of multiple viruses thus inducing resistance simultaneously to more than one virus. the new mechanisms should be designed to minimize their biological and financial costs. targeting of transgenes into 'neutral' regions of the genome may be one such strategy. the 'neutrality' of such regions can be tested by inserts of non-functional genes. the regions proven to be 'neutral' would be subsequently used for inserts of resistance genes. ideally the functioning of the new mechanisms should be triggered by the presence of the inducing virus, otherwise the mechanism should remain 'silent'. this type of mechanism would minimize its biological cost to the host. despite preliminary testing of transformed cells in culture, it will be essential to subject livestock carrying the resistance transgenes to a series of rigorous tests gama et al, ) . the tests need to prove the genetic potential of the new stock for economically important production traits, general viability, as well as resistance against the disease for which the transgene was designed. in instances of slight impairment of the production capacity of the transgenic, compared to the original stock, decisions on the practical usefulness of the modified animals will depend on comparison of the economic benefit derived from the transgene against the cost of the animals' reduced production performance. in this context, the prevalence of the pathogen in question and the damage it causes in the production areas for which the resistant animals are intended will be, no doubt, important considerations. based on considerations of the viral life cycle, and natural and genetically engineered resistance mechanisms that were already tested, several possible strategies can be proposed and are listed below according to stages of viral life cycle. the strategies are identified in a general manner, without reference to specific viruses. therefore, no description of details of their design and implementation is attempted. the aim of this list is to stimulate further activity in this area by outlining the opportunities that exist. without a doubt, a new resistance mechanism that would prevent viral attachment and penetration into host cells represents the most desirable approach. those acting on subsequent phases of viral life cycle are less desirable and should be considered if prevention of viral attachment and penetration is impossible. viral attachment and penetration into host cell transgenes that produce viral antireceptor (virion surface) proteins to block cellular receptors; -produce soluble receptors or their components to block virion surface proteins and prevent their interaction with cellular receptors; -replace host receptor genes by a modified form that is able to perform the receptor's physiological function but does not allow the attachment of the virus; -produce substances that interfere with viral penetration into host cells. transgenes that induce antisense rna to a part of the viral genome crucial for virus multiplication; -cause multiplication and accumulation of viral or modified viral rna in host cells; -disturb viral replicase or its function; -produce ribozymes attacking viral rna; -produce a defective viral protein that competes with the normal one to produce a high proportion of non-infectious virions. transgenes that induce and maintain a latent state of the virus; -do not allow activation of a virus from its natural latent state. transgenes that protect against perturbances of the host's immune system; -produce the vaccinating antigen only after the immune system is fully developed (self vaccinating transgenes). conclusions enormous variability of viral types in their strategies for life and survival will likely make it difficult to engineer generalized resistance to viruses. in their evolution, some viruses have developed strategies that do not harm the host sufficiently to cause extinction of the host -and the virus. nevertheless, in some instances virus-host coevolution has resulted in disease-producing relationships that cause economic losses and suffering of the animals and birds. conventional breeding methods will remain the principal approach to the improvement of disease resistance in livestock but in some instances, introduction of new genetically engineered resistance mechanisms may be justified. prerequisites for the design of new resistance mechanisms include good knowledge of the viral genome and life cycle (keeping to a minimum the biological cost of the new strategies to the host) and of the probability that the strategies will be overcome by viral evolution. a combination of gene targeting techniques with embryonic stem cells, when such cells become available for livestock, will greatly facilitate the introduction of new, genetically engineered virus resistance. all livestock with new resistance mechanisms will have to be subjected to thorough testing. there are several possible strategies for the development of new resistance mechanisms in livestock. the transgenes to be designed for such strategies can act at various phases in the viral life cycle. ideally, expression of the transgenes should be triggered by the presence of the inducing virus, otherwise the resistance mechanism should remain 'silent'. strategies that prevent viral entry to the host are expected to be most valuable as they could eliminate all damage to the host caused by the virus. hprs- (exogenous avian leukosis virus, subgroup j) has an env gene related to those of endogenous elements eav-o and e and an e element found previously only in sarcoma viruses isolation and characterization of a . kilobase-pair cdna fragment encoding the binding domain of the bovine leukemia virus cell receptor attachment of sa- rotavirus to erythrocyte receptors varmus he ( ) a receptor for subgroup a rous sarcoma virus is related to the low density lipoprotein receptor the cloned receptor for subgroup a avian leukosis virus maps to tva * s, the dominant gene for susceptibility to subgroup a virus natural variants of cytotoxic epitopes are t-cell receptor antagonists for antiviral cytotoxic t-cells vaccinia virus -kilodalton protein: relationship to several mammalian proteins, including two growth factors human proto-oncogene c-jun encodes a dna binding protein with structural and functional properties of transcription factor ap- genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues antisense therapeutics vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor long terminal repeat (ltr) sequences, env, and a region near the ' ltr influence the pathogenic potential of recombinants between rous-associated virus types and erythrocyte p antigen: cellular receptor for b parvovirus resistance to parvovirus b infection due to lack of virus receptor (erythrocyte p antigen) a sialoglycopeptide from human erythrocytes with receptor-like properties for encephalomyocarditis and influenza viruses resistance in transgenic tobacco plants expressing a nonstructural gene sequence of tobacco mosaic virus is a consequence of markedly reduced virus replication does the beta-andrenergic factor receptor function as reovirus receptor? development of transgenic sheep that express the visna virus envelope gene human coronavirus oc interacts with major histocompatibility complex class i molecules at the cell surface to establish infection ribozyme-mediated destruction of rna in vivo influence of endogenous viral (ev) gene expression and strain of exogenous avian leukosis virus (alv) on mortality and alv infection and shedding in chickens retroviral elements in the genome of the chicken: implication for poultry genetics and breeding host-range determinants of plant viruses characterization of rna-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants determinants essential for the transmissible gastroenteritis virus receptor interaction reside within a domain of aminopeptidase n that is distinct from the enzymatic site molecular characterization of gene-for-gene systems in plant fungus interactions and the application of avirulence genes in control of plant pathogens risk assessment: do we let history repeat itself? determination of the rate of base pair substitution and insertion mutation in retrovirus replication utilization of ribozymes in plants. plant viral resistance will transgenic crops generate new viruses and new diseases? early virion-associated suppression of cellular protein synthesis by herpes simplex virus is accompanied by inactivation of mrna epstein-barr virus receptor of human b lymphocytes is the cd receptor cr systems for production of calves from cultured bovine embryonic cells genetically engineered protection against viruses in transgenic plants the complementary genetic systems in flax and flax rust molecular mimicry: virus modulation of the immune response transgene effects, introgression strategies and testing schemes in pigs avian cells expressing the murine mxl protein are resistant to influenza virus infection 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plants of an extra gene members of the low density lipoprotein receptor family mediate cell entry of a minor group common cold virus trans-dominant inhibition of human immunodefficiency virus type rev occurs through formation of inactive protein complexes cellular expression of murine leukemia virus gp -related antigen on thymocytes of uninfected mice correlates with fv- gene-controlled resistance to friend leukemia virus infection inhibition of host protein synthesis and degradation of cellular mrnas during infection by influenza and herpes simplex virus herpes simplex virus glycoprotein d mediates interference with herpes simplex virus infection sense and antisense rna-mediated resistance to potato leafroll virus in russet burbank potato plants t-lymphocyte t molecule behaves as the receptor for human retrovirus lav virus-host-cell interactions haller ( ) inhibition of influenzal viral mrna synthesis in cells expressing the interferon-induced mx gene proct genetic control of host 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identification and chemical synthesis of a host cell receptor binding site on hepatitis b virus control of cellular and viral transcription during adenovirus infection cell killing by simian virus : variation in the pattern of lysozomal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus -transformed simian cell lines the cellular receptor for gibbon ape leukemia virus is a novel high affinity sodium dependent phosphate transporter rous-whipple award lecture: viruses and diseases of the twentieth century adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominantnegative mutant proteins bind to the viral replication origin ribozymes: principles and designs for their use as antisense and therapeutic agents genetics of cell receptors for avian leukosis viruses the interplay of microbes and their hosts regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thimidine kinase with alpha gene promoters protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense rna host susceptibility to endogenous virus: defective glycoprotein-expressing proviruses interfere to infections multiplication of viruses inhibition of translation of poliovirus: inactivation of a specific initiation factor artificial insertion of a dominant gene for resistance to avian leukosis virus into the germ line of the chicken the concept of parasite-derived resistance-deriving resistance genes from parasite's own genome protease activation mutants of sendai virus. activation of biological properties by specific proteases enhancers and eukaryotic gene transcription blocking of hiv- infectivity by a soluble, secreted form of the cd antigen on the use of transgenes in livestock improvement heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus interferon-regulated influenza resistance gene m! is localized on mouse chromosome molecular genetics of plant disease resistance virus evolution inhibition of retroviral replication by antisense rna the potential for effective antisense inhibition of retroviral replication mediated by retroviral vectors constitutive expression of the nef gene suppresses human immunodeficiency virus type (hiv- ) replication in monocyte cell lines pathogenesis of viral infections antigens and structure of the adenovirus a critical site in the cell surface receptor for ecotropic murine retroviruses required for amino acid transport but not for viral reception herpes simplex genes: the blueprint of a successful human pathogen the molecular biology of the gene structure of the influenza virus hemagglutinin complexed with its receptor, sialic acid strategies to protect crop plants against viruses: pathogen-derived resistance blossoms very virulent marek's disease viruses: importance and control cross protection of transgenic tobacco plants expressing a mild strain of tobacco mosaic virus mouse hepatitis virus receptors: more than a single carcinoembryonic antigen the author wishes to express his gratitude to the institut national de la recherche agronomique, for the provision of a pleasant, friendly, and stimulating working environment at the laboratoire de g n tique factorielle at jouy-en-josas, france, where he compiled this review during a six month stay in . helpful comments and suggestions were provided during the preparation of the manuscript by lb crittenden, ri hamilton, and jl spencer. key: cord- - iqpl p authors: mackay, ian m.; arden, katherine e. title: rhinoviruses date: - - journal: viral infections of humans doi: . / - - - - _ sha: doc_id: cord_uid: iqpl p picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide. hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons. aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing and with significant direct and indirect healthcare expenditure. ari incidence is highest in the first years of life, with up to thirteen episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month. picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide [ ] . hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons [ , ] . aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing [ ] and with signifi cant direct and indirect healthcare expenditure [ , ] . ari incidence is highest in the fi rst years of life, with up to episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month [ , - ] . in preschool-aged children, nearly % of general practitioner visits are for ari [ ] , many of which are self-limiting. aris can often be managed in the community with supportive care from parents, but complications can arise that require a medical visit for management of asthma, otitis media, or sinusitis [ ] . hrvs replicate in nasal cells, sinus cells, bronchial epithelial cells (becs) [ , ] , and smooth muscle cells [ ] but not in monocytes [ ] or dendritic cells (dcs) [ ] . the infl ammatory immune response they trigger very soon after infection has its greatest impact in the young, the elderly, those with asthma or chronic obstructive pulmonary disease (copd), and in the immunocompromised. first infections usually elicit a stronger response. antiviral interventions have been under development for decades; to date most have met with varying degrees of failure or unacceptability. vaccines have been considered unachievable because of the large number of diverse and distinct viral types. there are classically defi ned and recognized hrv serotypes grouped into two species, hrv-a and hrv-b, and a recently defi ned third species, hrv-c, containing more than genotypes identifi ed and characterized entirely by molecular means. their cousins, the four enterovirus species (ev-a, ev-b, ev-c, and ev-d), are also found in the airways at times. most systematic and mechanistic studies of hrv etiology and pathogenesis have been informed by studies in adults, mostly prior to the discovery of hrv-cs. adults exhibit reduced symptoms from hrv infections because of prior exposure and the resultant protective immune memory which that imparts (see sect. . ). furthermore, many modern studies ( ) draw conclusions about lower respiratory tract (lrt) disease using urt specimens and ( ) infrequently sample, doing so across small cross sections of time. these limitations have hampered attempts to associate virus detection and disease. current thinking is that hrv-cs may be key players in asthma exacerbations although our inability to culture them routinely has hindered our progress in understanding their role. the impact of the hrvs has been underestimated for decades, and the concept of the hrvs as a very large assemblage of genetically, immunogenically, antigenically, and temporally distinct and stable viral entities remains rare; they are more commonly considered a single variable virus, a view that science does not support. the disease most commonly associated with the airways and resulting from hrv infection is the common cold, a selflimiting coryzal illness [ - ] . the term dates back to ancient greece, but evidence that the syndrome and asthma, another disease most frequently due to hrv infection, has been with us since ancient times can be viewed in writings on the ebers papyrus, a medical document written in the sixteenth century bc [ , ] . in the common cold was considered either to be due to exposure to the elements or to infection by bacteria [ ] . it was later understood to be largely due to something in bacteria-free fi ltrates, and so the search for viral causes began [ , ] . the common cold unit (ccu) was established in salisbury, uk, to seek solutions to the mysteries of the common cold, mostly through adult volunteer infection studies and careful systematic science [ ] . the ccu functioned for years ( - ) , and it was here in that the fi rst in vitro culture of an hrv was achieved using lung tissue from a particular embryo ( fig. . ) [ , ] . propagation failed once this tissue was expended [ , ] . once hrv isolation was possible, viral serotyping developed and culture techniques were further refi ned. this leads to an international effort to characterize and name the hrvs [ - ] . in renewed interest in hrv research was triggered by the description of a distinct clade of hrv types [ ] found using molecular typing. the resultant fl urry of hrv research raised questions about many earlier paradigms of rhinovirology and of the role of established respiratory viruses in aris. the novel clade was proposed as a new species, hrv-c, which was taxonomically confi rmed in [ - ] . prior to the discovery of the hrv-cs, the genus rhinovirus had been abolished and the hrv-a and hrv-b species assigned to the genus enterovirus within the family picornaviridae [ ] . the hrv-cs have been assigned a new naming scheme based on genetic sequence in the absence of antigenic or serological data. while the sequencing of all serotyped hrv genomes was completed in , few of the hrv-cs or apparently novel hrv-as or hrv-bs have been similarly characterized, so the full spectrum of hrv genomes, the rhinovirome, remains incomplete. in this chapter we have described individual serotyped hrvs as the "classical" types, a type being the description for a single, genetically stable, stand-alone hrv. methods for epidemiologic analysis the original clinical defi nition of an hrv infection was written using data from cell and tissue culture and adult human infection studies. after in vitro isolation methods employed a virus interference test to more easily determine successful isolation; cultures suspected of infection with an uncharacterized hrv prevented infection by another, readily titratable virus [ ] . later, price ( ; the jh strain) and then pelon and co-workers ( ; , strain) developed culture systems that permitted hrv replication to be more easily identifi ed [ , ] . the early hrvs were initially classifi ed as echoviruses (echo ; later hrv- ) [ ] . at the same time, propagation of the hgp (hrv- ) strain resulted from using increased acidity, lowered cultivation temperatures, and constant motion (rotation) [ , ] . despite the challenges [ ] , virus isolation was a more sensitive indicator of infection than an antibody rise in paired sera [ ] . it was found that several cell lines and methods were required to encompass virus concentrations ranging from to tcid /ml [ - ] and growth differences among the different virus types. additionally, cell age after plating (< h), inoculum volume (relevant to the culture vessel), medium ph ( . - . ), and cell density were important factors for the reproducible appearance of hrv-induced plaques and for higher virus yields [ - ] . the hrvs can grow at temperatures above °c (some prefer that under certain conditions) [ ] , but rolling at °c, preceded by a - -h stationary incubation period [ ] , has historically provided the highest yield and fastest in vitro hrv growth [ , , , ] . serodiagnosis grew increasingly impractical as the number of serotypes increased [ , ] . however, antibody-based methods were essential for type-specifi c neutralization of infection [ ] from which early epidemiology data were derived and around which the hrv nomenclature system evolved in [ ] . the fi rst classical strains were officially named in [ ] , the last in [ ] . today we know that cell culture-based methods are unreliable for accurately representing respiratory virus epidemiology; although enhanced by immunofl uorescence, they are still used [ ] . the hrv-cs have not been successfully cultured in any cell lines or primary cell culture, although many attempts have been described [ , - ] . in hrv-c and w (another hrv-c) were shown to grow using organ culture [ ] . sinus tissue hosted increasing levels of viral rna, as did adenoid, tonsil, and nasal polyp tissue, but much less effectively, as measured by in situ hybridization [ ] . the sinus organ culture system also allowed testing of the fi rst reverse engineered hrv-c (pc ) [ ] . isolation identifi ed hrvs in ~ % of adults with aris, associated with . illnesses per year [ ] . because culture is ineffi cient and subjective and requires expertise, even for the culturable hrv types, it is becoming an art lost to clinical laboratories the world over. it is unsurprising that pcr-based methods now prevail, providing a much improved understanding of the nature and scope of hrv infections. the virological and immunobiological cost of this improvement is a paucity of low passage "wild" hrv isolates to work with; thus, many research fi ndings from recent years have employed easy to grow highly passaged and adapted hrv isolates. the impact of virus adaptation on the reliability of data from use of such viruses is unknown. pcr-based assays have dramatically increased the frequency of hrv detection [ - ] . the improved sensitivity and reduced turnaround time have shown that hrvs, as a group, are usually the predominant viruses in ari cases [ - ] . with reliable detection levels that extend from as few as tcid /sample to well above clinically relevant loads, pcr can detect virus levels which are commonly shed during all stages of experimental infection studies [ , ] . the common understanding of the systemic [ - ] or symptomatic [ , ] context of hrv detections was established during the era of culture detection, and pcr has challenged these paradigms by detecting virus more often than culture. hrvs are sometimes found in "healthy controls"; however, it is likely that with more thoughtful defi nitions of "healthy," these detections would reduce. it is not uncommon to experience a feeling that one is "coming down" with something that never develops further. this is likely due to a transient infection or reinfection by an hrv or other respiratory virus that is eliminated quickly by the host response. it is possible to correlate viral nucleic acid load at the sampling site with disease severity; however, this is made diffi cult by the highly variable sampling effi ciency of respiratory tract specimens which only permit the generation of reliable quantitative pcr (qpcr) data if serial specimens are available [ ] . the ′ untranslated region (utr; figs. . and . ) is the most common target for diagnostic oligonucleotides since the fi rst hrv rt-pcr in [ ] , and the region has retained relevance for virus detection by its adaptation to reverse transcriptase real-time methods (rt-rtpcr) [ , , , , - , , - ] . the ′utr is comprised of a number of conserved sequence "islands" (fig. . ) that permit the robust detection of the majority of hrvs and those "respiratory evs" which can be regularly detected in the respiratory tract [ , ] . the detection of respiratory evs in no way detracts from the importance of supporting clinical decision making using these assays. however, repositioning [ ] . the pcr primers of broadly reactive conventional rt-pcr [ , ] and rt-rtpcr [ , ] assays are shown these primers or changing the method of employing them [ - ] may undermine assay performance, as evidenced by predicted hybridization mismatches, uncommonly low detection frequencies [ ] , and by comparison of multiple primer sets using the same specimens [ ] . the addition of an oligoprobe rtpcr method increases amplicon detection sensitivity and specifi city, identifying -fold fewer tcid /ml or fold fewer genome copies than agarose gel detection of amplicon [ , , ] . other molecular tools, capable of detecting multiple targets, have evolved in recent years [ , , - ] , and some have gone on to be approved for clinical laboratory use [ ] . microarrays can detect thousands of viral targets, but are expensive for routine use (usd - per sample) and not sensitive enough to avoid a pre-hybridization pcr amplifi cation when using clinical specimens. at their most robust, microarrays, like pcr, rely on the existence of conserved regions of sequence to detect unknown viruses allowing them to detect previously unknown hrv types [ ] . highthroughput or "deep" sequencing platforms have become less expensive and more readily available, and they have succeeded in fi nding new diversity within the hrv species [ ] . the experiments remain costly so have not yet found a place for regular screening tasks and remain coupled to a need for pre-pcr steps. rapid protein-or virion-based assays are not (yet) adequately sensitive [ , ] . because of the high number of hrvs and the high frequency of infections, genotyping methods have become an essential accompaniment for understanding hrv epidemiology. nucleotide sequencing of the vp , ′utr+vp +vp (called hereafter vp /vp ), or ′utr region has replaced traditional serological methods, because of its speed and need for fewer specialized reagents compared to serotyping. vp yields the most comprehensive subgenomic genotyping information and is essential for the minimal defi nition of a new hrv type [ ] . the vp /vp region (fig. . ) is considered easier to use because it encompasses suffi cient genetic diversity to confi rm the identity of a clinical hrv type while also providing broad enough sensitivity to amplify the ~ hrvs from a challenging biological substrate, clinical specimens [ ] . screening of airway specimens for hrvs is not routine [ ] due to factors including cost and the perceived low clinical relevance of detection. genotyping is mostly relegated to research facilities. because of this, hrv molecular epidemiology studies tend to be smaller and focused on a specifi c disease or research question. most in-depth molecular studies of hrv replication have focused on a single hrv type. generally, it is presumed that results can be extrapolated to the other hrv types and to the in vivo situation. hrvs replicate in the cytoplasm (fig. . ) [ ] with membrane-associated replication structures containing double-stranded rna (dsrna) replicative intermediates (ri) which are formed in cells h after infection [ , ] . single-stranded infectious rna forms after ris start to accumulate [ ] . genomic rna (plus strand) is the template for complementary minus strand synthesis which in turn is the template for new genomic plus strands that become incorporated into virions [ ] . virions are synthesized from to h after infection and reach maximum release levels at - h [ ] . hrv replication in epithelial cells may shut off host cell transcriptional activity via direct cleavage of transcription factors and nuclear pore complex components. protease a ( a pro ) of hrv-b may directly cleave eukaryotic initiation factor g (eif g) when bound to eif e [ , ] . the eifs have key roles in initiation and rate control of host cell translation [ ] . host cellular protein production is virtually replaced by hrv-b proteins after only h of infection [ ] . hrv-b -infected cells also display reduced nuclear importing and degraded nuclear pore complex (npc) components [ ] . this may represent another hrv strategy for limiting the host response by preventing or reducing key signaling pathway molecules (e.g., irf- , stat , nf-κb) and shutting down host cell protein synthesis. protease c ( c pro ) from hrv-a targets the nucleus and can disrupt active and passive nucleocytoplasmic transport [ , ] . recombinant a pro protein from hrv-a ,hrv-a , hrv-b , hrv-b , hrv-c , and hrv-c exhibited differing specifi cities and kinetics against eif g as well as npc components demonstrating functional diversity between hrv types [ ] . this fi nding underscores the functional diversity within the hrv species and the risk of extrapolating too greatly from the study of single hrv types. it is apparent from a wealth of immunobiological data that hrvs still effi ciently trigger a proinfl ammatory immune response that has considerable clinical impact among at-risk groups, and that their putative interruption of host cell machinery does little to hinder this. the virion encapsulates an approximately kb positive sense rna genome (fig. . ), which tends to be more adenine and uracil (a+u) rich than the ev genome [ ] . in particular, a+u more frequently occupies the third or "wobble" genome replication in association with membranes produces the viral polyprotein which is co-and posttranslationally processed by a pro and c pro into the proteins ( p -p ) and structural peptides ( vp -vp ; vp and vp derive from the vp precursor protein) that assemble into protomers, pentamers, and fi nally capsids. nonstructural proteins are also released in these cleavages as well as through autoproteolytic cleavage. mature hrv virions packaged with an ssrna genome escape by cell lysis (adapted with permission from arden et al. [ ] ) codon position. the single rna "gene" acts as messenger rna to encode the single multi-domain, proteolytically processed "polyprotein." the coding region is bracketed by utrs which perform regulatory functions necessary for genome duplication [ ] . these are very similar genomic, transcriptional, and translational features to those of their close cousins, the evs. most of the information currently required for virus identifi cation by the international committee on taxonomy of viruses (ictv) can be found through analysis of the genetic features of hrvs ( fig. . ). there are complete hrv polyproteins on the genbank database ( fig. . ). the fi rst complete hrv genome sequence (hrv-b ) was described in [ ] followed by hrv-a in [ ] and hrv-a b in [ ] (fig. [ ] . sequencing of the vp /vp region was completed for all classical strains in [ ] , and the complete set of d regions were available in [ ] . currently there are at least named hrv-c vp regions the current spectrum of complete hrv complete polyprotein amino acid sequences available on the genbank database. the alignment was conducted using mafft within geneious pro v . [ ] . the phylogenetic and molecular evolutionary analyses were con-ducted using mega version (poisson model, bootstraps with consensus support shown at the nodes where space permitted [ ] ) (reprinted with permission from miller and mackay [ ]) available and complete hrv-c genomes. many more genomes are appearing as part of the rhinovirus consortium's efforts to complete and study the rhinovirome using highthroughput sequencing technologies to genetically characterize hrvs from their combined clinical specimen stores ( http://www.international-rhinovirus-consortium.org/ ). many ′utr and vp /vp sequences reside on the genbank database, most of which are labeled using in-house laboratory schemes rather than an approved nomenclature. analysis of the full-length genomes supports the use of ′utr, vp , and vp /vp subgenomic regions for useful representation of hrv species and types [ , ] . recombination, the process of genetic exchange which results in a chimeric genome [ ] , can only be detected in mature viruses after the fact, and it must therefore be inferred indirectly through genomic analysis and comparison. predictions of infrequent recombination among the hrvs [ ] have been made based on examination of the available set of hrv coding and noncoding regions [ ] . intensive analyses reported that recombination is not a driving force for the evolution of hrv types [ , , ] . some discrepancies are likely because of the different number of sequences used, the different origins of the viruses used for sequencing, and the analysis methods employed. hrv-c evolution seems to have been more affected by prior recombination, than is apparent for members of hrv-a or hrv-b. this is similar to the ev species but with far fewer predicted recombination events than for ev evolution [ , , , ] . most of the recombination proposed to have affected the hrvs occurred between hrv-c and hrv-a and is often found within the ′utr or at the ′utr/vp junction [ , , ] but rarely in coding sequence ( a [ ] or c [ ] ). the high sequence diversity among the individual hrv polyprotein coding sequences may keep recombination events to a minimum in order to retain viral fi tness [ ] . the ability of hrvs to recombine in practice awaits empirical evidence; the extent of recombination among all hrv or ev types and the frequency with which viable recombinants arise are entirely unquantifi ed. the - nm hrv virion has been visualized for only a handful of hrv-a and hrv-b types (including hrv-a a, hrv-a , hrv-b , hrv-b , and hrv-a ), but no hrv-c structures have been empirically determined to date. the fi rst, hrv-b , was described in [ ] followed by hrv-a a in [ ] , hrv-a in [ ] , hrv-a in [ ] , and hrv-a in [ ] . hrv-c structure has only been predicted using computer modeling, but their basic structure seems to be that expected of an hrv ( fig. . ) [ ] . the hrv capsid shell is composed of protomers, each comprising one copy of the viral proteins vp , vp , vp , and vp . vp , vp , and vp (each ~ kda) are to some extent exposed on the capsid surface, whereas vp (~ kda) is internalized and associated with viral rna. five protomers come together at a point around a fi vefold axis, and this cluster is called the pentamer. the fi vefold axis is circumscribed by a cleft referred to as the "canyon." vp , vp , and vp are each formed by a convoluted set of protein sheets and loops [ ] . the loops protrude beyond the external capsid surface and contain discontinuous antigenic sites. of the hrv types studied, four neutralizing antibody immunogenic (nim) regions have been identifi ed on hrv-b and hrv-a : nim- a (located in vp ), nim- b (vp ), nim-ii (vp and vp ), and nim-iii (vp and vp ) [ ] . antigenic sites identifi ed on hrv-a are called a, b, and c [ ] . the scope and location of antigenic and immunogenic moieties among the hrv-cs is unknown. using known receptor binding sequence as a guide for computer modeling ( fig. . ), it has been predicted that when discovered, the receptor for the hrv-cs will differ from the major and minor receptors defi ned for the hrv-as and hrv-bs [ ] . the three hrv species within the genus enterovirus are a genetically, immunogenically, and antigenically diverse assemblage of > viral types (table . ). this accounts for the combination of hrv-a a and -a b, exclusion of hrv- , which is actually ev-d despite confusion over acid liability [ - ] and combination of hrv-hanks which is actually hrv-a [ ] . serological studies indicate that some hrv-a and hrv-b types may not be distinct enough to deserve a unique identity [ ] . species within the genus share > % amino acid (aa) identity in the polyprotein and in c+ cd and > % aa identity in p ( fig. . ) as well as their host cell receptors, a limited natural host range, a genome base composition (g+c) that varies by no more than . %, and a similar compatibility of proteolytic processing, replication, encapsidation, and genetic recombination [ ] . a variant of the same hrv type shares - % aa identity or more in vp [ ] . much of the nongenetic criteria remain undefi ned for the hrv-cs. in the genera enterovirus and rhinovirus were offi cially combined, retaining the former genus name enterovirus with the human enterovirus c as the prototype species. a genus in the order picornavirales , family picornaviridae , is at least % different in its amino acid identity from any other genus. in a proposal establishing the species human rhinovirus c was ratifi ed by the ictv. formal hrv-c numbering commenced in , and type numbers were initially assigned based on the date of submission of relevant sequences to genbank (hrv-c , formerly nat ; hrv-c , f. nat ; hrv-c , f. qpm; hrv-c , f. c , etc.; table . ) [ ] . a clinical detection of an hrv-c can be considered a novel type principally based on its vp sequence or provisionally ("c_pat," table . ) based on vp /vp [ ] and could be confi rmed as a variant of a previously characterized hrv-c by identity thresholds to either region. the ′utr can be and still is used [ , ] for hrv genotyping, but it is a more problematic region than vp or vp /vp because of the recombination activity that affects this region, especially among the hrv-cs [ ] . this is presented as phylogenetic intermingling of some hrv-a and hrv-c types [ ] . nonetheless, careful application of sequence identity thresholds when comparing clinical sequences to the genbank database (≥ % identity required before assigning a clinical detection to a particular type) succeeds in characterizing hrv species and types [ ] . there are currently types within hrv-c (which includes the types once grouped together under hrv-"a ," hrv-x, and hrv-ny clades), hrv-a types, and hrv-bs. the most up-to-date information on current taxonomic trends can be found at the ictv picornaviridae study group website ( http://www.picornastudygroup.com/ ). ( c ) hrv-c versus hrv-a simplot data projected onto the hrv-c pentamer. the domains of interest are mostly shown within a single asymmetric unit. ( d ) a minor group pentamer (hrv-a , gray ) including antigenic sites (sites a -c , green ) and very-low-density-lipoprotein receptor (vldlr) footprint ( red ) [ ] . attachment of the vldl-r involves adjacent vp molecules. magnifi ed vp area represents one half of a vldl-r footprint [ ] . amino acid substitutions ( arrowed ) contributed to the differences between minor group sites b and c (adapted with permission from mcerlean et al. [ ] ) historically a key feature distinguishing the hrvs from the evs was the instability of the hrv capsid in the presence of acid and their lower preferred laboratory propagation temperature ( - °c versus °c for evs). over time hrvs have been subclassifi ed in different ways. the fi rst was based on tissue tropism and host range. hrvs that preferred growth using monkey cells were called "m" strains and those (the majority) that grew only in human cell cultures, "h" strains [ , - ] . these two groups correlate with receptor usage [ ] (table . ) and possibly with the titer of the inoculum employed [ ] . in it was proposed to abandon this terminology in favor of a sequential numbering system [ ] . picornaviruses recognize a variety of cellular receptors [ , , ] . hrv types are also subdivided into major and minor groups defi ned by use of one of the two main receptor molecules [ , ] . the capsid of the majority of classical hrvs ( n = ) [ ] interacts with the amino-terminal domain of the kda intercellular adhesion molecule (icam- ; cd ) [ - ] . receptor binding destabilizes the hrv capsid, probably by dislodging the "pocket factor," and initiates uncoating [ , , ] . icam- interacts with its receptor, leukocyte function antigen- (lfa- ), and plays a role in recruitment and migration of immune effector cells [ ] . the minor group [ ] of classical viruses employ members of the low-density lipoprotein receptor (ldlr) family to attach to cells [ ] . binding of vldl-r occurs outside of the canyon employing a different destabilizing and uncoating mechanism. heparan sulfate may act as a receptor under specifi c conditions [ , , ] . in andries et al. defi ned, and laine et al. refi ned, two "antiviral groups" (a and b) based on their susceptibility to a panel of antiviral molecules [ , ] . these groupings refl ected the nature of the amino acid (and hence nucleotide) sequence of the region interacting with the antiviral molecules. these antiviral groups can also be visualized using phylogeny [ ] . when sequences from other subgenomic regions, including p , c, and cd, were examined by phylogeny, the species were found, in most cases, to inversely correlate with antiviral grouping labels (table . ). m and h indicate early cell tropism-based classifi cation (monkey, human) abandoned in favor of a sequential numbering system [ ] . hrv types were later divided into the major and minor groups defi ned by receptor tropism [ , ] . receptor-designated minor group hrv types are underlined, and major group types are shown in bold. antiviral groups (a and b) are labeled [ , ] . hrv-a and hrv-a are also likely the same serotype [ ] . a full list of genetically close serotype pairings was presented by ledford et al. [ ] hrv-c nomenclature was defi ned in and currently includes a number of p rovisionally a ssigned t ypes (pat) which are confi rmed once preliminary vp /vp data can be confi rmed with vp sequence and the provisional number removed (e.g., c_pat to c_pat have already been reassigned) today, sequencing and phylogeny play a central role in species classifi cation within the genus, and together, they are surrogates for the important biological classifi cation criteria [ , , , - ] . for the hrv-cs, fi rst described as the "hrv-a " clade (not to be confused with the single virus, hrv-a , this naming scheme appeared after the hrv-c clade's name was proposed) of viruses in [ ] , sequencing of ′utr and vp /vp has provided the bulk of hrv information from clinical studies. while culture in primary sinus tissue has been reported [ ] , no receptor is yet defi ned. hrvs are the most numerous and frequently detected of all the "respiratory viruses," so-called because of their predominant detection in and tropism for the human urt or lrt ( fig. . ). the circulation of hrvs varies with population age, underlying disease, immunocompromise, over time, and across distance. circulation is infl uenced by the nature, strength, distinctiveness, and memory of the immune response hrvs trigger and by the nature and prevalence of other concurrently circulating respiratory, and perhaps nonrespiratory, viruses. with the recent discovery of the unculturable hrv-cs came the realization that previous hrv epidemiology was only reliable if conducted by one or more suitably broad-spectrum hrv pcr assays [ ] ; hence, prior to , detection of the full spectrum of ≥ hrvs did not occur. after , the ability to detect all types very much depended on the nature of the pcr primers and detection methods used. the great number of distinct hrv types has burdened the search for answers to epidemiology-related questions. however, as for other important respiratory viruses including human respiratory syncytial virus (hrsv) and the infl uenza viruses (ifvs), the virus types within a species show evidence of being both distinct and discrete viruses that are independently recognized by their host and consequently independently infect their hosts. each hrv type is also genetically stable [ ] . the hrv species circulate variably from year to year with evidence of epidemics of distinct types. a prospective longitudinal cohort study over months examined hrv frequency and diversity in specimens from healthy children ( - years of age) [ ] . a median of three hrvs and a maximum of six were detected per child. a similar outcome resulted from an australian cohort study [ ] . genotyping reveals more of the hrv diversity at a single site than culture ever could with molecular studies fi nding between and distinct hrvs at a single location [ , , ] . the number of additional hrv cases that occur in children outside of specifi cally defi ned symptomatic periods remain to be defi ned, with current studies indicating that a much higher number of hrv infections may occur. more comprehensive investigation of hrv type and illness will be undertaken during analysis of data from the australianbased observational research in childhood infectious diseases (orchid) study ( http://clinicaltrials.gov/show/ nct ). interestingly, the hrv-bs are often underrepresented, even when accounting for the smaller number of known hrv-b types [ ] . a number of studies have not found any robust patterns between the circulating hrv types or species and clinical outcome, but the majority of studies seeking this information are short and sample infrequently, limiting their ability to fi nd the patterns they seek [ ] . studies into the relative sensitivities of nasopharyngeal aspirates (npa) and swab sampling methods produce differing results, but generally, if seeking the best diagnostic yield for as many respiratory viruses as possible (i.e., seeking a laboratory diagnosis to support clinical decision making), npas are the sample of optimal choice. one study reported similar clinical sensitivities between swabs and npas for human coronaviruses (hcovs), ifvs, and hrsv, but reduced sensitivities using swabs for hrvs, human adenoviruses (hadvs), human metapneumovirus (hmpv), or parainfl uenza viruses (hpivs) [ ] . a second study reported no difference in sensitivities for hrvs, hadvs, and hpivs but a reduced sensitivity for hrsv and ifvs when using swabs [ ] . nasopharyngeal washes also yield more viral culture success than either nasal or pharyngeal swabs. nonetheless, many studies use nasal swabs as the sample of choice because they allow self-collection and involve much less discomfort than npas, and pcr has meant that infectious virus is not required, only viral nucleic acid which relaxes some limitations imposed by the need for rapid, careful, temperaturecontrolled, and expensive transport requirements [ , , ] . bronchoalveolar lavage samples are best for seeking lrt etiologies, especially in adults where nasal wash viral loads can be low compared to those in children, but this is an invasive method with some risk attached [ ] . hrvs infect all people, all around the globe. spread of hrvs is most obvious and frequent from child to child and from child to parent [ ] . in populations of mixed age, the majority of hrv detections occur in children [ ] . among specimens from healthy children, over a third ( %) were hrv positive. children less than years of age ( % of whom were hrv positive) were shown to have more hrv infections and a wider diversity of hrv types than children more than years old ( % hrv positive) [ ] . healthy adults in the military [ , ] , at university [ ] , at home [ - ] , and in the workplace [ ] have also featured prominently in historical, culturebased, and volunteer infection studies and heavily infl uenced our view of hrv infection outcomes [ , ] . although studies of children in hospital-based populations usually report more signifi cant clinical outcomes (relating to the lrt) [ ] than community-based studies, these data are still broadly applicable. hospital populations originate from the community and refl ect the more serious and perhaps fi rst exposures to the virus. hospital-based populations defi ne the potential of a virus to cause severe clinical outcomes. disease at this end of the spectrum has the strongest infl uence on future prioritization of therapeutic research and developments [ ] . modern air travel contributes to the rapid spread of respiratory viruses as seen in their often frequent detection among travelers [ ] including those with febrile illnesses [ ] . apart from children, hrvs are found with the great clinical impact in the elderly (described as - years of age) with % of aris positive for an hrv, sometimes with a greater burden of disease than ifvs [ ] . those with asthma or copd are also affected by the ari triggering exacerbations of wheezing illness (see sect. . ). it is thought that this is not a different type of infection but rather a different response to infection by the host. wheezing can also result from infection in atopic people who do not have underlying asthma or copd. hrvs cause signifi cant impact in the immunocompromised, and this group is the only population to date that has been found to host truly persistent hrv infections (see sect. . ). because the hrvs are the largest group of viruses to infect humans, it is not surprising that they confuse differential diagnoses during pandemics and have key roles in co-detections and asymptomatic disease. the study of hrvs is the study of all respiratory viruses; while each can be considered in isolation, this will likely be detrimental to a greater understanding of respiratory virus pathogenesis. hrvs circulate throughout the year but usually with a bimodal peak in temperate locations in both hemispheres. the highest peaks, mostly defi ned using adult populations, are in the autumn (fall) and spring [ , , ] (and, peculiarly, on a monday [ ] ). the major winter dip in hrv prevalence closely coincides with the peaks of other respiratory viruses, particularly ifvs [ ] and hrsv [ ] . one hypothesis states that a miasma exists in the school classroom, of particular relevance to those who suffer asthma exacerbations, and this miasma maintains immune stimulation, which subsequently wanes among school children during holidays, to be challenged anew upon return to school [ ] . it is clear that an interplay or interference takes place between viruses at the population level, particularly evident among rna viruses. there is a correlation between spiking spring and autumnal hrv case numbers and an asthma exacerbation "season" - days after return to school from holidays, in a range of climates [ - ] . this was particularly obvious among asthma hospitalizations of children ( - years of age) in ontario, canada, which peaked at weeks - across a decade [ ] . upon investigation, hrvs were the most prevalent of the viruses found in a -year analysis of emergency room presentations in ontario [ ] . hrvs also predominate during "hay fever season" [ ] . although a defi ned seasonality is not always found in the tropics [ ] , this may sometimes be due to testing that does not include hrvs [ , ] or only some hrvs [ ] . all the hrv types continue to circulate today, including those named in the earliest of the nomenclature assignments. at a single site during - months, or more types can co-circulate [ , ] [ ], dropping [ , ] if the study time frame at the site is shortened. a recurring hrv type, defi ned using molecular tools, accounted for . % of any virus detected in a birth cohort followed for months [ ] and, in another cohort, occurred twice in two children, within a -month period [ ] . within a given year and across different years, it is apparent that hrv species exchange predominance [ , , , - ] . no evidence exists to satisfactorily explain this; however, herd immunity may be a factor. the use of cell and tissue culture underestimated the frequency of multiple infections in patients, most likely because the dominant virus out-replicated any others, or due to viral load differences, specimen quality issues, differing cell tropisms, or the triggering of an antiviral state by the fi rst virus. when the majority of respiratory viruses are sought using pcr techniques, multiple virus-positive specimens can comprise a third of those tested [ ] , dropping to around a fi fth of ari episodes when fewer viruses are sought [ ] . there is sometimes an emphasis on the high number of hrv cases that are identifi ed in the presence of another virus, and including hrv testing does raise the frequency of pathogen detection above one per sample [ ] . coinfections, or, more correctly for pcr-based studies, co-detections (since pcr cannot determine infectivity), have been found to either increase [ , - ] or have no impact on the clinical outcome in their host [ - ] , and so the issue of clinical relevance of co-detections is still uncertain. in extreme cases, half of all hrv detections can be found concurrently with another virus. on the surface, this is a signifi cant fraction, and yet % or more of hrsv, hmpv, ev, and ifv detections and % of hcov-nl detections can be found in the company of another virus [ ] . other studies fi nd different, but still higher proportions of co-detections involving non-hrvs [ ] . whether co-detections represent a particular synergism between the involved viruses, a differential capability to manipulate the host immune response, a sign of innocuousness for the most frequently involved virus [ ] , or a chance due to overlapping seasons remains unclear. it is clear, however, that co-detections are not an anomaly or an error due to "overly sensitive" pcr tests; they are evidence of further biological complexity that, until recently, remained hidden from us. recent studies have shown that the initial impression of hrvs being overrepresented in these cases was incorrect. closer analysis of viral co-detections has revealed patterns [ , ] . these became clear when codetections were examined bidirectionally, not just how many hrvs were positive for virus x but also how many of virus x cases were positive for an hrv. whether in a hospital or a community setting, hrvs more often occur as the sole virus detected in aris [ , ] . considering their ubiquity, it is interesting that relatively low numbers of concurrent detections occur [ , ] , supporting the concept that hrvs have a direct role in the clinical outcome of their infection [ ] . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens, while the host provides a vessel for hrv replication and transmission. studies of single respiratory viruses without being in the context of the respiratory virome are of limited value in drawing conclusions about clinical impact. much of the longitudinal epidemiology data previously relied upon to form assessments of hrv signifi cance was acquired using culture-based techniques. with improved and more comprehensive testing, patterns can be seen among the interactions of hrvs and other respiratory viruses. virus interference is a type of virus-virus interaction (vvi) that has been known for decades. vvi has recently been categorized into types [ ] . at the population level, it has been noted that during trials of live attenuated ifv (laiv) vaccines, an interferon (ifn) response was triggered that protected vaccinees against off-target viruses for days postvaccination [ ] . this study went so far as to suggest such effects could be maintained for a prolonged period using a regime of consecutive schedule vaccinations, each separated by days or more, during times of a prolonged epidemic [ ] . a similar effect was produced using live ev vaccines (lev) to replace pathogenic ev types and interrupt outbreaks [ ] . orally administered levs succeeded in their principal task but also reduced the incidence of aris during epidemics by % overall [ ] . this shows that immune activation in the gastrointestinal system generates an anatomically distinct protective effect and there may be a similar effect on the gut's infl ammatory status after respiratory virus infection. in contrast to the laiv results, the offtarget protective effect was reversed in a study using a trivalent inactivated ifv vaccine [ ] . the mechanism underneath these opposing outcomes is unclear. during the heyday ( s) of tissue culture for virus studies, a common biological assay for infection with hrv involved attempted infection of the culture with an enterovirus (ev) or hpiv- [ , ] . failure of the superinfecting virus to grow heralded the likely presence of a noncytopathogenic hrv. virus interference has been used to measure ifn in specimens through its inhibition of hrv growth [ ] . more recently hrv-hadv dual pcr-positive cases were found less often than expected and harbored lower viral loads of hrv than did specimens from cases of sole hrv infections [ ] . signifi cantly, the majority of these instances of vvi involve rna viruses [ ] . it has been shown that dual infections of peripheral blood mononuclear cells (pbmcs) with viruses other than hrsv (including hrvs) induced immune responses similar to those of single infections, but coinfections including an hrsv resulted in reduced ifn-γ responses [ ] . vvis are affected by the ability of each to moderate the host response against them. virus interference has also been identifi ed in virus positives as a series of patterns among respiratory specimens tested for up to respiratory viruses (fig. . ) [ , ] . statistical analyses supported that many of the co-detections occurred in patterns, in particular that fewer co-detections involved an hrv than would have been expected by chance alone ( p ≤ . ). for some period, rna virus infection, especially the hrv group, may render the host less likely to be infected by other viruses and, by extrapolating to the community level, help constrict the epidemic periods of other viruses by reducing the number of fully susceptible hosts. virus interference as a feature of respiratory virus epidemiology can also be seen in results of other studies [ ] . during an -week period that spanned peak h n pandemic infl uenza season in wisconsin, it was infl uenza a virus (ifav) that seemed to dominate hrv in children with asthma who were sampled weekly [ ] . whether this refl ects all ifv-hrv interactions or just those involving a novel ifv such as h n is unclear. it was found that pbmcs from these children exhibited normal immune responses [ ] . reports of subjects with continuous and extended (greater than - weeks) periods of hrv positivity [ , ] increased as pcr methods replaced cell culture for hrv detection. this had only rarely been recorded using culture [ ] . hrv rna has been detected days prior to symptoms commencing and for as long as or more weeks after they cease [ , - ] . studies that only defi ne the period between aris in children as that time when specimens are rt-pcr negative [ ] will not detect overlapping serial infections (fig. . ). epidemiology that incorporates hrv typing generally does not fi nd chronic shedding [ ] . hrv shedding normally ceases within - days, after signs and symptoms have stopped [ , , , , , ] . thus, the perception of persistence is probably due to serial or overlapping infections by multiple untyped strains [ , , , ] . few studies [ ] have suitably addressed persistence in hrv infections involving healthy subjects since pre-and post-sampling clinical data are rarely described [ , ] . to date, true persistence-an ongoing detection of a single confi rmed hrv type-has been limited to individuals with underlying immunosuppression or immune dysfunction [ ] . hrv-cs were detected more than three times longer in immunocompromised young patients than in immunocompetent children, with a mean of versus days [ ] . multiple detection of the same hrv type ( % identical hrv- a sequence in each patient over time) extended to months in hematopoietic stem cell transplant recipients. the proof of causality is as diffi cult to achieve as the proof of innocuousness when it comes to respiratory viruses and aris. the defi nition of "well" subjects prior to or at the time of sampling or inoculation is sometimes not clear, especially for young children who cannot reliably report symptoms [ , , ] . often parents notice a symptomatic illness before an infection is detected in the laboratory [ ] , supporting the importance of diaries in longitudinal home-based community studies. nonetheless, even with the support of telephone interviews and home visits, milder cold symptoms may be missed. it is not uncommon for an asymptomatic control to subsequently become symptomatic or have been symptomatic before sampling [ , ] . some studies employ sensitive symptom scoring systems [ ] , but the criteria for being symptomatic are usually designed to describe and clearly discriminate overt or more "severe" illnesses, those with obvious and measurable signs. strict defi nitions help improve patient management and the commencement or better direction of treatment or cohorting. however, in research studies the arbitrary degree of severity required for reporting a symptomatic event often overlooks very simple changes in host biology due to a virus's replication. these changes to the norm are mild but nonetheless represent disease (a disorder of structure or function that produces specifi c symptoms or that affects a specifi c location and is not simply a direct result of physical injury) in the literal sense. such minor or short-lived, often unrecorded [ ] , indications of infection include sinus pain, headache, sore throat, earache, watery eyes, fatigue, muscle aches and pains, and mood changes. within families, hrvs are frequently transmitted from vsig activated "shields up" a b children who are usually symptomatic [ ] . infants frequently exposed to other children have more asymptomatic viral infections [ ] . among infected adult family members, asymptomatic infections are more likely [ ] . among older parents, whether their children live at home or not, asymptomatic infections are more frequent following hrv challenge than among adults without children or in younger parents [ ] . in a study of viral species in age-stratifi ed cases and controls, signifi cantly lower viral loads were found in those without the required symptoms [ ] . qpcr may prove useful to determine viral load cutoffs to address this issue in the future, although the respiratory tract is a diffi cult tissue for qpcr [ ] . the high sensitivity of pcr-based methods has raised concerns over the clinical relevance of a virus-positive result [ ] . it is clear that a proportion, around fi ve to % of study-defi ned asymptomatic control populations [ , , ] , are virus positive using sensitive pcr-based methods. this may vary up to nearly % of cases when stratifi ed by age, virus, and season or when including highrisk populations [ , ] . every respiratory virus, even ifvs and hrsv, can be found in cases without symptoms at the time of specimen collection even after specifi c inoculation of adults [ , , ] . this is a complex and incomplete story in need of more research, and so it is frustrating that positivity in asymptomatic people is often used to rank viral importance. better data are required from asymptomatic controls for any conclusion to be drawn about causality [ ] , but this requirement often disregards the memory of a normal functioning protective host immunity. it is the host response that defi nes the degree of clinical severity for the infl ammatory disease that is the hallmark of an ari [ ] . it is well known that previous exposure to a virus affords protection from the full clinical spectrum of disease upon repeat exposure to that virus. it should come as no surprise then that hrvs, which usually cause brief infection anyway, could well produce only minor signs and symptoms upon reinfection. the unique and extremely personal infection history of each member of a control group cannot be determined unless they are part of a longitudinal cohort. so, what do cohort studies, supported by comprehensive pcr-based testing, tell us about asymptomatic virus infections? some cohort studies do not look in asymptomatic children, seeking samples only at times of symptomatic illness [ , , ] . a birth cohort of children enrolled and sampled when ill and every months for months identifi ed hrvs - % of infants and toddlers who had no nasal symptoms (defi ned solely by the presence of rhinorrhea) [ ] . the childhood origins of asthma (coast) birth cohort followed infants at high risk for allergies and asthma for months and identifi ed hrv infections as preceding (mean age of fi rst detection, months) those of hrsv (mean age at least months), and hrvs were found in % of asymptomatic versus % of moderately to severely ill patients; the most frequently symptomatic children also had the greatest proportion of asymptomatic infections [ ] . in a study of children with asthma sampled weekly for weeks during each of two peak hrv seasons, nearly two-thirds who were virus positive but not sensitized to at least one allergen showed no asthma symptoms, and nearly half showed no ari symptoms; in the children who were sensitized, less than one-third showed no asthma symptoms, and only a fi fth had no ari symptoms [ ] . a convenience population of healthy children ( - years old) without asthma were followed during at least three seasons, and picornaviruses were detected in % of specimens ( % of infections) not associated with symptoms, the impact of hrv typing and of sampling based only on symptoms. the example provided here diagrammatically represents a single, hypothetical monitoring period, starting at time = , for a single individual. the period of potentially detectable hrv is indicated by an open box. if sampling occurred at each time point ( - ) and hrv positives were genotyped, it would be apparent that three different strains infected the individual, although discerning hrv-x from hrv-z at time point would require a molecular cloning approach. illness, in different forms, may have continued over the entire period depending on the symptoms required/recorded and the period of time represented by the monitoring period. in this case a clinical diagnosis may record only a single symptomatic episode. genotyping may not be performed, and sampling may be intermittent, and so association between viral type or species and disease is impossible. in the study examples indicated by ( a ) start and fi nish sampling or ( b ) symptomatic sampling, ( asterisks mark sampling times in fi lled bars), the laboratory data would have made only one or two identifi cations, respectively. in the third example, ( c ) frequent sampling of this type has previously led to conclusions of hrv persistence or chronic shedding; when combined with genotyping, it becomes apparent that different hrv types are present although of the infections came from households with an infected sibling [ ] . in summary, there is clear evidence for the presence of hrvs in asymptomatic controls. a precise proportion cannot yet be defi ned. some study controls show signs of a "lead-in" period where rna positivity precedes an ari defi ned on follow-up, while others may have been defi ned as symptomatic if more symptoms had been accounted for. mechanisms and routes of transmission hrvs have been found at extra-respiratory sites. viremia was determined in the blood of children with lrt infection or pericarditis [ , ] , and hrv-c was more commonly associated with viremia than was hrv-a, supporting possible increased pathogenicity [ ] . blood was also positive for hrv rna and infectious virus from infants at necropsy [ , ] , and hrv rna was detected in the plasma of children with asthma, bronchiolitis, or common cold [ ] . an hrv was once isolated from feces [ ] , and more recently higher than expected loads of hrvs were detected in fecal specimens from children with suspected meningitis and fever of unknown origin [ ] , with gastroenteritis [ ] , and in a child with pericarditis [ ] . nonetheless, the nasopharynx is still considered the main site of focal virus production [ ] , regardless of inoculation route [ ] , and most studies of transmission routes have centered on the urt. in contrast to ifv and hrsv, hrv infection involves less destruction of tissue. ciliated epithelial cells are sloughed off in proportion to the severity of an hrv ari, but this damage is minimal and does not occur during the viral incubation period or with subclinical infections [ , ] . the incubation period between infection and onset of virus shedding into nasal secretions is - days with shed viral titers peaking in adults between days and [ , ] . the time until successful hrv transmission among adults in a childless family setting is usually - days and requires the donor to be shedding at least tcid at some stage, to have recoverable virus on the hands and in the nares, enough shared time, and a moderate to severe ari [ ] . the lungs have been shown to host replicating hrv [ ] , and the reader of such reports may be left with the perception that detection of hrv replication in the lrt explains all lrt symptoms. however, relatively few studies seek or identify true hrv replication in the lrt. while the overwhelming majority of lrt cases detect hrv from the urt, a correlation between urt positivity and lrt disease does exist [ ] . it is well known from experimental inoculation studies that hrv infection can result from inoculation of the conjunctival sac after virus is moved through the nasolacrimal duct [ ] . in these studies virus was commonly delivered by aerosol or intranasal instillation of . ml to ml of suspension [ - , , - ] . in the laboratory, hrvs can retain infectivity for hours to days on suitable, nonporous solid surfaces, especially if the inoculum remains damp [ , ] , which supports direct self-inoculation especially in the family setting and indirect inoculation via fomites [ ] . in a trial to defi ne the movement of virus from a contaminated donor to a recipient via multiple surfaces or by hand-to-hand contact, % (donor to objects to recipient) and % (donor to recipient fi ngers) of the virus recoverable from the donor's fi ngertips were recoverable from the recipients' [ ] . even under observation, eye rubbing ( . h − - . h − ) and nosepicking ( . h − - . h − ) occur frequently [ , ] , suggesting self-inoculation could outpace personal hygiene, particularly in the young. it was once thought strange that aris were so common, but isolation rates for the expected viruses were so low [ , ] . with a better understanding of the importance of preexisting antibody (something common among the predominantly adult volunteers used by many studies), the discovery of a third, unculturable species of hrv (still causing aris but impossible to isolate or detect using antibody-based systems for which no reagents existed), and a vastly improved diagnostic sensitivity, this is much less confounding. in the past, household cross infection, determined by ari, was low, about fi ve exposures to infected members required for infection [ ] despite viral loads in nasal washings peaking at . × tcid /ml [ ] . experimental transmission was also reportedly ineffi cient [ ] . in contrast, "naturally" close-quartered military populations, interacting over - weeks, experienced rapid spread of hrvs to > % of the group [ ] . the use of pcr recently clarifi ed this discrepancy, confi rming that frequent transmission in families is more common than culture-based studies had identifi ed, often resulting in asymptomatic infection among older siblings and parents [ ] . pcr has helped defi ne the scope of viral rna, if not actual infectious virus, survival, and spread. transmission studies require infectious hrv, and so the hrv-cs do not contribute to the historical data. under crowded or intimate conditions and with more severe colds, transmission reaches - % [ , ] . in some studies, both large-and small-particle aerosols proved ineffi cient, supported by a low isolation rate from saliva ( % compared to % of hand washes and % of nasal swabs) [ , , ] and from only . % of participants exposed to large-particle aerosols [ ] . in other human donor-recipient model studies however, aerosol proved to be the main transmission route among antibody-free adults [ , ] . the discrepancy may have been due to insuffi ciently long or intense exposure in the earlier aerosol experiments [ , ] . apart from particle size, spread of virus by aerosol is affected by existing nasal obstruction which can divert secretions from the nares to contaminate saliva, the presumptive source of virus in coughs and sneezes [ ] . when exposed to liters of a small-particle aerosol, tcid of hrv- was associated with fever and prominent tracheobronchitis in antibody-free (< : ) adult volunteers but not when delivered via nasal drops or a coarse aerosol [ ] . it has also been found that simple breathing releases hrv rna (the same type was also identifi ed from nasal mucous) from at least a third of adults and children with symptomatic aris and infectious hrv could be isolated from a fi fth [ , ] . it is apparent that hrvs accumulate at sites with heavy human traffi c, potentially forming a secondary source of infection. hrv rna can be detected from % of ~ -hourold fi lters placed to sample air in offi ce buildings [ ] . in aircraft, high effi ciency particulate air (hepa) fi lters have been found to harbor hrv rna more than days after they were removed for servicing [ ] . hrv infections trigger a vigorous proinfl ammatory immune response that is thought to drive the symptoms experienced as illness [ , , ] , but they do not seem to actively prevent or interfere with the host's immune response the way most other viruses have evolved to do. there may be a role for repeated challenge by hrvs and other respiratory viruses leading to infl ammation and tissue remodeling. the host response to hrv infection can be broadly broken into the innate (very fast, encoded in the germ line, nonadaptive) and adaptive (slower to develop, reliant on t cells, b cells, and the generation of antibody) responses. while the innate system is "always watching," it is signifi cantly amplifi ed by virus infection. the adaptive response is initiated by the host's fi rst infection with a particular virus and then functions to limit subsequent infections through the production of neutralizing antibodies and amplifi cation of existing cell-mediated immunity. after virus-receptor binding and internalization, the earliest host cell immune response to an hrv infection is elicited by the innate immune system (fig. . ). epithelial cells represent the front line against hrv invasion although alveolar macrophages and dcs are better equipped to respond [ ] and do so despite not hosting hrv replication directly [ ] . virus detection is mediated by pattern recognition receptors (prrs) that have evolved to recognize conserved molecular structures shared among diverse pathogens. internal-or surface-mounted prrs include sentinels that specifi cally recognize picornavirus rna and protein and, in doing so, trigger an immune circuit that results in the production of ifns and subsequently hundreds of ifn-stimulated gene products. the innate response to viral infection hinges on inducing two type i ifns (initially ifn-ß then ifn-α), secreted cytokines that produce antiviral, antiproliferative, and immunomodulatory outcomes [ ] . the type iii ifns (ifn-λ or il- , ifn-λ or il- a, and ifn-λ or il- b) are also produced in response to viral infection in a range of cells, although their receptor is not as widespread [ ] . the type ii ifn, ifn-γ, is produced by activated t cells and natural killer cells rather than in direct response to virus [ ] . detection of viral components triggers protein signaling cascades that regulate ifn synthesis through the activation of viral stress-inducible genes (vsigs) [ , ] . these are sometimes expressed constitutively but upregulated after ifn induction following hrv infection [ ] . released ifn-ß binds to the ifn-α/ifn-ß receptor in an autocrine (the same cell) and paracrine (neighboring cells) manner, starting a positive feedback loop for type i ifn production, the "second wave." vsigs include the antiviral proteins protein kinase r (pkr), ′ ′oas/rnasel, and the mx proteins [ ] . ifn-α upregulates expression of mxa, ′ ′-oas, and pkr [ ] . the mx pathway is also induced after virus infection but is not constitutively expressed [ ] . depending on the sentinel system stimulated, there are different pathways to vsig activation. those vsigs with antiviral properties (e.g., mxa, pkr, ′ ′oas/rnasel) inhibit different stages of virus replication and strengthen an antiviral state in the host. while this state is well known, the nature of its induction by different respiratory viruses and the impact of induction upon the replication of other respiratory viruses are topics for considerable ongoing research. one pathway to ifn induction relies on the ifn-upregulated cytosolic sentinels retinoic acid inducible gene rig-i-like receptors (rlrs) rig-i (specifi c for ifav and others) and melanoma differentiation-associated gene (mda , specifi c for picornaviruses and others) [ , ] . these rna helicases recognize either rna with a ′-triphosphate or distinct dsrnas, which results in activation of nf-κb leading to "classical" type i ifn induction [ , ] . studies into the innate response to hrv infection have been limited to the use of a very few easily cultured types. it is presumed that the result can be extrapolated to most if not all types. this is yet to be tested. rig-i is degraded by hrv-a [ ] , ifn regulatory factor (irf)- homodimerization is interfered with hrv-b which limits ifn-β induction [ , ] , and mda is degraded by hrv-a a but not hrv-a [ ] . another pathway for recognizing hrv infection involves the toll-like receptors (tlrs), transmembrane prrs that terminate in an intracellular signaling region. the endosomally localized tlr , tlr , tlr , and tlr recognize nucleic acids and are also involved in innate antiviral responses. tlr and tlr identify g/u-rich ssrna from endocytosed viruses, while tlr recognizes unmethylated cpg dna present in dna viruses [ , ] . tlr and tlr are found on the cell surface and recognize hrv or hrsv proteins, respectively [ , ] , and tlr recognizes dsrna. tlrs operate mainly, but not exclusively, in plasmacytoid dc [ ] . the particular tlr that notifi es of an hrv incursion may depend on the method of virus approach [ ] . tlr activation can reduce ′ ′oas and mxa mrna expression and ip protein in adolescents with asthma compared to healthy controls [ ] . tlr activation did not result in a similar disparity [ ] . it has been suggested that hrvs may have evolved with humans to such an extent that their symbiotic relationship serves to help train the human immune system [ ] . intriguingly, within the hrv species, there are differences in the type and level of host response induced [ ] which may refl ect receptor usage, route of entry and cell type infected, hrv species, or the degree of laboratory-adapted virus used during in vitro studies. after initial hrv infection, the innate response results in production of proinfl ammatory cytokines, vasoactive peptides, and chemokines that attract leukocytes, granulocytes, dcs, and monocytes (table . ) [ , , ] . the t-lymphocyte response to viral intrusion can be broadly categorized as t h - -like and t h- -like. other t-cell subsets exist, but most work in relation to hrv has been conducted on the earliest defi ned subsets. the t h - cellular response is important in managing cellular immunity and producing interleukin (il)- and ifn-γ. the t h - cellular response manages humoral immunity and stimulates b cells via il (initiating production of ige), il (infl uencing eosinophils), and il (crucial component of allergen-induced asthma). these two t-cell responses act in concert with epithelialderived chemokines (e.g., eotaxin) to promote the recruitment and activation of eosinophils and mast cells, contributing to chronic airway infl ammation and the hyperresponsiveness of airways to a variety of nonspecifi c stimuli [ ] . t h- lymphocytes, opposing t h- lymphocytes, contribute to an allergic infl ammatory cascade, akin to what occurs to rid humans of parasites [ ] . the t h- response can also be repressed by binding of microrna, which leads to an altered balance favoring a t h- state in mice and probably in humans [ ] . regulatory t cells (t reg ) suppress allergic infl ammatory pathways and are therefore fundamental in protecting the airway from allergen sensitization [ ] . considerable immunobiological research has focused on asthma exacerbation, with which hrvs are intimately involved. although upregulated by hrv infection, the t h - response is comparatively defi cient in people with asthma [ , ] . this is problematic as an increased t h - -like cytokine response, deduced from higher sputum mrna ifn-γ/il values, speeds clearance of hrv and symptom amelioration [ ] . one possible cause of the t h - defi ciency in people with asthma is inadequate maturation of type i and iii ifn responses due to reduced exposure to infections early in life [ ] . the "hygiene hypothesis" [ , ] posits a pathway for an asthma etiology described [ ] in terms of the young, unchallenged immune system, dependent on infections to stimulate the development of its t h- -like functions. one theory suggests that hrvs play a central role in developing that effi cacious antiviral immunity, particularly in infancy, via their frequent, usually mild self-limiting infections [ ] . genome-wide expression analysis of becs from healthy and asthmatic adult subjects after hrv-a a infection revealed some signifi cant differences that were found between cell types and response to infection [ ] . these included immune response genes (il b, il , il , il f , il ) and airway remodeling genes (loxl , mmp , fn ) and an overall proinfl ammatory response and metabolic slowdown consistent with proteolytic cleavage of transcription factors by some hrvs [ , - ] in the infected cells. this study further noted some similarities to gene expression changes observed in brushings from people with mild asthma after allergen exposure and in bal cells from subjects with corticosteroidresistant asthma [ ] . overall, hrv replication and the host transcriptional response to it were similar in normal or asthmatic bec cells [ ] . this indicated, at least in adults, that something beyond the epithelial cell is an important contributor to more severe clinical outcomes in asthma. the application of inactivated hrv-b was found to promote release of il from monocytes (an immunosuppressive cytokine) and to inhibit the stimulation of il (drives t h - development) [ ] . however, neither il nor il was signifi cantly induced in asthmatic adult volunteers in response to hrv-a compared to healthy subjects [ ] . while ifn-α was detected after transfection of dcs with hrv-b ssrna, low tnf-α and il levels were also noted [ ] . it was posited that the reduced il could indicate negatively affected local immunity possibly predisposing to secondary infections [ ] . infection of stromal lung cells by hrv-b triggered exaggerated levels of the pleiotropic il (an il type cytokine), akin to those triggered by hrsv, which were also detected in nasal secretions from children with wheezing [ ] . other cytokine changes have been identifi ed in atopic adult volunteers challenged with hrv-a . g-csf and il (chemo-attractant for neutrophils) levels rose in the urt (as examined by protein detection in nasal lavage) and lrt (mrna detection in sputum) with concomitant rises in blood and nasal neutrophil numbers [ , , ] . the nasal epithelial cells of atopic individuals, especially in season, express more icam- than those of nonatopic adults [ ] as do normal subjects infected by the major group hrv-b [ ] . by contrast, ifn-γ and il , which appear later postinfection, downregulate icam- expression in infected cells [ ] and encourage infi ltration of neutrophils [ ] , respectively. changes in icam- levels may modify participates in creation of an antiviral state; produced by and infl uences the maturation of dcs il- β proinfl ammatory properties; enhances adhesion molecule expression including icam- ; induces il- receptor gm-csf a granulocyte and monocyte growth factor il- stimulates growth and differentiation of t and b lymphocytes and cytotoxic activity of nk cells and monocytes il- t h differentiation, promotes ige synthesis il- activation, differentiation, and proliferation effects on t and b lymphocytes; induces c-reactive protein stimulating pyrexia il- /cxcl- neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and degranulation activity il- anti-infl ammatory factor produced by monocytes that acts by inhibiting proinfl ammatory cytokines il- , il- , and tnf-α irf a master hub, regulating antiviral immunity ip /cxcl chemoattractant for activated t h and nk cells tnf-α proinfl ammatory activity similar to il- β; activates neutrophils; induces vascular permeability mpc- a monocyte attractant bradykinin potent infl ammatory mediator, increases vascular permeability tslp an il- -like cytokine that activates myeloid dcs to induce naive t cells into t h cells producing il- , il- , and tnf-α; induced by hrv in the presence of il- bec bronchial epithelial cells, dc dendritic cell, irf interferon regulatory factor, ifn-γ inducible cytokine protein, nk natural killer, pbmc peripheral blood mononuclear cells, il interleukin, tnf tissue necrosis factor, tslp thymic stromal lymphopoietin t-lymphocyte-mediated cytotoxic or t h interactions with hrv-infected cells, upregulating receptor expression and encouraging eosinophil and t-cell infi ltration into the lower airways of asthmatic individuals [ , ] . before an hrv can enter a cell, it must pass through a defensive barrier of secreted anti-hrv antibody, mostly iga. the ease with which this passage occurs is proportional to the progression of clinical disease. healthy adult volunteers were found to develop iga by at least days to weeks after inoculation-about the same time as serum antibody-and retain peak levels for at least weeks [ , - ] , falling faster than serum levels [ , ] . there is also some evidence for a degree of nasal immune memory [ ] . volunteers with pre-study serum antibody could still be infected in some studies [ , , ] , but not in others [ ] . infection is more clear in volunteers without preexisting nasal antibody to experimental challenge virus; they become infected, exhibit more severe ari, and shed more virus for longer [ , ] . iga does not seem to modify illness severity or virus shedding, but high levels prevent reinfection by the initiating virus type. low levels or absence of iga does not prevent reinfection by the same hrv type, which may manifest as symptomatic or asymptomatic disease [ ] . older children, adolescents, and adults have greater amounts of hrv-neutralizing antibody than young children [ ] , accompanying a trend toward decreasing numbers of symptomatic aris with increasing age [ , ] . this feature raises an issue: did the use of older subjects in many common cold studies underplay the pathogenic potential of the hrvs because protective or partially cross-protective antibodies moderated the impact of infection? consequently, quantifying levels of type-specifi c serum antibody became routine practice prior to some studies. adult volunteer studies determined that no infections resulted if preexisting neutralizing antibody titers ≥ : existed; as levels grew from , so did levels of resistance to infection [ , ] . adults were protected by serum titers of : - : [ , ] . the trend was interrupted by adults in the - year age group, presumably because they had begun families and their young children acquired and amplifi ed currently circulating types from the community and transmitted them into a household that was either immune naïve or lacking suffi cient antibody or cell-mediated memory for protection [ ] . traditional vaccine strategies were quickly ruled out as a prophylactic intervention for hrv illness because of the extensive antigenic variability that is a hallmark of the genus enterovirus [ , ] . however, if it were possible to identify "master" strains [ ] that exhibit suffi cient antigenic cross-reactivity to induce broad heterotypic responses against many other hrv strains, then an effective vaccine could still be possible. in fact, boosting host immunity to an hrv type by repeat infection does heighten immunity to one or more other types [ , ] . the highest of these heterotypic antibody titers develop against those types with the highest preexisting antibody levels [ ] . the fi rst description of a unifying hrv numbering system recounted the appearance of minor serological cross-reactions, which were removed by modifi cation of the technique [ ] . subsequently, cross-reactions were better defi ned during experimental inoculation when multiple hrv immunogens and antigens were used to deduce the extent of heterotypic responses [ , , , ] . less promising for hrv vaccinology was the description of antigenic variation within hrv types which suggested that immunity to one variant of the type might not protect against infection by other variants [ , ] . the "prime strain" is a specifi c antigenic variant of a prototype hrv type that is neutralized to a lesser extent by antisera from the prototype, while yielding antisera that effectively neutralize both itself and the prototype [ ] . another form of this cross-neutralization is ascribed to the "intertypes," which are hrv isolates that share a lower-level serological relationship with a pair of hrv strains, which themselves share neutralizing reactivity, e.g., hrv-a and hrv-a [ ] . the low-level reciprocal neutralizing activity was not equivalent in both directions; anti-hrv-a sera had a higher titer for hrv-a than anti-hrv-a sera did for hrv-a [ ] . over strains were linked directly by such one-or two-way cross-reactions or indirectly through two or more strains. hrv-a and hrv-a are linked via hrv-a , hrv-a , and hrv-a (anti-a serum neutralizes hrv-a , anti-a neutralizes hrv-a , and anti-a neutralizes both hrv-a and hrva-a [ , ] ). a surrogate molecular method which provided insight into these interrelationships, perhaps expanding upon them to identify useful patterns for vaccine immunology purposes, would be most welcome. in summary, heterotypic immunity and hrv intertypes might be exploitable features of hrv immunobiology that could confer maximum protection upon the host from the minimum number of hrv types [ ] . hrvs circulate in great numbers, and any specifi c roles for distinct hrv types in initiating disease remain to be defi ned. the relatively inconsequential common cold is the most frequent manifestation of viral infection in humans, with to > % of colds positive for an hrv [ , , ] . furthermore, aris due to hrv infection can exacerbate or result in a much greater burden of disease in those with asthma, copd, or cystic fi brosis. other complications include otitis media, pharyngitis, and wheeze in atopic people without asthma. the role of viruses in the origin of some of these diseases or their exacerbation is still unresolved. the lrt disease may mask the urt nature of the infection, favoring clinical diagnosis of an lrt illness. interestingly, during the h n pandemic, much of the parentinitiated healthcare visits from a birth cohort in the united states were not due to pandemic virus but hrv and hrsv [ ] . there is no known natural murine rhinovirus on which to base a small animal model of hrv infection, and mice are not natural hosts for hrvs. a recently developed model of airway disease using mengovirus (a picornavirus infecting rodents) may yield valuable in vivo airway infection and infl ammation data [ ] . hrvs are often detected in neonates and infants with lrt signs and symptoms because the very young have narrow, immature airways and are more signifi cantly affected by airway swelling, excessive secretions, and smooth muscle contraction [ ] . this may also be due to the relatively naive immunity of very young children. much of the more severe disease in hrv-positive children occurs in the youngest of them. some key examples are addressed below. for the common cold, as for any illness, accurate epidemiology and burden of disease data underpin the prioritization of preventing, treating, and further researching the etiological agent. to assign funds for researching the agent, health policy makers also need to understand how effi cacious and costeffective the development of an intervention will be [ ] . the host immune response to hrv replication is the main cause of the signs (quantifi able fever, rhinorrhea) and symptoms (feeling of fever, myalgia, headache, fatigue, and mood change) of a cold that the host experiences [ , , ] . a feature of common colds is increased vascular permeability which, enhanced by kinins, results in increased plasma protein (albumin and immunoglobulin [ig] g) levels in mucus, approaching the levels in serum [ ] . histamine levels do not rise in nasal secretions of otherwise healthy cold sufferers [ ] . during the resolving phase of the ari, glandular proteins (lysozyme, siga) predominate [ ] . the common cold syndrome is also described as rhinosinusitis (the agglomeration of rhinitis and sinusitis since they frequently clinically coexist) [ , ] . this consists of nasal discharge or rhinorrhea, nasal obstruction, sore throat, sinus pain, headache, sneezing, watery eyes, cough, fever, fatigue, muscle aches and pains, and mood changes [ , ] . these are caused directly or indirectly by viral infection; cough is the result of vagus nerve irritation by mucus; sneeze results from trigeminal nerve irritation; sore throat is likely due to the action of prostaglandins and bradykinins; and fever, psychological effects, fatigue, and myalgia are mediated by cytokines [ ] . hypertrophic adenoids have also been found to have a high proportion of viral, especially hrv, occupation regardless of host symptomatic state [ ] . observation of natural culture-confi rmed hrv colds in adults noted that cough usually started by day and was more persistent up to days later [ , ] . rhinorrhea, sneezing, and sore throat were reported by half or more of patients and headache by at least a quarter of cases [ , ] . as neutrophils accumulate at the site of primary urt infection, the myeloperoxidase in their azurophilic granules creates the yellow-green coloration of nasal mucus that was once considered a sign of bacterial superinfection [ , ] . a common cold caused by an hrv cannot be clinically distinguished from one that caused by any of the other respiratory viruses [ , ] . as is likely for a single hrv type, once the host has been infected by an hmpv, hpiv, ifv, etc., a secondary exposure to that same virus type will produce less severe clinical outcomes due to pre-primed host immunity. asthma is a clinical diagnosis made on the basis of patient history, physical examination, assessment of airway obstruction or reversibility, and response to bronchodilators [ ] . it is a complex chronic respiratory disease involving airway infl ammation, airfl ow obstruction, and airway hyperresponsiveness, which manifests as recurrent reversible attacks with deteriorating asthma control that are generated by interactions between infectious agents and other environmental and genetic factors that remain incompletely characterized [ ] . the mechanistic role for hrvs in asthma inception and exacerbation is not yet defi ned [ , ] but is being revealed as the extremely complex interplay between infl ammation due to virus versus that due to atopy is explored [ ] . possible virus-host interactions include (i) severe hrv infection of healthy infants which may result in subsequent development of asthma; (ii) hrvs may trigger asthma in children with a genetic predisposition toward atopy; (iii) repeated mild infections may protect against more asthmogenic/cytopathic viruses or the overdevelopment of the t h type response; and (iv) hrvs may simply exacerbate that which already exists [ ] . it is unclear if the risk of atopic asthma during infancy is increased by aris which affect the development of the immune system, or whether aris lead to asthma development in children with a genetic predisposition to more severe responses to infection [ , , ] , or a mix of both. in children with asthma, viruses have been detected in at least % of exacerbations ( % picornaviruses, probably hrvs [ ] ) and in % of adults [ ] . acute wheezing episodes (including bronchiolitis and acute asthma) are a frequent, epidemic, and seasonal lrt manifestation of urt respiratory virus infection of children from all ages, especially during the fi rst year of life [ , - ] . bacteria are not major factors in wheezing exacerbations [ ] . wheezing is blamed for high socioeconomic and healthcare costs, overuse of antibiotics, being the primary cause of hospitalization among children, and, rarely, for death [ , , ] . traditionally hrsv infection has most often been the virus causally associated with expiratory wheezing, wheezy bronchitis, or asthma exacerbations because of the virus's well-known ability to infect the lrt, its more frequent detection in some studies [ ] , and the low perceived likelihood of urt viruses such as hrvs replicating in the warmer lrt. nonetheless, periods of epidemic wheezing in the absence of high rates of hrsv detection are common [ , ] . hrvs even predominated in some culture-based studies of wheeze [ , ] . the coast study used sampling criteria that were intentionally designed to investigate the role of hrsv in illness, but instead indicated that hrvs were the most important predictor of subsequent wheezing in early childhood, and this is supported worldwide [ , , ] . the asthmatic airway is characterized by an infi ltration of eosinophils and th -type t cells (th cells) [ ] . in those with an atopic background, eosinophilia was more common, and the virus isolation rate was higher than in the nonatopic group [ ] . the cytokine and eosinophil activation profi les for hrsv-induced wheezing differ from those induced by hrv in which il is signifi cantly higher in serum and nasal aspirates than for hrsv [ ] . ip was the only cytokine signifi cantly elevated in all symptomatic wheezing groups [ ] . signifi cantly higher rates of hrv detection with more obvious lrt symptoms are more common in children with asthma than in non-asthmatic populations [ , , , ] . exacerbations of asthma are often preceded by a symptomatic rather than asymptomatic hrv infection [ , , , - ] although, in some instances, an exacerbation is the only sign of infection [ ] . reduced peak expiratory volume in children is especially associated with detection of respiratory picornaviruses [ ] . severe "wheezy bronchitis," a historical term describing an acute illness with preceding ari and characterized by cough, wheezing, breathlessness, and mucous production, was more often positive for a virus than mild disease [ ] . even the use of culture found that hrvs predominated in both urt and lrt (sputum containing becs) or combined respiratory tract samples [ ] . bacteria were often present with ifv, but not with hrvs [ ] . the airway epithelial cells form a physical barrier in addition to their roles in immune surveillance and regulatory control [ ] . however, the asthmatic bronchial epithelium is compromised by incomplete tight junctions that are more sensitive to airborne pollutants [ ] and most likely to allergens and respiratory viral infections. this is further specifically disturbed by hrv infection which reduces expression levels of tight and adherens junction proteins [ ] . in those with asthma, the presence of an hrv can induce illness that, while often more severe than in non-asthmatics, has been associated with signifi cantly different hrv load or duration of hrv rna detection in people with asthma compared to those without [ ] . hrv-c types are often detected in more serious clinical outcomes than hrv-a or -b [ ] although hospitalizations may be fewer for hrv-cs than the other species [ ] . aom is diagnosed by middle ear effusion (otorrhea) with simultaneous signs and symptoms of ari including fever, earache, rhinitis, cough, sore throat, chest wheeze, nocturnal restlessness, irritability, poor appetite, diarrhea, and vomiting. transient abnormal (negative) ear pressure upon tympanometry occurs in two-thirds to three quarters of uncomplicated colds among healthy children [ , ] . aom is a frequent reason for outpatient antibiotic therapy which can reduce the time to resolution of symptoms in infants and has been attributed to reducing the overall hospital burden of aom [ - ] . since a longitudinal day-care study in , the association between aom and viral urt infection has been coalescing, and it is now clear that aom often occurs with or shortly after a viral ari, most frequently in the young and occurring more often during winter than summer [ , ] . the use of infl uenza vaccines reduced aom occurrence by a third during an epidemic period [ ] , but the use of pneumococcal vaccine did not reduce the occurrence of aom overall, just that relatively small fraction ( %) due to the target bacteria [ ] . the isolation by culture and pcr detection of viruses from middle ear fl uids and the refractory nature of some aom cases to antibiotic therapies confi rmed that viruses play an important role in this illness [ , , ] . studies relying on underperforming culture-based techniques underestimated the role for viral aris [ , ] , but other studies using pcr techniques and including hrvs found them to be the most frequently detected virus in middle ear fl uids and nasopharyngeal secretions [ , ] . the use of pcr has identifi ed respiratory viruses, most often hrvs, in nasal secretions of - % of children with aom [ , ] . because virus is often detected in the nasopharynx at the same time as the middle ear fl uid, the question of the relevance of a pcr positive is a valid one [ ] . picornaviruses have been detected in % of nasopharyngeal swabs taken during cold season from aom-prone infants and young children, and large quantities of hrv rna have been detected by in situ hybridization of adenoid tissues from % of children with recurrent aom and/or adenoid hyperplasia [ , ] . in a cohort of children followed from to months and using culture-rt-pcr, hrvs in the urt were the second most frequent pathogens associated with aom, after hrsv [ ] . viruses, most often hrvs ( . % of aom with ari), were also detected concurrently with non-ari periods associated with aom episodes ( % of aom without ari) [ ] suggesting that aom may be the only manifestation of some hrv aris, just as wheezing sometimes is. in the united states, subjects were enrolled and followed in a birth cohort until the fi rst aom episode or between and months of age ]. hrvs accounted for % of viruses detected and % of specimens with a single virus detected. this dominance was maintained even through the h n infl uenza pandemic [ ] . in the day-care aom study mentioned above, primary acquisition of streptococcus pneumoniae or haemophilus infl uenzae had minimal importance as an initiation factor for aom with effusion, but nasopharyngeal colonization was important [ ] . animal studies have shown that virusbacteria interactions have a role in nasopharyngeal colonization and aom development [ ] . positive correlation has been made between hrv detection in aom-prone children and moraxella catarrhalis infection as well as a tendency toward the copresence of streptococcus pneumoniae [ ] . the presence of hrv-b was shown to increase adherence of s. pneumoniae in human tracheal epithelial cell cultures [ ] . it is believed that these three bacterial pathogens can colonize without symptoms until a viral ari shifts the balance toward a cytokine-mediated infl ammatory state [ ] . other diseases in which hrvs are often detected this disorder of older patients encompasses emphysema (alveolar destruction) and chronic bronchitis (large airway infl ammation with chronic mucous production) and describes a long-term obstruction to airfl ow in the lung (compared to asthma which is a reversible obstruction with normal fl ow between exacerbations). while bacteria are found in half of all exacerbations, antibiotic therapies have often yielded poor outcomes [ ] . hrv infections result in more copd exacerbations (~ % of cases [ ] ) than any other virus identifi ed to date [ , ] . an experimental human model of hrv infection in copd provided preliminary evidence that hrvs cause exacerbations [ ] . viral culture associated symptomatic hrv infections with exacerbations among chronic bronchitics, including cases of isolation from sputum (lrt sample) in the absence of hrv in the urt [ ] . adding the measurement of an infl ammatory marker in the serum, like il- , further improves the speed of predicting an infectious etiology for exacerbations of copd [ ] . pneumonia is a disease that often occurs early in life, is responsible for millions of deaths each year [ ] , and is caused by viral and/or bacterial infections. a diagnosis of pneumonia requires a radiologically confi rmed infl ammatory infi ltration of the lung tissue. childhood communityacquired pneumonia (cap) is common in developing countries [ ] . cap also complicates existing chronic medical conditions and takes advantage of immunosenesence [ ] . the role of hrvs in contributing to the development of bacterial pneumonia is likely underestimated [ , ] . determining an etiology is confounded by the rarity of obtaining lrt specimens, by short-term studies, and by the complex milieu of viruses and bacteria involved. less invasive sampling of the urt permits more routine sampling and screening, and so convenience and reduced risk have led to the detection of putative pathogens in the urt with the general assumption that they account for lrt disease, especially in children under the age of years [ ] . pneumonia studies are complicated by the lack of a suitable control group; sputum is not produced from the healthy lower airway and needle aspiration, while a gold standard is also a hospital procedure with some risk [ ] . studies that are comprehensive and use sensitive molecular testing are also rare for the study of cap etiology. when used for cap investigations, pcr methods almost double the microbiological diagnoses over conventional culture and serology techniques, especially improving the identifi cation of mixed infections and fastidious viruses [ ] . rapid diagnosis aids management and helps make decisions about treatment, while prolonged searching for an etiological agent leads to further invasive testing [ , ] . at least a quarter of clinical cap cases remain unsupported by microbiological fi ndings [ , ] . infections causing pneumonia vary with age and vaccination status [ ] . viruses can be detected in up to % of infants ( - months of age) with pneumonia, and these cases follow a seasonal pattern [ , ] . bacteria can also be detected in over % of infants and older children, the elderly, and those with severe cap [ , ] . studies that predated the use of pcr pronounced hrsv, followed by hrvs, the major viral contributors to cap, with viruses comprising - % of childhood pneumonia cases [ , ] . in the pcr age, the role of hrvs has received increasing attention, and they are increasingly the major viral group detected from both urt and lrt (sputum) specimens of children with cap. this holds true even when studies extend across or more years, which presumably would account for seasonal variation in virus prevalence [ , , , ] . it is suspected that viruses such as hrv prepare the way for subsequent bacterial infection in some direct or indirect fashion [ , , , ] . there are laboratory data which support this [ ] as well as observational data showing a high proportion of hrvbacterial co-detections [ , ] . mixed infections including viruses are a possible cause of antibacterial treatment failure and sometimes a puzzle for physicians. mixed infections occur frequently in lrt diseases such as pneumonia, which is not surprising since new techniques make it clear that the lungs are not the sterile environments we once thought [ , , , ] . viral-bacterial coinfections can comprise % of patients, while viral-viral ( - %) and bacterial-bacterial ( - %) are much less common [ , , , , ] . hrsv or hrv is often co-detected with s. pneumoniae in urt samples [ , , ] . hrv detections dominate in younger children with pneumonia during peak hrv seasons, although frequently in co-detections with other viruses [ ] . acute bronchitis (less than -week duration in children) is defi ned as a sudden cough that often results from large airway infection and frequently involves viruses. croup or laryngotracheobronchitis (viral or recurrent [ ] ) is a common lrt illness in children that includes the trachea and larynx as well as the larger airways, resulting in a barking cough. patients with croup most often have a viral infection with some role for hrvs, although the extent of this is unclear [ , ] . despite testing, a third of cases remain without a viral etiology [ ] . tracheobronchitis resulted from some hrv-a infection of volunteers [ ] . chest pain and cough have been reported in half or more of adults with hrv infection [ ] as well as in children and adults with hrvs detected during exacerbations of bronchitis, with or without an associated ari [ , ] . bronchiolitis occurs seasonally, especially in winter, in infants ( - months of age), affecting the small peripheral bronchioles. winter is the peak season for hrsv circulation, but not usually for hrv. bronchiolitis is a clinical diagnosis encompassing various disease entities and is most often reported in association with detection of hrsv, a winter virus [ , ] . however, hrvs make up the majority of hrsv-negative bronchiolitis cases [ ] , and hrvs are co-detected with hrsv for which hospitalization is prolonged compared to cases positive for either virus alone [ ] . those children positive for an hrv during a clinically diagnosed bout of bronchiolitis have a signifi cantly higher risk of recurrent wheezing in the subsequent year than those in whom another virus is detected [ ] . hrvs were reported in over fi vefold more cases of bronchiolitis than hrsv among patients in a -year prospective cohort of very low birth weight infants in buenos aires, argentina [ ] . after a viral ari, some proportion of infections may be complicated by sinusitis (infl ammation of the sinus mucosa), the extent of which may be underestimated in children if the ari is mild and unattended by parents [ ] . symptoms may include sinus pain, headache, facial pain, discolored nasal discharge, postnasal drip, cough, sore throat, malaise, and sometimes fever (more so in children) [ , ] . the precise role for viruses and bacteria in sinusitis is still unclear [ ] . sinusitis is a common comorbidity in those with asthma [ ] . the in situ presence of hrv-b rna in maxillary sinus epithelium was reported in seven of adults with acute sinusitis [ ] . hrvs were also detected by pcr in half of adults with acute maxillary sinusitis; half of the hrv positives were negative for any bacteria [ ] . the common cold is often associated with computed tomographically confi rmed sinus cavity occlusion or abnormality in adults with self-diagnosed aris [ , ] . magnetic resonance imaging identifi ed reversible abnormalities of the paranasal sinuses in a third of healthy adult volunteers following challenge with hrv-a [ ] . further evidence of the tropism of hrvs for sinus tissue comes from it being, so far, the only successful host for in vitro hrv-c replication [ ] . culture-and serology-based testing has shown that virus infections in cystic fi brosis (cf) patients occur with the same prevalence as the general community, but the consequences of infection are more obvious or severe. these include deterioration of lung function, cough, increased expectoration and weight loss, and a synergistic increase in bacterial growth or acquisition of new bacterial infections [ , - ] . the mechanism behind the acquisition of new bacteria is still unknown and not always observed [ ] , but may involve a reduction in the host's immune response or viral damage to the respiratory epithelium. there is circumstantial evidence that hrv infections have been associated with respiratory exacerbations in cystic fi brosis patients [ , ] , albeit in very low numbers by nonmolecular studies [ ] and without a significantly different clinical outcome from non-hrv aris in these patients [ ] . molecular methods have not yet been applied regularly, thoroughly, and systematically, but they generally fi nd hrvs to be prominent among cf children with ari-associated respiratory exacerbation and involved in mixed viral-bacterial infections [ ] . hand washing and disinfectant wipes have been shown to be effective methods of interrupting transfer from fomites to the nose or to conjunctivae [ , , ] . however, with eye rubbing, face touching, and nose-picking occurring frequently [ , ] , self-inoculation often outpaces personal hygiene, particularly in the young. hand disinfection is frequently recommended for prevention of hrv infection but has not been supported by controlled clinical trials in a natural setting [ ] despite good results in experimental tests [ ] . ethanol-containing disinfectants were more effective than simple hand washing with soap and water for removal of hrv-a inoculum, as assessed by culture, and the inclusion of organic acids afforded a residual antiviral effect [ - ] . however, continual hand washing with extra ingredients resulted in skin irritation [ ] . the experimental testing [ , ] may have been biased by short study periods, the absence of a mucus carrier to mimic natural surface deposition and overly stringent control over virus application/hand disinfection compared with the natural study. additionally, the natural setting study used pcr [ ] which detects hrvs more often than culture. the disparity between outcomes may also refl ect the contribution of airborne hrv transmission. because of the absence of a vaccine or specifi c antiviral, the most popular method of intervention in uncomplicated hrv aris is treatment of the symptoms. this is achieved using analgesics, decongestants, antihistamines, and antitussives. due to a lack of studies, data are limited on the effectiveness of over-the-counter common cold medications for children [ ] . anticholinergic agents have proven useful to reduce rhinorrhea [ ] . for controlling symptoms in those with exacerbated asthma, most of which do not require hospitalization, bronchodilators and oral corticosteroids are the main treatments [ ] . the interruption of proinfl ammatory immune responses or specifi c signaling pathways using steroids, or other novel therapeutics, may prove to be a more robust approach for treating hrv infections; they have not been successful for hrsv [ ] . when initiated early in the illness, a combination of antiviral (ifn-α b) and anti-infl ammatory (chlorpheniramine) components showed promise for interrupting nasal viral replication and symptoms [ ] . antiviral agents (table . ) require early application to effectively precede the pathogenic immune response to hrv infection [ ] , but they often fail to reproduce their in vitro successes in vivo. most antirhinoviral drugs are based on capsid-binding agents (fig. . ) . additionally, oral delivery can complicate drug safety because this route increases the risk of systemic side effects compared to a nasal or topical route, but these risks must be considered alongside the disease to be treated; drug side effects are disproportionately severe compared to a common cold than to a severe asthma exacerbation. a systemic route is benefi cial if an effect is sought on hrv replication sites that are otherwise inaccessible, such as those not associated with respiratory tract illness [ ] . the recent discovery of the new species, hrv-c, has shone a bright light on how little was known about the hrvs. the hrv-cs and also the newly discovered hrv-as and hrv-bs are fastidious in culture, with a single report of hrv-c growth in primary sinus tissue, and the identity of a cellular receptor still unknown. thus, it is diffi cult to proceed in many areas, including basic virology, seroepidemiology, immunobiology, and antiviral testing. determination of the receptors for these new hrvs would aid the search for a more accessible culture system. there would be great interest in a vaccine for some or all of the hrvs, but with increasing evidence of the interactions between hrvs, their hosts, and other respiratory viruses, it may not be wise to interfere before we fully understand what the impact of losing a constantly circulating hrv challenge would be. antivirals specifi cally targeting the hrvs may be a better bet, but routine hrv testing and genotyping will fi rst need to be more widespread as surveillance for antiviral resistance will be an important component of monitoring the success of any intervention. studies to determine whether there are differences in clinical and immunobiological impact between the many different types are lacking but would greatly improve our ability to plan future routine testing, understand all the clinical responses to the diverse hrvs and to outbreaks of ari, and improve hrv epidemiology. it is interesting to note that the hrv-bs are signifi cantly underrepresented in hrv detections. we do not yet know their niche or clinical impact. it may be possible that hrv-bs are the most well adapted of the hrvs, causing little to no detectable clinical impact, or they may create a different impact than that which we expect, or they may be a species in decline. the jury remains out on whether hrvs cause or are involved in the development of asthma or merely trigger exacerbations once asthma is established. with a very high healthcare impact from asthma around the world and atopic conditions that may be exacerbated by hrvs on the rise, this is an important area for 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picornavirus infection: a review of clinical outcomes rhinitis, sinusitis, and asthma rhinovirus rna in the maxillary sinus epithelium of adult patients with acute sinusitis computed tomographic study of the common cold physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds viral infections of the respiratory tract in patients with cystic fi brosis respiratory infections in cystic fi brosis patients caused by virus, chlamydia and mycoplasma -possible synergism with pseudomonas aeruginosa clinical manifestations of exacerbations of cystic fi brosis associated with nonbacterial infections association of respiratory viral infections with pulmonary deterioration in patients with cystic fi brosis the role of respiratory viruses in cystic fi brosis rhinovirus c and respiratory exacerbations in children with cystic fi brosis infective respiratory exacerbations in young adults with cystic fi brosis: role of viruses and atypical microorganisms a randomized trial of the effi cacy of hand disinfection for prevention of rhinovirus infection effectiveness of hand sanitizers with and without organic acids for removal of rhinovirus from hands effi cacy of organic acids in hand cleansers for prevention of rhinovirus infections virucidal hand treatments for prevention of rhinovirus infection over-the-counter cold medications: a critical review of clinical trials between and ipratropium nasal spray: a new treatment for rhinorrhea in the common cold a -versus -day course of oral corticosteroids for children with asthma exacerbations who are not hospitalised: a randomised controlled trial combined antiviralantimediator treatment for the common cold in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus c protease treatment of picornavirus infections the common cold: control? uncommon(ly considered) manifestations of infection with rhinovirus, agent of the common cold intranasal interferon-a treatment of experimental rhinoviral colds human tolerance and histopathologic effects of long-term administration of intranasal interferon-a safety and effi cacy of intranasal pirodavir (r ) in experimental rhinovirus infection combating enterovirus replication: state-of-the-art on antiviral research rhinovirus chemotherapy a comparison of the anti-rhinoviral drug binding pocket in hrv and hrv a a new oral rhinovirus inhibitor bta human rhinovirus c protease as a potential target for the development of antiviral agents in vitro resistance studies of rupintrivir, a novel inhibitor of human rhinovirus c protease effi cacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofl oxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells pellino- selectively regulates epithelial cell responses to rhinovirus azithromycin induces antiviral responses in bronchial epithelial cells suggested reading newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? human rhinoviruses: the cold wars resume proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types cold wars: the fi ght against the common cold acknowledgments we wish to sincerely thank the following for valuable discussions: john upham, anne chang, danielle wurzel, michael nissen, ron turner james gern, and stephen b. liggett. we are grateful for the extreme patience of corin and ronan mackay, slightly less so for their frequent provision of our fi rsthand experience in hrv clinical symptoms. key: cord- -n majqes authors: modrow, susanne; falke, dietrich; truyen, uwe; schätzl, hermann title: historical overview date: - - journal: molecular virology doi: . / - - - - _ sha: doc_id: cord_uid: n majqes “poisons” were originally considered as the causative agents of illnesses that we know as viral diseases today. at that time, there were no standard methods to detect pathogenic (disease-causing) organisms such as bacteria and protozoa in the supposed “poisonous materials”. only animal experiments performed by louis pasteur at the end of the nineteenth century, in which no dilution of the poisonous properties was achieved even after several passages, suggested that the disease-causing agent was able to multiply in the organism. therefore, there was talk of a reproducible “virus” (latin for “poison” or “slime”) in living organisms, and later also in cells. in st. petersburg in , dimitri i. ivanovski demonstrated that tobacco mosaic disease is caused by an “ultrafilterable” agent, whose size is significantly smaller than that of bacteria: tobacco mosaic virus (bacteria filters have a pore size of approximately . μm, however, most viruses are smaller than . μm). soon afterwards, martinus willem beijerinck came to the same conclusion: he developed, for the first time, the notion of a self-replicating, “liquid” agent (contagium vivum fluidum). the discovery of foot-and-mouth disease virus by friedrich loeffler and paul frosch in greifswald in was the first evidence of an animal pathogenic virus. since when have we known of viruses? "poisons" were originally considered as the causative agents of illnesses that we know as viral diseases today. at that time, there were no standard methods to detect pathogenic (disease-causing) organisms such as bacteria and protozoa in the supposed "poisonous materials". only animal experiments performed by louis pasteur at the end of the nineteenth century, in which no dilution of the poisonous properties was achieved even after several passages, suggested that the disease-causing agent was able to multiply in the organism. therefore, there was talk of a reproducible "virus" (latin for "poison" or "slime") in living organisms, and later also in cells. in st. petersburg in , dimitri i. ivanovski demonstrated that tobacco mosaic disease is caused by an "ultrafilterable" agent, whose size is significantly smaller than that of bacteria: tobacco mosaic virus (bacteria filters have a pore size of approximately . mm, however, most viruses are smaller than . mm). soon afterwards, martinus willem beijerinck came to the same conclusion: he developed, for the first time, the notion of a self-replicating, "liquid" agent (contagium vivum fluidum). the discovery of foot-and-mouth disease virus by friedrich loeffler and paul frosch in greifswald in was the first evidence of an animal pathogenic virus. however, it can be retrospectively documented that as long as , years agowithout knowledge of the nature of the pathogens -practices were implemented which today would be described as vaccinations against viral diseases. in ancient china, india and egypt, devastating smallpox epidemics must frequently have occurred; pharaoh ramses v -as his death mask shows -most likely died of an infection of smallpox virus. as observed at that time, people who had survived the disease were spared from the disease in further epidemics; therefore, they had to have developed some kind of protection caused by the first disease -they were immune. this protective status could also be induced artificially; when dried scabs of smallpox were transmitted to healthy people, they were at least partially protected against smallpox -a measure that we now denominate variolation (the medical term for smallpox is "variola"; ▶ sect. . ). historical descriptions indicate that smallpox was used as a biological weapon at that time. in the eighteenth century, it was discovered in england and germany that overcoming milker's nodule disease, which is triggered by a virus related to smallpox, confers protection against genuine smallpox. edward jenner must have been aware of these observations in when he transmitted swinepox and cowpox material, as a sort of vaccine, initially to his first-born son and later to james phipps, a young cowherd. both boys remained healthy following exposure to the human pathogenic smallpox virus by inoculation of smallpox pus; in fact, a protective effect was generated by this first deliberate "virological experiment". knowledge of this vaccination spread very rapidly from england to the european continent and the usa. the term "vaccination" is derived from the latin vacca, which means "cow". vaccinations were soon prescribed by law and this led to a gradual reduction of the dreaded disease. in the former german reich, a first vaccination law was enacted in . however, it still took about years until a human, ali maov maalin, was naturally infected with smallpox (in somalia) for the last time (in ), after the who had conducted a worldwide vaccination programme. today, the disease is considered eradicated. on a similar basis, i.e., without precise knowledge of the nature of the pathogen, louis pasteur developed a vaccine against rabies (▶ sect. . . ) in paris in . he transmitted the disease intracerebrally to rabbits in , seeing the causative agent rather in unknown and invisible microbes. as he demonstrated, the pathogen lost its disease-inducing properties by continuous transmission in these animals. in this way, pasteur achieved the basis for a vaccine virus (virus fixe), which, in contrast to the wild-type pathogen (virus de rue), was characterized by a constant incubation period. rubbed and dried spinal cord of rabbits that had been inoculated with the virus fixe was no longer infectious, but caused (initially in dogs) protection against rabies. for the first time, in , pasteur inoculated a -year-old alsatian boy named joseph meister with this material. the boy had been bitten by a rabid dog days before, and finally survived, by virtue of the protective effect induced by the vaccine. they especially noticed the striking ability of these agents to lyse bacteria and, therefore, called them bacteriophages -according to the greek word phagein, which means "to eat". the exploration of the nature of bacteriophages has provided virology with important findings and impulses both in methodological and in conceptual terms. many of the steps that characterize a viral infection were first discovered in experiments with bacterial viruses: such processes include attachment and penetration, the reproduction-cycledependent regulation of gene expression that results in early and late synthesized proteins, and lysogeny, which is associated with the existence of prophages. the study of viruses and their attributes was particularly difficult because they, in contrast to bacteria, could not be propagated in artificial culture media. however, it could be ascertained that some of the pathogens isolated from diseased people were transmissible to animals, in which they were able to reproduce. for example, human herpes simplex virus was transmitted from human skin blisters to the cornea of rabbits by wilhelm gr€ uter in marburg in . the extraordinary susceptibility of ferrets allowed the isolation of influenza a virus by christopher andrews, wilson smith and patrik laidlaw from pharyngonasal fluid of a sick person for the first time in . animal experiments also provided many insights into the pathogenesis of viral infections from another point of view. richard e. shope discovered rabbit papillomavirus in , and thus the first tumour virus, which -as was later shown -contains a dna genome. he suspected that such a virus could exist in a latent form as a provirus in the organism. in addition, the discovery that skin cancer can develop from benign skin papillomas is attributed to him. hence, malignant tumours develop in two or more steps -nowadays a universally accepted notion. shope further observed that the incidence of cancer differs in different rabbit breeds, and thus genetic traits of the host also influence the development of cancer. in the framework of animal experiments, erich traub made an important observation while studying the virus of lymphocytic choriomeningitis in princeton in : when pregnant mice were infected with the virus, the virus was transferred to the embryos; mother animals became sick from meningitis, and produced protective antibodies in the further course of the disease. by contrast, the newborn mice remained healthy, but secreted large quantities of the virus for life without developing a specific immune response against the pathogen. this discovery was the first example of an immune tolerance induced by a virus, but the general significance of this phenomenon was not recognized, and the now popular term was not coined (▶ sect. . . ). later, lymphocytic choriomeningitis was shown to be an immunologically related disease. the restriction of cytotoxic t lymphocytes by certain genetically determined types of mhc proteins was demonstrated for the first time in this model by rolf m. zinkernagel und peter c. doherty in . the above-mentioned experiments of traub evidenced also for the first time the intrauterine transmission of a virus. this raised the question of similar ways of infection in humans. in fact, after a severe rubella epidemic in australia in , sir norman gregg observed embryopathies when pregnant women were affected by the infection. as demonstrated later, these malformations were the result of intrauterine transmission of rubella virus. in , coxsackievirus was discovered after transmission of virus-containing stool extracts into newborn mice (coxsackie is a small town in the us state of new york). later in new york, ludwik gross isolated murine leukaemia virus from blood cells. besides the importance for tumour virus research, these observations aroused interest in the question concerning the basis of the high susceptibility of newborn animals to viral infections, and suggested investigations on the innate resistance of an organism to infections as well as the time and the causes of its formation. laborious and time-consuming experiments were initially the only way to prove the existence of viruses: therefore, there simpler methods were sought. one way involved the observation of so-called inclusion bodies in virus-infected tissues, which were soon judged as an indication for proliferation of the pathogen; as we now know, inclusion bodies are the accumulation of viral proteins and particles in the cytoplasm or the nucleus. the first inclusion bodies were discovered by dimitri i. ivanovski; at the same time, guiseppe guarnieri discovered similar deposits in cells infected with smallpox virus, then in , adelchi negri found inclusion bodies in ganglion cells of rabid animals, which were later named after him. thus, there were at least simple dye detection methods for some viral diseases. however, virus culture methods became available later. in the s, it was found that embryonated chicken eggs are appropriate for the propagation of some viral species. between and , a pandemic emerging viral disease, spanish flu, claimed more than million lives, i.e., more than in the first world war. after cultivation of the virus responsible in embryonated chicken eggs in , their haemagglutinating properties were discovered in (i.e., their ability to agglutinate red blood cells), thereby laying the basis for the development of haemagglutination tests to detect viruses. another important step in the history of modern virology was the development of the first ultracentrifuges, which became available at about the same time. they made possible the sedimentation and concentration of the minute virus particles. however, the breakthrough in the elucidation of the pathogenetic mechanism of influenza viruses was only possible by the development of molecular biological techniques that allowed the investigation of the genetic material of this pathogen, which exists in the form of singlestranded rna. its sequencing revealed the genetic reasons for the previously not understood ability of influenza viruses to change their antigenic properties at periodic intervals (▶ sect. . . ). however, it was particularly the donation-funded research of poliomyelitis (▶ sect. . . ) which provided crucial new insights. retrospectively, it represents the actual transition to molecular biological research of viral infections. the strong increase in the incidence of polio and the number of deaths -a result of enhanced hygiene standards and the shift of infection rates into later years of life -brought about in the usa the establishment of the national polio foundation by franklin d. roosevelt, himself a victim of this disease, in the early s. with the funds raised, a major research programme was initiated whose coronation was the discovery of the cytopathic effect by john f. enders, thomas h. weller and frederik c. robbins in . in , hugh b. and mary c. maitland had already introduced the method of tissue culture, in which the cells of small tissue pieces were cultivated in serumcontaining liquid media and infected with viruses. successful viral replication was then demonstrated in animal experiments or by detecting the presence of inclusion bodies. when antibiotics became available in the s, it was then possible to largely prevent bacterial contaminations in cultures, which led to much simpler handling in this method. polioviruses were cultivated in embryonic human cells of fixed kidney tissue fragments, and thereby cellular alterations were easily identifiable. this diagnostically valuable cytopathic effect drove the development of virology forwards. it was the basis for the plaque test that was developed by renato dulbecco and marguerite vogt in , which rendered possible, for the first time, the quantitative determination of the number of infectious particles in cell culture. the capability to cultivate polioviruses under controlled conditions in vitro was the basis for the development of the two polio vaccines: the inactivated vaccine developed by jonas e. salk and the live vaccine with attenuated, i.e., weakened polioviruses, developed by albert b. sabin. both vaccines are still in use today. later, vaccines against measles, rubella and mumps were also produced following the principle applied by sabin. by using the method of cell culture, it was possible to cultivate even yellow fever virus, vaccinia virus and rabies virus in vitro. wallace p. rowe isolated adenoviruses from cultures of human tonsil tissue after a long cultivation period in . a further development of viral cultivation in vitro provided the method of co-cultivation, which consists in the addition of indicator cells to the tissue cultures, which indicate viral replication by the occurrence of a cytopathic effect. in this manner, the existence of herpes simplex virus was verified in latently infected human dorsal root ganglia in , whereas direct virus detection was not possible at that time. until then, it had generally been assumed that in the course of viral infections the pathogen would be eliminated completely from the body by the resulting antibodies. the occurrence of herpes blisters as recurrent disease in people with antibodies -known as herpes immunological paradox -refuted that notion (▶ sect. . ). ernest w. goodpasture had previously suggested that the trigeminal ganglia should contain a "latent" form of the virus. after such a virus had been detected by co-cultivation, it was recognized that there are a number of infections with latent or persistent viruses, which -independently of the illness symptoms -are excreted either intermittently (e.g., herpes simplex virus) or permanently (such as epstein-barr virus). the primary isolation of human immunodeficiency virus (hiv) was also accomplished by co-cultivation of lymph node biopsy material from an aids patient with suitable t lymphocytes. concurrently with the rather practical benefits from developments in viral cultivation, interest in general biological issues became increasingly important. the crystallization of tobacco mosaic virus from liquid media by wendell stanley in california in stimulated discussions as to whether viruses are dead or living matter. the main question concerned, however, the nature and structure of the genetic material, which was found to be nucleic acid by the seminal experiments of oswald t. avery, colin mcleod and maclyn mccarty with pneumococci in . in , alfred d. hershey and martha chase proved that during bacteriophage t infections only dna, but not the protein shell of the virus, penetrates into the bacterial cell. this demonstrated that nucleic acids are the carrier of genetic information. a few years later, in , gerhard schramm and heinz fraenkel-conrat showed independently in tobacco mosaic virus that rna can also be infectious. schramm and his colleagues had already described in germany in that tobacco mosaic virus is composed of rna and proteins; however, these findings attracted initially only little attention. the base ratios in dna molecules (a ¼ t and c ¼ g) that were discovered and developed by edwin chargaff enabled james d. watson and francis h. crick, in connection with the rosalind franklin's x-ray structural analysis, to develop their model of the dna double helix in . in , matthew meselson and franklin w. stahl demonstrated that dna is replicated semiconservatively during cell division. these fundamental insights created the way for the elucidation of basic molecular biological processes which are nowadays generally familiar. the now common molecular genetic, biochemical and immunological methods allow the detection of viruses in the organs and the study of their spread in the organism. the function and effect of viral genes can be explored in isolation and in interaction with other viral or cellular components. today, many viruses can be cultivated in large quantities in vitro in order to resolve their morphology and particle structure as well as to sequence their genetic information. in the case of non-cultivable viruses, modern molecular biological methods are available which make possible the investigation of the pathogens. virus-cell interactions can be explored, and provide important insights into the mechanisms of viral replication. on the other hand, many of the molecular processes in eukaryotic cells have been elucidated by using viruses as cell research tools. in this way, the process of rna splicing was originally described in adenoviruses, in which widely separated gene segments are assembled into single messenger rna molecules after transcription. the fact that dna is arranged with histone proteins into nucleosome structures within the nucleus was also first discovered in a virus, simian virus . in addition, even enhancers were originally described in viruses, i.e., the specific dna regions that increase the expression of certain genes in a localization-and orientationindependent manner. several of these viral regulatory elements have been used for alternative purposes: for example, the most frequently used promoter/ enhancer sequences to control the expression of heterologous genes in commercially available vectors are derived from cytomegalovirus. this immediate-early promoter/enhancer region actually regulates the expression of early genes of the virus (see ▶ sect. . ). furthermore, the transfer of nucleic acid sequences and foreign genes by viral transduction, e.g., using vector systems based on the functions of adenoviruses or retroviruses, is today an indispensable constituent of molecular and cell biology, and has essentially contributed to the development of gene therapy procedures. what is the importance of the henle-koch postulates? the study of the epidemiology and pathogenesis of infectious diseases raises the fundamental question of how can it be proved that an illness is caused by infection with a bacterium or a virus. robert koch derived four postulates from his work with anthrax bacteria between and , which his teacher jacob henle had previously developed as a hypothesis from the study of so-called miasma and contagions, i.e., the animate or inanimate disease and infection agents: . a pathogen must be detected in all cases of a certain disease, but it must be absent in healthy organisms. . the pathogen must be cultivable on culture media or in suitable cell cultures in the form of pure cultures. . healthy animals must develop the same disease after inoculation of the pathogen. . the causative agent must be reisolated from the infected animals. koch noted that the postulates do not comply in all cases. he acknowledged that there are healthy and long-term carriers and that a normal flora of facultative pathogenic bacteria exists. in the realm of virology, not all pathogens comply with these postulates. positive examples are measles virus (▶ sect. . . ), human poxviruses (▶ sect. . . ), canine and feline parvoviruses (▶ sect. . . ) and classical swine fever virus (▶ sect. . . ). with regard to viral diseases, charles river proposed modifications of the postulates in . the exceptions concern preferentially latent or persistent viral infections and the fact that tissue and organ damage or tumour formation cannot always be reproduced as consequences of infection. taken from epidemiology, the "evans postulates" are a worthy supplement (▶ table . ). they show the aetiological importance of a pathogen for a clinical picture if, among others, the pathogen is significantly more frequent in an exposed group and the disease in this population is commoner than in a non-exposed group. similarly, an immune response should be detectable in the affected collectives. the evans postulates are valuable particularly for multifactorial infectious diseases, such as canine kennel cough and porcine circovirus infection (▶ sect. . . ). the further development of virological and immunochemical detection methods in recent years has allowed the use of additional criteria for the causal relationship between a virus and a disease. these include the detection of a specific humoral or cellular immune response against the pathogen, i.e., igm or igg antibodies and specific stimulatable lymphocytes, and the detection of viral proteins, enzyme activities, dna or rna by in vitro and in situ methods. the specific detection of viral nucleic acids in tissues is especially important for the aetiological correlation between persistent viral infections and cancer. the fulfilment of koch's postulates or their modifications is still essential for the development of aetiological relationships between the pathogen and the host. what is the interrelationship between virus research, cancer research, neurobiology and immunology? as early as in , it was demonstrated that rous sarcoma virus can cause cancer. in , margarete vogt and renato dulbecco observed the transformation from benign to malignant cells after infection with murine polyomavirus in vitro. after animals had been inoculated, these cells generated tumours. shortly afterwards, it was also discovered that rous sarcoma virus can transform cells in vitro. thus, tumour virus research became a driving force of virology. it has enriched the realm of cancer research with decisive impulses both in conceptual and in methodological terms. experiments with the oncogenic polyomavirus also showed that its dissemination within mouse populations can be followed by serological methods. this aroused the hope that it would be possible to reduce cancer development to viral agents and to study its nature using the classical methods of epidemiology such as virus isolation and antibody detection. in particular, the study of oncogenic retroviruses in animal systems provided seminal insights into the molecular processes that lead to carcinogenesis (▶ sect. . ). by investigating oncogenic retroviruses in , howard temin and david baltimore discovered reverse transcriptase -an enzyme that transcribes the single-stranded rna of retroviruses into double-stranded dna. after integration of the viral genetic information into the genome of the host, these viruses lose their individual existence. a few years earlier, temin had described that an inhibitor of dna synthesis prevents replication of rous sarcoma virus, which should not be the case in a typical rna virus. the integration of a viral genome, which is then present as a provirus, has been associated with tumour development. this event interrupts the continuity of the genome of the cell, as cellular and viral genes can be amplified and destroyed, or their expression can be activated by recombination with viral promoters. as mentioned above, shope had already described that carcinomas arise from papillomas by a two-stage or multistage process. the development of cervical carcinoma caused by human papillomaviruses and that of primary liver cancer caused by hepatitis c virus or hepatitis b virus are similar. even epstein-barr virus exerts its tumorigenic effect in a complex way: the viral dna is detectable in nasopharyngeal carcinoma tumour cells and in various lymphomas (african burkitt's lymphoma). the cells are infected and immortalized, but do not produce infectious virus particles. furthermore, chromosomal translocations are found in b-cell lymphomas (▶ sect. . ). however, malignant transformation develops in a multistep process by interaction with other factors, such as malaria, which contributes to a chronic stimulation of cells. research on the molecular processes that occur in infections with papillomaviruses, hepatitis b virus and retroviruses has led to the development of vaccines which induce protection against the respective viral infection and are capable of preventing the development of cancer as a long-term consequence. the vaccination strategy in southeast asia that was initiated and promoted by the who years ago has led to a significant decrease of primary liver carcinoma, which is caused by hepatitis b virus infections (▶ sect. . ). in veterinary medicine, vaccines against feline leukaemia virus have proved that cats are protected against infections by this exogenous retrovirus and that tumour formation can be prevented (▶ sect. . ). the detailed investigation of the molecular biology and pathogenesis of human papillomavirus infections by the research group of harald zur hausen at the german cancer research center (dkfz) paved the way for the development of appropriate vaccines. these have been available for several years and protect against infections with the highly oncogenic papillomaviruses: they prevent the possible development of cervical carcinoma -one of the commonest cancers in women (▶ chap. , ▶ sect. . ). in september , harald zur hausen was awarded the nobel prize in physiology or medicine for his work concerning the role of papillomaviruses in cervical cancer. the term "slow virus infections" was initially coined by björn sigurdsson for maedi disease of sheep in iceland in . maedi-visna virus causes respiratory symptoms in a slow and progressive disease after very long incubation and latency periods. maedi-visna virus thus became a model for a range of pathogens that cause diseases with a similar protracted course (▶ sect. . . ). the exploration of its pathogenesis revealed that most slow virus infections are caused by pathogens which are usually associated with other diseases. slow virus infections principally affect the central nervous system and are caused, for example, by measles virus and jc polyomavirus. subacute sclerosing panencephalitis is probably caused by mutations in measles virus genes, which lead to the emergence of defective virus particles (▶ sect. . . ) . in progressive multifocal leucoencephalopathy, which is triggered by jc polyomavirus, the virus seemingly enters the brain very early and persists there for a long time before the disease breaks out as a result of damage to the immune system (e.g., by infection with hiv; ▶ sect. . . ). infections with hiv can also be considered as a slow virus disease. similarly, prion diseases also progress along the lines of a slow virus infection, but they are caused by non-viral pathogens and have a fundamentally different pathogenesis (▶ chap. ). working on yellow fever virus, m. hoskins, g.m. findlay and f. maccallum discovered the phenomenon of interference in : if an avirulent virus was injected into an experimental animal, the animal was protected from the consequences of infection by a virulent strain when it was applied within the next h, i.e., before the onset of an immune response. in , alick isaacs and jean lindenmann showed that interferon is responsible for the interference effect. it is species-specific, inducible and belongs to a group of substances that are known as cytokines today. interferons play an important role in the primary, non-specific defence against viral infections and in stimulating the immune system. the observation that antiviral interferon preparations also have tumour-inhibiting effects was surprising. generally, cytokines are synthesized when a suitable inducer binds as a ligand to its receptor on the cell membrane, thus triggering specific signal transduction processes in the cell (▶ chap. ). attempts have been made to develop antiviral chemotherapeutic agents since about . in retrospect, this search can be divided into three stages: the first successful experiments for therapy of a viral infection were performed by josef wollensak and herbert e. kaufman around in herpetic keratitis. they used substances that inhibit viral replication in vitro and were known from experimental cancer therapy. however, the selectivity of these substances, i.e., their ability to selectively influence viral and not cellular processes, was only slight because of the high cytotoxicity of the compounds. after the discovery of virus-coded enzymes such as thymidine kinases, dna polymerases and proteases, it was possible to address the development of specific inhibitors. antiviral drugs such as amantadine against influenza a virus (▶ sect. . ) and adenine arabinoside and acyclovir (acycloguanosine) against herpes simplex virus (▶ sect. . ) were found by targeted empiricism, i.e., by attempting to find a compound that selectively influences viral reproduction among many compounds with similar effects. the achievement of gertrude elion and her staff to use acyclovir as a systemically applicable and selective antiviral drug in herpes encephalitis was an important milestone in chemotherapeutic research. she was awarded the nobel prize in physiology or medicine in for her work. after the development of dna sequencing techniques, the experimental chemotherapy of viral infections entered its third stage. virus-encoded enzymes were discovered such as the retroviral protease and neuraminidase of influenza viruses, and it was possible to build structural models of enzymes by comparison with proteins of similar functions and known three-dimensional structures. this allows one to identify potential active centres and to develop compounds, also known as "designer" drugs, which accommodate within the active centres and inhibit the viral enzymes. that means deviating from purely empirical research, and is a first step towards a more rational development of antiviral compounds (▶ chap. ). molecular virology has achieved significant successes in recent decades: many infectious diseases can be prevented through the use of modern vaccines or have been completely eradicated (▶ chap. ). this ultimately made possible the global elimination of infectious agents such as smallpox virus. poliovirus, which causes poliomyelitis, is no longer found on some continents, and is currently confined to fewer than ten countries worldwide. in cases in which no preventive vaccination is possible today, e.g., against hiv and some herpesviruses such as cytomegalovirus and herpes simplex virus, a large number of antiviral drugs are available. although these drugs do not provide a cure, they substantially allow the control of symptoms. these successes might tempt one to assume that virus research has become unnecessary. the assessment of the epidemiological situation by the who and the many sensationalistic headlines in newspapers and the media with which we are repeatedly confronted imply the opposite. because of their frequent and high rates of mutation, viruses are subject to continuous change and development: viruses are permanently compelled to cope with the infected organism and its immune defence systems, always trying to undermine and circumvent them. in particular, viruses that persist in the organism are capable of evading the host immune defence systems by very skilful strategies. the worldwide increase in travel leads not only to contact with new human pathogens, but also to their rapid dissemination. this is demonstrated, for example, by sars virus infections (▶ sect. . ), the pandemic with the new influenza a virus variant (mexican flu, "swine flu") and the threatening potential with regard to humans of new highly pathogenic influenza viruses (▶ sect. . ). new and novel viral diseases which have their origin in the animal kingdom (zoonoses) are also expected owing to increased environmental changes and their serious consequences. outbreaks of infection with ebola virus, nipah virus and hendra virus are examples. deforestation of rainforests has led to a change in living conditions for bats, which then infect horses and pigs and, via these intermediate hosts, also humans. birds carried west nile virus from africa to north america, and avian flu virus h n was transported from asia to europe by migratory birds. the aids pandemic that was induced by human immunodeficiency viruses was originally the result of a zoonotic transmission from monkeys to humans, followed by efficient further dissemination within the human population. the threat from both new and already well-known viral infections will not decrease because of reduced vaccination, particularly in industrialized countries; therefore, scientists who conduct research in the field of molecular virology will continue to have an ample sphere of activity. behbehani am ( ) the smallpox story in words and pictures. university of kansas medical center, kansas city causation and disease. the henle-koch postulates revisited helmut ruska and the visualisation of viruses mikrobenj€ ager. ullstein, frankfurt levine aj ( ) viruses. palgrave macmillan m€ uller r ( ) medizinische mikrobiologie. parasiten, bakterien, immunit€ at, th edn history of virology key: cord- - snefs authors: strodtbeck, frances title: viral infections of the newborn date: - - journal: j obstet gynecol neonatal nurs doi: . /j. - . .tb .x sha: doc_id: cord_uid: snefs viral infections of the newborn result in significant morbidity and mortality each year. the fetus and newborn are particularly wlnerable to viral infection. the range of expression may vary from no clinical disease to devastating illness and infection occurring before, during, or after birth. nursing management is determined by the specific viral infection, the severity of the illness, and the unique conditions of the newborn and his/her family. promising new therapies are on the horizon that may lessen the severity of viral disease. until such time, the major thrusts of management of neonatal viral disease are prevention of infection and supportive care for the acutely ill newborn. ical infection can vary from recovery to persistent infection with or without sequelae to death (overall, ; smith, ) . this article reviews the unique pathogenesis of newborn viral infection and the response of the neonatal immune system to viral attack; current information on the clinical features of specific viral diseases is summarized. the diagnosis, management, and nursing care of the newborn with a viral disease are addressed. the first goal and ultimate management of neonatal viral disease is prevention of the infection iral infections of the newborn are a serious con-v cern; such infections result in significant morbidity and mortality each year. the incidence of viral infections in the newborn is estimated at - % of all live births (smith, ) . the fetus and newborn are particularly vulnerable to viral infection for numerous reasons, including a developing immune system that is inadequate for preventing infection and containing the spread of viruses, lack of immunologic experience with viruses, and the presence of rapidly growing cells and tissues (strodtbeck, ) . viral infection in the newborn can vary from no clinical disease to devastating illness. infection of the newborn with a virus produces a variety of clinical presentations, ranging from absence of symptoms to infection before (congenital), during (natal), or after birth (postnatal) (overall, ) . because of the affinity of viruses for rapidly growing cells, newborn infection results in multiple outcomes that are determined by the specific virus and the gestational age at the onset of infection. congenital viral infections may result in miscarriage or stillbirth, congenital defects of various organ systems, clinical infection, or asymptomatic infection. natal and postnatal infections can result in asymptomatic infection or clinical infection. outcomes of clin- viruses are unique microorganisms in that they are obligatory intracellular parasites that must gain access to the inside of a host cell for replication. physical contact is necessary for this process, and many factors influence the attraction between the host cell and the invading virus. the structure of an individual virus and the presence of certain enzymes determine the mechanism by which the virus replicates inside the host cell. viral replication follows five basic steps. proteins using host cell components and metabolism; . assembly of new virions in the cytoplasm; and . release of the new virions into the intracellular environment. release of the virus can occur quickly or slowly and frequently results in destruction of the host cell (strodtbeck, ; voyles, ) . or lysis. latency occurs when the provirus reproduces itself along with the host cell such that all daughter cells contain the viral genetic material. these cells can remain latent or be triggered into one of the other two outcomes at a future time. a classic example of latency is the herpes virus, which can remain dormant until a trigger such as stress activates the virus and a characteristic herpetic skin lesion is produced. in the controlled growth outcome, the host cell is not destroyed but becomes committed to the production of new virions, which are gradually released into the surrounding interstitial space. the last outcome, lysis, involves destruction of the host cell upon release of the newly formed virions. this process is referred to as the cytopathic effect and often results in patterned tissue damage that can be used in the clinical laboratory for identification of the virus (strodtbeck, ; voyles, ) . from this discussion, it should be clear why viral infections can result a such a wide array of presentations. because of the intracellular, parasitic nature of viruses and the resulting dependence on host cell metabolic machinery for proliferation, the use of antiviral drug therapy is limited. many of the drugs developed for viral disease also have an effect on rapidly growing cells, which limits their use in the newborn. thus, the newborn is not only vulnerable to infection, but also is disadvantaged for viable treatment options. the newborn infected with a virus is capable of mounting an immune system response. the response depends upon the gestational age of the newborn and is not as efficient or effective as the response of an adult. clinical viral infection in newborns usually results in a more rapid progression to full-blown disease and earlier onset of symptomatic organ involvement than would be seen in adults with the same infection (overall, ; smith, ; strodtbeck, ) . entry of the virus into the newborn triggers activation of the developing immune system. local antibodies such as iga are stimulated. because of their inability to contain the virus, a primary focus of infection is established. the virus replicates itself and releases new virions into the interstitial space, where they are carried away via the lymphatics or the bloodstream to secondary foci of infection. if tissue damage occurs at this point, the inflammatory and cell-mediated responses are activated. escaping virions in the bloodstream stimulate the production of circulating igm and igg antibodies. igm tends to be produced earlier if the virus is new to the immune system, whereas igg tends to be produced first if the virus is a repeat challenger. the rise in fetal igm during intrauterine infection allows fetal or cord blood levels to be used in the diagnosis of congenital viral infection. igm production peaks early in the infection and rapidly declines. igg peaks gradually and functions longer than igm. both of these immunoglobulins attempt to neutralize the invading virus by binding with the virus to activate complement. this mechanism is designed to prevent virus absorption to host cells (overall, ; strodtbeck, ) . once the virus gains entry to the host cell, it becomes the responsibility of the cell-mediated system and the inflammatory response to contain the infection. infected host cells develop new virus-induced, antigenic determinants on their cell membranes that alert these defenses. the new antigens trigger t lymphocytes to respond along with macrophages and other components of the immune system (overall, ; strodtbeck, ) . activation of the immune response at this point may result in the destruction of some host cells, rather than the virus, by the immune system. in the developing fetus, this can worsen the severity of birth defects associated with specific viral diseases, such as rubella or cytomegalovirus (cmv). for example, neurons coated with cmv may be destroyed by lymphocytes which, when added to the number of neurons undergoing viral lysis, increases the risk for microcephaly. antigen-antibody complexes also are formed that liberate chemotactic factors and cytokines. histamine and kinin release results in vasodilation, heat production, and increased capillary permeability. this results in increased temperature (fever) and stagnant anoxia locally, which decreases absorption of the virus because most viruses are susceptible to increased temperature and acidic environments (strodtbeck, ; voyles, ) . a side effect of this process is a less-than-ideal environment for the growth of healthy tissue. the final consideration in the newborn's response to viral infection is the production of interferon. interferons are highly active proteins produced by virus-infected cells that act upon neighboring cells to prevent the spread of the viral infection. they also are produced by cells of the immune system. interferons disrupt viral infectivity in a variety of ways that are still being studied. newborn interferon production appears to be decreased via one pathway and normal via the other (strodtbeck, ) . despite the deficiencies of the newborn immune system to viral attack, only a few of the hundreds of viruses known to cause human disease actually result in infection of the fetus or newborn (overall, ) . viral infection of the newborn usually is classified as congenital, acquired, or nosocomial (hospital-acquired) in origin. congenital viral infections occur when an infected mother transmits the virus across the placenta to the fetus. theoretically, all viruses are capable of causing congenital infections, but most do not (overall, ) . this may be attributable in part to maternal and placental immune defenses that protect the fetus or to the nature o f the maternal illness. most viral infections in pregnancy are mild respiratory or gastrointestinal infections that do not pose a serious threat to the health of the fetus (overall, ) . the viruses implicated in congenital infection are cytomegalovirus, herpes simplex virus, rubella (german measles), and varicella (chickenpox) (overall, ; smith, ) . there is debate within the medical community about a congenital viral syndrome caused by the human immunodeficiency virus (hiv). acquired viral infections occur after exposure of the newborn t o maternal vaginal tract flora and breast milk. genital secretions may be contaminated with cmv, enteroviruses, hepatitis b virus, and herpes simplex virus. contaminated breast milk has been implicated in newborn infection with hiv, herpes simplex, and other viruses (overall, ) . nosocomial viral infections may be acquired in the newborn nursery or the intensive care nursery (guerina r goldmann, ; strodtbeck, ) . current trends of short length of stay and rooming in minimize the risk of hospital-acquired infection in the healthy infant assigned to the newborn nursery. high-risk or premature infants transferred to the intensive care nursery are at risk for nosocomial viral infections from members of their family, health care providers, other infants, treatment therapies (such as blood transfusions), or contaminated objects (fomites) in the environment. specific features about the most common viral infections of concern to the newborn are summarized in table . pertinent information on the incidence, epidemiology, and characteristics of the virus; transmission risk; and effect(s) on the newborn are identified. diagnosis and clinical manifestations of the infection, along with treatment, prognosis, and prevention strategies, are discussed (arvin r maldonado, ; cherry, ; gershon, ; mueller r pizzo, ; torok, ; whitley & arvin, ) . diagnosis of viral infection in the newborn usually is prompted by a strong suspicion based on physical characteristics of the newborn, history of exposure or maternal illness, or failure of sepsis testing to yield positive results. careful review of the maternal history for evidence of viral illness and serum antibody determinations can assist in making the diagnosis; however, definitive diagnosis is based on neonatal studies (overall, ; smith, ) . accurate diagnosis often is difficult. because many infected infants have no symptoms or the clinical manifestations are nonspecific, laboratory diagnostic tests must be used to determine the virus responsible for the illness. laboratory diagnosis may involve direct isolation of the virus in culture; detection of viral antigen or antibodies by immunologic, genetic, or electron microscopic study; or histopathologic methods (guerina & goldmann, ; overall, ; smith, ) . the best method of diagnosis varies, depending upon the specific characteristics of the virus. recovery of virus from clinical specimens usually is more difficult than recovery of other microorganisms (strodtbeck, ) . a negative result may mean an unsuccessful recovery of virus present. clinical judgment is important for determining the likelihood for additional management and treatment of the suspected viral illness. the most common management strategy for newborn viral infection is supportive care. the availability of specific chemotherapies such as antiviral drugs is limited. promising new therapies are being developed, and several are undergoing evaluation in clinical trials with select populations (filippell & rearick, ; kinney & eiden, ; overall, ; stagno, ) . such therapies include ganciclovir for cmv disease, hyperimmune intravenous globulins for cmv and respiratory syncytial virus (rsv) infections, and new vaccinations for rotavirus. most of these therapies, if approved for use in newborns, will most likely lessen the severity of the disease and subsequent sequelae, rather than cure the infection. other strategies include improvements in nutrition for the most susceptible newborns, interferon administration to enhance viral-specific immune defenses, and lymphokine enhancement of the neonatal immune system. the first goal and ultimate management of neonatal viral disease is prevention of the infection. diligent hand washing; improved screening of visitors and health care providers who come into contact with susceptible newborns; increased efforts to immunize susceptible populations, such as childbearing women and children, to known viral pathogens, such as rubella and measles; and immunization of health care providers who work with immunocompromised patients are strategies that can be used to achieve this goal. the american academy of pediatrics ( ) recommendation that all neonatal intensive care unit (nicu) staff members receive annual influenza immunizations to prevent the spread of infection to infants with chronic lung disease is routinely ignored by most units across the country (eisenfeld et al., ) . nursing care of the newborn with a viral illness is determined by the specific viral infection, the severity of the illness, and the unique conditions of the newborn and his/her family. newborns with a known viral infection who have no symptoms require no special nursing care while in the hospital. newborns with viruses such as rubella and cmv who have no symptoms should be followed up closely for the development of late onset se- cherry, ; cooper, preblud, r alford, rearick gershon, ; giacoia, ; kinney bz eiden, azt: zidovudine; cns: central nervous system; ddc: dideoxycytidine; ddi: dideoxyinosine; dic: disseminated intravascular coagulation; nec: necrotizing enterocolitis; mueller l(r pizzo, ; overall, ; smith, ; stagno, ; torok, ; whitley r arvin, nicu: neonatal intensive care unit. egies are important for protecting the ill newborn from other infections and preventing nosocomial spread of the usually is prompted by a strong suspicion based on physical characteristics of the newborn, history of exposure or maternal illness, or failure of sepsis testing to yield positive results. quelae (cooper, preblud, tk alford, ; overall, ) . family members should be provided with available educational materials. brochures, such as those on cmvfrom the children's biomedical research institute in st. paul, minnesota, can be helpful for some families (children's hospital, ). the first action of nursing care of the newborn with symptoms often is to raise the index of suspicion for viral disease. the presence of physical findings, such as characteristic rashes or vesicles, microcephaly, intrauterine growth retardation or small size for gestational age, should alert the nurse to the possibility of congenital infection. obtaining a detailed, accurate maternal history, including immunizations received, signs/symptoms of possible viral illness during the pregnancy, and exposure to possible sources of viral infection, is important. collecting correct specimens for diagnostic tests and cultures is as important as prompt processing by the clinical laboratory (strodtbeck, ) . nursing care of the newborn with severe viral disease is complex and varies according to the clinical presentation of the neonate. presentation of specific management plans for each viral infection is beyond the scope of this article. general nursing care may involve ventilator therapy, pharmacologic support of cardiovascular status, correction of acidosis and shock states, correction of coagulopathies, and treatment of severe neurologic disorders, such as meningitis and seizures. skin care and nutrition are other areas of concern to nursing because many infants may have rashes or be at risk for secondary infections. care of the family is important and can be complicated by maternal or paternal feelings of guilt regarding congenital infection or infection acquired at the time of delivery. the need for special care such as isolation also can present problems and increase the family's stress. meticulous attention to hand washing, aseptic technique, and the use of personal protective equipment are additional important aspects of nursing care. these last strat- within recent years, there has been a developing awareness on the national level of the role of viruses as nosocomial pathogens (giacoia, ; guerina & goldmann, ; strodtbeck, ) . although viral infections are uncommon in the newborn nursery, the potential for outbreaks remains a constant concern, especially during peak seasons of viral infections within the community. infected visitors and health care workers may unknowingly expose healthy newborns to respiratory viruses such as rsv, influenza, and adenovirus; enteric viruses such as rotavirus and enteroviruses; herpes simplex virus; and varicella virus (overall, ) . early discharge results in newborns being discharged before the end of the minimum incubation period for most viruses. these infants often have symptoms develop at home. routine telephone follow-up of discharged newborns may alert the nursery staff to the presence of a problem. the nicu is especially susceptible to nosocomial viral infections for a variety of reasons, including extensive use of invasive technology for the care of sick infants, a fragile and immunocompromised patient population, and large numbers of health care workers and visitors who come in contact with the patients (guerina & goldmann, ; strodtbeck, ) . newborns with underlying cardiac and pulmonary dysfunction are particularly at risk for the development of nosocomial viral infections, especially rsv and cmv (guerina & goldmann, ; smith, ) . many viruses have been implicated as etiologic agents of nosocomial infections in the nicu. the most commonly identified viruses are the respiratory viruses (rhinovirus, adenovirus, rsv, parainfluenza, and influenza virus); the enteric viruses (rotavirus and coronavirus); the enteroviruses; and the herpes viruses (cmv and herpes simplex). nosocomial viral infections are of concern because they prolong hospital stays and increase health care costs, increase neonatal morbidity and mortality, and are difficult to recognize and diagnose. new evidence suggests that infection with cmv or influenza virus may complicate matters by predisposing the critically ill newborn to bacterial superinfection because of viral-induced changes in polymorphonuclear leukocyte function (abramson & wheeler, ) . reports of nosocomial infection range from case studies to unit outbreaks (singh-naz, brown, & ganeshananthan, ; strodtbeck, ; watson et al., ) . risk factors linked to nosocomial viral infections include intubation and assisted ventilation; transfusion with cmv-positive donor blood; ingestion of contaminated breast milk; direct and indirect contact among patients, visitors, and hospital personnel; contact with contaminated objects in the environment; and inadequate hand washing (guerina & goldmann, ; strodtbeck, ) . nursing care of the newborn with severe viral disease is complex and varies according to the clinical presentation of the neonate. each year significant neonatal morbidity and mortality occur as a result of viral infections. the presence of a developing immune system inexperienced in response to viruses in a rapidly growing host make the newborn especially vulnerable to viral disease. the effects of viral infection are varied and range from no symptoms to mild or severe disease. although the newborn immune system is capable of mounting a defense to the virus, the response often is inadequate for preventing infection. the outcome of viral infection depends upon the specific virus, the gestational age of the newborn at the onset of infection, and the severity of the infection. in addition to limited specific antiviral drug therapy, many of the available drugs are not recommended for use in newborns. several new therapies are being tested and show promise; eventually they may lessen the severity of disseminated disease and minimize the long-term sequelae. until such time, the major thrusts of management of neonatal viral disease are prevention of the infection and supportive care for the acutely ill newborn. virus-induced neutro-phi dysfunction: role in the pathogenesis of bacterial infections the red book other viral infections of the fetus and newborn enteroviruses infectious diseases of the fetus and newborn infant rubella respiratorysyncytial virus chickenpox, measles and mumps uncommon pathogens in newborn infants neonatal nosocomial infections: prevention and management current therapy in pediatric infectious disease enteric infectious disease in neonates: epidemiology, pathogenesis, and a practical approach to evaluation and therapy acquired immunodeficiency syndrome in the infant viral infections of the fetus and neonate nosocomial adenovirus infection: molecular epidemiology of an outbreak congenital viral and protozoan infections cytomegalovirus the epidemiology of nosocomial viral infections in infants who are long term residents of the neonatal intensive care unit human parvovirus bl the biology of viruses vertical transmission of hepatitis a resulting in an outbreak in a neonatal intensive care unit herpes simplexvirus infection key: cord- -u k pds authors: mason, jay w.; trehan, sanjeev; renlund, dale g. title: myocarditis date: journal: cardiovascular medicine doi: . / - - - - _ sha: doc_id: cord_uid: u k pds viruses are the most common cause of myocarditis in economically advanced countries. enteroviruses and adenoviruses are the most common etiologic agents. viral myocarditis is a triphasic process. phase is the period of active viral replication in the myocardium during which the symptoms of myocardial damage range from none to cardiogenic shock. if the disease process continues, it enters phase , which is characterized by autoimmunity triggered by viral and myocardial proteins. heart failure often appears for the first time in phase . phase , dilated cardiomyopathy, is the end result in some patients. diagnostic procedures and treatment should be tailored to the phase of disease. viral myocarditis is a significant cause of dilated cardiomyopathy, as proved by the frequent presence of viral genomic material in the myocardium, and by improvement in ventricular function by immunomodulatory therapy. myocarditis of any etiology usually presents with heart failure, but the second most common presentation is ventricular arrhythmia. as a result, myocarditis is one of the most common causes of sudden death in young people and others without preexisting structural heart disease. myocarditis can be definitively diagnosed by endomyocardial biopsy. however, it is clear that existing criteria for the histologic diagnosis need to be refined, and that a variety of molecular markers in the myocardium and the circulation can be used to establish the diagnosis. treatment of myocarditis has been generally disappointing. accurate staging of the disease will undoubtedly improve treatment in the future. it is clear that immunosuppression and immunomodulation are effective in some patients, especially during phase , but may not be as useful in phases and . since myocarditis is often selflimited, bridging and recovery therapy with circulatory assistance may be effective. prevention by immunization or receptor blocking strategies is under development. giant cell myocarditis is an unusually fulminant form of the disease that progresses rapidly to heart failure or sudden death. rapid onset of disease in young people, especially those with other autoimmune manifestations, accompanied by heart failure or ventricular arrhythmias, suggests giant cell myocarditis. peripartum cardiomyopathy in economically developed countries is usually the result of myocarditis. jay w. mason, sanjeev trehan, and dale g. renlund • viruses are the most common cause of myocarditis in economically advanced countries. • enteroviruses and adenoviruses are the most common etiologic agents. • viral myocarditis is a triphasic process. phase is the period of active viral replication in the myocardium during which the symptoms of myocardial damage range from none to cardiogenic shock. if the disease process continues, it enters phase , which is characterized by autoimmunity triggered by viral and myocardial proteins. heart failure often appears for the first time in phase . phase , dilated cardiomyopathy, is the end result in some patients. diagnostic procedures and treatment should be tailored to the phase of disease. • viral myocarditis is a significant cause of dilated cardiomyopathy, as proved by the frequent presence of viral genomic material in the myocardium, and by improvement in ventricular function by immunomodulatory therapy. • myocarditis of any etiology usually presents with heart failure, but the second most common presentation is ventricular arrhythmia. as a result, myocarditis is one of the most common causes of sudden death in young people and others without preexisting structural heart disease. • myocarditis can be definitively diagnosed by endomyocardial biopsy. however, it is clear that existing criteria for the histologic diagnosis need to be refined, and that a variety of molecular markers in the myocardium and the circulation can be used to establish the diagnosis. • treatment of myocarditis has been generally disappointing. accurate staging of the disease will undoubtedly improve treatment in the future. it is clear that immunosuppression and immunomodulation are effective in some patients, especially during phase , but may not be as useful in phases and . since myocarditis is often selflimited, bridging and recovery therapy with circulatory assistance may be effective. prevention by immunization or receptor blocking strategies is under development. • giant cell myocarditis is an unusually fulminant form of the disease that progresses rapidly to heart failure or sudden death. rapid onset of disease in young people, especially those with other autoimmune manifestations, accompanied by heart failure or ventricular arrhythmias, suggests giant cell myocarditis. • peripartum cardiomyopathy in economically developed countries is usually the result of myocarditis. the difficulty of diagnosing and treating myocarditis was recognized by senac in : "the inflammation of the heart is difficult to diagnose and when we have diagnosed it, can we then treat it better?" after sobernheim in defined myocarditis as any inflammation or degeneration of the heart, the term myocarditis was used for nonvalvular myocardial diseases, including ischemic and hypertensive cardiomyopathies. nearly a century later, white suggested that the term myocarditis be restricted to "true inflammation of the myocardium." the last half-century has seen the development of endomyocardial biopsy techniques, histologic criteria, and serologic methods to diagnose myocarditis. as our knowledge of the immunopathologic mechanisms evolves, new therapeutic strategies are developing. the world health organization/international society and federation of cardiology task force on cardiomyopathies classified cardiomyopathies whenever possible by etiologic/pathogenetic factors. this classification recognizes chronic viral, postinfectious autoimmune, and primary autoimmune forms of dilated cardiomyopathy (dcm). the classification states that "myocarditis is diagnosed by established histological, immunological and immunohistochemical criteria." the dallas criteria provide consensus-derived histologic criteria: "an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of ischemic damage associated with coronary artery disease." however, many have speculated that less pronounced histologic abnormalities may be present and that additional molecular, immunologic, and immunohistochemical diagnostic criteria can be used productively. [ ] [ ] [ ] [ ] [ ] [ ] myocarditis, irrespective of the etiopathologic factors, remains an inflammatory cardiomyopathy associated with cardiac dysfunction. a wide variety of infectious and noninfectious causes are associated with myocarditis (tables . to . ) . several epidemiologic observations linking these agents with myocarditis have been corroborated by serologic, polymerase chain reaction (pcr), or in situ hybridization methods. the incidence of infectious myocarditis in the general population is largely unknown. in a prospective study over several years, in a predefined subpopulation, an incidence of . % was found. these cases were confirmed by myocardial enzyme leak and characteristic electrocardiographic (ecg) changes. the ecg abnormalities suggesting asymptomatic myocardial involvement, in the absence of enzyme release, have been noted in . % of military conscripts during the course of other acute infectious diseases. during an epidemic of influenza a, the incidence rose to . %. in a prospective trial of consecutive patients admitted to a large infectious disease hospital in sweden, % showed ecg abnormalities suggestive of myocarditis. approximately % of a virus-infected population may experience symptoms or findings suggestive of cardiac involvement. the incidence of myocarditis associated with nonviral infections is even more difficult to estimate. although the list of possible etiologic agents is large, the enteroviruses, specifically coxsackievirus b, over decades have been the most commonly identified etiologic agents of inflammatory cardiomyopathy. among healthy active adults, at least % have detectable serum antibodies indicating prior infection with coxsackievirus b. , the world health organization has surveyed viral infections related to cardiovascular disease globally. in a year period from to , coxsackievirus b had the highest incidence of cardiovascular disease ( . cases per population), followed by influenza b ( . cases), influenza a ( . cases), coxsackievirus a ( . cases), and cytomegalovirus (cmv) ( . cases). the predominance of enteroviruses among myocarditisassociated agents has been substantiated by several laboratory and clinical studies. [ ] [ ] [ ] using serologic methods, vikerfors and associates reported that nearly % of consecutively studied myocarditis patients had enterovirus immunoglobulin igm. frisk and coworkers found a similar incidence of coxsackievirus b igm antibodies by reverse herpes simplex in two, and cmv in one patient. the control group did not demonstrate any viral genome sequences. just as the incidence of specific viral infections varies over time, so should the relative proportion of agents responsible for myocarditis. in a recent study, bowles and colleagues supported the observation by martin and coworkers that adenovirus is the most common agent associated with myocarditis in children, but they also found that adenoviruses predominated over enteroviruses in adults. figure . shows the dominant role of adenoviruses and enteroviruses in myocarditis. note that parvovirus was detected in young people. parvovirus b- has recently been identified as a cause of myocarditis and, in some regions, it has been found in adults as well as children. [ ] [ ] [ ] [ ] [ ] these differences between previous and newer studies are due, at least in part, to geographical and temporal variation in the incidence of specific viral infections. cytomegalovirus is a recognized cause of acute infectious myocarditis, although it is rare in healthy individuals. , maisch and associates demonstrated, using in situ hybridization techniques, cmv-specific nucleotide sequences in % of patients with acute myopericarditis. certainly in transplant recipients, cmv infection is fairly common and has been reported to affect the transplanted heart. , hepatitis c virus infection is frequently noted in patients with dcm, and hepatitis c virus rna has also been recovered from lymphocytes infiltrating the myocardium in chronic active myocarditis. matsumori and colleagues , found a high incidence of hepatitis c viral genomic material in a wide variety of cardiac disorders in japan. myocarditis is a well-recognized complication of corynebacterium diphtheriae infection, although this is now rare in the western world. myocardial dysfunction is also seen in association with salmonella septicemia, although it is rarely clinically severe. , myocardial dysfunction is primarily related to the toxemia of the severe infection, which is also observed in meningococcal and nonrheumatic streptococcal infections. perhaps the best-recognized bacterial agent thought to be responsible for myocarditis is the β-hemolytic streptococcus that causes rheumatic fever. fortunately, rheumatic fever is seen in the western world with only a low frequency of sporadic cases in regional clusters. the incidence in the united states is less than per , , but in the developing world, rheumatic heart disease continues to be the leading cause of cardiac hospitalization in the -to -year-old age group. although the inflammatory component of rheumatic carditis is largely restricted to the valves, it has been believed to cause myocardial dysfunction. myocarditis is a well-documented complication of borrelia burgdorferi infection (lyme disease) and is reported in up to % of cases. cardiac involvement is often characterized by the development of atrioventricular (av) block and rarely progresses to left ventricular dysfunction and cardiomegaly. mycoplasma pneumoniae infection has also been associated with myocarditis. lewes and coworkers demonstrated asymptomatic myocardial involvement as documented by ecg changes in a third of the cases with acute mycoplasma infection. six percent of military conscripts with clinical myocarditis were found to have active m. pneumoniae infection. chlamydia infections have also been associated with myocarditis, especially among small children, often having fatal outcomes. c. pneumoniae infection has also been noted in a few cases of mild myocarditis and has been found with respiratory infection associated with myocarditis, resulting in sudden death in a young athlete. chlamydia psittaci infection may be associated with myocarditis in % to % of those affected, usually with minimal clinical signs or symptoms. pericarditis is more frequent and likely to cause cardiac morbidity with ornithosis. other causative infectious agents rickettsial infections, like rocky mountain spotted fever and scrub typhus, are frequently accompanied by myocardial involvement, although vasculitis is more prominent with these infections. q fever may also be associated with myocarditis. trypanosoma cruzi is a well-recognized cause of myocarditis and cardiomyopathy in south america (chagas' disease). toxoplasma gondii poses a significant problem among cardiac transplant recipients because a large number of the recipients lack antibodies against this agent, which may cause myocarditis. toxoplasmosis also poses a major threat to patients with aids. myocarditis has frequently been seen in human immunodeficiency virus (hiv)-infected populations with or without concomitant toxoplasma infection. , in two autopsy studies of patients with aids, myocarditis was found in almost half of the cases; in another study, % of prospectively studied patients with aids had echocardiographic evidence of myocardial dysfunction. , myocarditis may also occur in patients with aids as a result of t-cell restitution after antiviral therapy. myocarditis can also be seen with parasitic infections such as trichinella spiralis, which has an affinity for striated muscle, including the heart. other noninfectious causes noninfectious causes of myocarditis include druginduced hypersensitivity, - direct toxicity of specific pharmaceutical agents, , [ ] [ ] [ ] and systemic collagen vascular disorders. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] eosinophilic myocarditis [ ] [ ] [ ] [ ] and giant cell myocarditis (gcm) [ ] [ ] [ ] [ ] [ ] [ ] [ ] are distinct forms of inflammatory myocarditis of uncertain etiology. microorganisms are rarely isolated or demonstrated in heart muscle; hence, identification of a specific infectious etiologic agent depends on recognition of its systemic manifestations. once specific noninfectious and nonviral infectious agents are excluded, myocarditis is often assumed to be of viral etiology. although definitive serologic evidence of viral infection can be obtained in many patients, it is absent in the majority of patients with presumed myocarditis. a significant number of cases of myocarditis is due to autoimmune phenomena either induced by a viral infection or resulting from systemic autoimmune disease. since the establishment of definitive etiologic diagnoses is ambiguous, the terms viral myocarditis, idiopathic myocarditis, lymphocytic myocarditis, autoimmune myocarditis, and interstitial myocarditis are frequently used interchangeably. the pathophysiologic mechanisms of myocarditis in humans are not fully understood. clearly, multiple mechanisms exist, including direct infection by viruses, bacteria, and other organisms; noninfectious causes, such as toxins and drug hypersensitivity; and parainfectious etiologies, resulting from the immune response to infection. most cases of overt heart failure due to myocarditis in north america, europe, and japan are thought to arise from the latter type of mechanism, during and after viral infection of the heart. a triphasic disease process is observed , (fig. . a ). in the first phase, active viral infection of the myocardium results in a variable extent of muscle damage, which often may not be clinically apparent. phase develops in an unknown proportion of infected individuals after partial or complete resolution of active infection and is characterized by further myocardial damage, both by ongoing immune activation and by newly developed autoimmune activity. a small proportion of patients develops dilated cardiomyopathy, the third phase of disease, resulting from the cumulative damage caused by infection, immunity, and autoimmunity. during this phase, a considerable body of evidence suggests that the immune and autoimmune processes persist, in part as a result of viral persistence. figure . b depicts the three roles virus may play in bringing about dilatation and chronic heart failure. after the initial injury that occurs during active viral replication, latent, nonreplicating viruses can still alter myocyte function through viral genomic expression. even if the virus is completely eliminated and the immune response ceases, through the various mechanisms of adverse remodeling, the cardiomyopathy may progress inexorably. the most widely accepted models for the study of human myocarditis are those of enteroviral myocarditis induced by coxsackievirus b (cvb ) and the encephalomyocarditis virus. induction of chronic murine myocarditis by cvb requires the virus to have a cardiovirulence capacity and murine strains of certain genetic background. , infection of syngeneic weanling mice with cvb results in brief cardiac infection lasting about a week, beyond which the virus cannot be cultured. however, viral rna persists for several months after the initial infection. , several mechanisms have been hypothesized to explain the initiation of chronic inflammatory response in myocytes by the viral infection: . although antibodies to these antigens are frequently identified in association with myocarditis, the clinical significance and causal relationship are yet unresolved. cytotoxic lymphocytes (ctls) from mice with cvb induced myocarditis possess the ability in vitro to recognize and kill neonatal myocytes, fibroblasts, and endothelial cells infected with the same strain of the virus, suggesting that the recognition of a novel tissue antigen is induced by the infection. cross-reactive, concurrent recognition of unrelated cardiac epitopes also occurs because ctls also lyse uninfected myocytes in vitro. the production of perforin, a pore-forming protein, has been proposed as one of the mechanisms for the cytolysis induced by lymphocytes. perforins, when inserted into myocyte membrane, induce a lethal augmentation in cell permeability that results in cellular edema and death. perforin-independent mechanisms have also been proposed, including a fas (cd /apol)-based inositol- , , -triphosphate-mediated cytolysis that can be demonstrated in perforin-deficient gene-knockout mice. coxsackievirus-infected mice also develop additional immune sensitization to cardiac heavy chain myosin, possibly owing to the release of the sequestered myosin antigens from the virus-damaged cells. immunization of mice with the heavy chain myosin and an adjuvant produces a histomorphologically similar picture to cvb -induced myocarditis. adoptive transfer of splenocytes can also produce experimental autoimmune myocarditis after myocardial infarction in syngeneic rats. the sensitized lymphocytes when transferred to normal rats cause cardiac-specific cellular infiltration with accompanying myocyte necrosis. the genetic susceptibility, kinetics, and cellular composition of the infiltrates in these models are similar and suggest the role of endogenous antigens as an epitope for the inflammatory response. the pathways and cellular participants in the immunopathogenesis of experimental viral myocarditis are well recognized. the replicating viral particles can be readily identified in cardiac myocytes within a few hours of inoculation of cvb into mice. , the viral particles reach a numerical peak in to days, and usually at to days, they are no longer detectable. the inflammatory infiltrate is detectable by day and reaches a plateau by days to . the early inflammatory infiltrate consists of lymphocytes, macrophages, neutrophils, natural killer cells, and the associated cytokines and humoral effectors. [ ] [ ] [ ] the natural killer cells are the first to appear and are detected in the activated state in to days. these cells are capable of lysing virusinfected cells in vitro. the t lymphocytes and macrophages follow the natural killer cells in the temporal sequence and become the predominant cells infiltrating the myocardium in to days. although cvb replicates readily in myocytes in vitro, the cells are resistant to lysis in comparison with other cultured cell lines. direct myocytolysis appears to play a minimal role in cell lines derived from normal mice. the immunodeficient severe combined immunodeficiency (scid) mouse model has provided valuable insight into the early immune activity in response to the viral infection. the scid mice lack mature t-and b-lymphocyte function and develop extensive myocardial necrosis with pleomorphic infiltrates, rapid viral proliferation, and profound virus-associated myocytolysis when inoculated with cvb . the macrophage and natural killer cell activity is unaffected in the scid mouse model and may participate in the myocytolytic activity, although direct viral myocytolysis predominates. pharmacologically immunosuppressed mice demonstrate similar characteristics, with higher viral loads, delayed clearance, and extensive myocyte necrosis, although direct viral myocytolysis is not frequent in immunocompetent mice. , , - even noncardiovirulent strains may have sufficient time to replicate and transform into quasicardiovirulent species in the absence of a functional antiviral immune response, which can then result in fatal myocarditis. this may also explain the clinical observation that many severe and fatal cases of myocarditis develop in young children with immature and incompletely developed immune systems. virus-specific ctls play a major role in the inflammatory response to viral infection of the myocytc. , the inflammatory response can be diminished significantly by t-lymphocyte depletion with either antithymocyte globulin or thymectomy and irradiation. , the ctls must recognize the foreign antigen in association with the syngeneic major histocompatibility complex (mhc) class i antigen that is found on immune-derived cells. the cvb -infected cells can readily express mhc class i antigens. the mhc class i molecules provide peptide-binding sites that evoke effector responses on recognition of the foreign peptide by the antigenspecific receptors of the t lymphocyte. however, tlymphocyte depletion and specific immunosuppression using cyclosporine have varying effects, depending on the murine model, the virus, and the time of therapy, and are not uniformly beneficial. [ ] [ ] [ ] the virus can no longer be cultured from cells after to days; however, areas of inflammatory infiltrate and myocyte necrosis do demonstrate persistence of viral rna, and the virus-specific ctls may continue to see these as immunologic targets and, hence, perpetuate the myocyte damage. the infected myocyte can still remain a target for the ctls, even if the viral antigens are cleared, owing to expression of "neoantigens" either induced by the virus or unsequestered due to the injury. , even nonviral antigens on infected myocytes can react with ctls, such as those induced by actinomycin d, and new glycoproteins have been identified on the surface of cvb -infected cells that can be recognized by ctls from other syngeneic-infected mice. recent observations suggest that co-stimulatory molecules b - , b - , and cd- may be expressed on myocytes in patients with myocarditis and may make the myocytes into antigen-presenting cells for ctls and natural killer cells, thereby playing an important role in the direct myocardial damage by these lytic cells. another mechanism for ongoing myocyte damage is the antibody-mediated autoimmune response. since the majority of the proteins identified as cardiac autoantigens are intracellular, it is unclear how these antibodies could harm normal intact myocytes. several mechanisms are proposed. one suggests that after the antibody response is initiated, the circulating antibodies to intracellular antigens crossreact with the native membrane cardiac tissue proteins. thus, after a small number of myocytes are damaged by the viral infection and release intracellular antigens, the resulting antibody response may affect normal myocytes, leading to global myocardial dysfunction. this hypothesis is supported by the demonstration of a number of cross-reacting antibodies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] also, the antibodies against the intracellular mitochondrial adenine nucleotide transferase protein cross-react with the myocyte sarcolemmal calcium ion channel protein, and binding of these channels can physiologically alter the metabolism and contractile function of the myocyte. another theory holds that ctls and antibodies target uninfected myocytes by recognition of self-antigens that were previously sequestered from immune surveillance. the processing and presentation of the self-immunogenic peptides complexed with the mhc is a prerequisite for this hypothesis. normal human cardiac myocytes do not express detectable levels of mhc class ii antigens, and their constitutive expression of mhc class i molecules remains controversial. a significant increase in the expression of mhc class i and class ii antigens by the myocytes has been demonstrated in association with myocardial inflammation, such as that seen with viral myocarditis or transplant rejection. [ ] [ ] [ ] the increased mhc expression has also been demonstrated in endomyocardial biopsy specimens from patients with idiopathic dcm and myocarditis, [ ] [ ] [ ] and immune regulatory dysfunction may have a genetic predisposition. there is also evidence for aberrant expression of intracellular antigens, such as adenine nucleotide translocator (ant) and branched-chain α-keto acid dehydrogenase (bckd), on the surface of the myocytes. the formation of antiidiotypic antibodies is an additional mechanism of immune regulation in which an antibody is formed to the idiotypic determinants (antigen recognition site) of the primary antibody. the antiidiotypic antibody may cross-react with unoccupied viral receptor sites on uninfected myocytes. this phenomenon has been reported with the reovirus, polyomavirus, and coxsackievirus b models of myocarditis. [ ] [ ] [ ] the passive transfer of antiidiotypic b cells from a cvb myocarditic mouse to a syngeneic mouse can cause nonviral myocarditis. the presence of a complex, cytokine-rich microenvironment is suggested by the heterogeneous inflammatory cell populations in the hearts of infected mice. the cytokines perform myriad immunomodulatory functions, including regulation of antibody production, preservation of self-tolerance, , conscription of ancillary cells in the inflammatory milieu, , and maintenance of clonal expansion of ctls. , certain cytokines regulate the collagenogenic and collagenolytic activity of fibroblasts. although mounting evidence supports the negative inotropic effects or the blunting of catecholamine response in myocytes exposed to various cytokines, there is no direct evidence to suggest that the cytokines are directly responsible for myocytolysis. in an in vitro model, barry demonstrated that high concentrations of interleukin (il)- , tumor necrosis factor-α (tnf-α), interferon-γ (ifn-γ), and il- have no effect on myocyte survival over hours, whereas the ctls from a mixed lymphocyte reaction cause virtually % killing. gulick and colleagues demonstrated that cultured neonatal myocytes, when exposed to macrophage-derived il- and tnf-α, have reduced levels of cyclic adenosine monophosphate and have a reduced inotropic response to catecholamines. the mechanism for decreased responsiveness to catecholamines is believed to be modulated by increases in nitric oxide production mediated by increased inducible nitric oxide synthase (inos) activity, and the blunting of the catecholamine response can be inhibited by the l-arginine analogue n g -monomethyl-l-arginine (l-nmma). the decreased contractile response of cardiac myocytes to β-adrenergic agonists following induction of inos also requires the presence of insulin and the co-induction of enzymes responsible for the production of tetrahydrobiopterin, a cofactor for nitric oxide synthase. the role of inos remains controversial because increased expression of inos mrna and that of other proinflammatory cytokines is evident and is associated with contractile dysfunction. there is evidence to support the idea that inos induction is crucial for the host response to cvb infection, and inos-deficient mice have significantly increased viral loads with extensive myocardial damage. inhibition of inos through suppression of nuclear factor (nf)-κb induction has recently been shown to prevent encephalomyocarditis virus myocarditis. other investigators have suggested that inflammatory cytokines may have direct negative inotropic effects, independent of the responsiveness to the β-adrenergic agonists. high doses of il- during chemotherapy have been reported to result in depression of myocardial function. exposure of cardiac myocytes to endotoxin results in increased nitric oxide production and direct depression of contractility owing to increased levels of cyclic guanosine monophosphate. further, tnf-α may induce direct negative inotropic effects by decreasing the ca + transient, with no change in the l-type ca + current and independent of nitric oxide synthesis. although the extent to which cytokines cause direct negative inotropic effects or attenuation of endogenous β-adrenergic agonist activity remains unclear, they do produce myocyte dysfunction and cardiac decompensation. transgenic mice with overexpression of tnf-α develop biventricular dilatation and cardiac failure, resulting in premature death. pathologic specimens from these mice reveal globular dilated hearts and transmural myocarditis with myocyte apoptosis. increased levels of intracellular adhesion molecule (icam- ), il- α, il- β, tnf-α, and macrophage-stimulating factor have been demonstrated in patients with myocarditis and idiopathic dcm. , furthermore, the susceptibility of mice in the cvb myocarditis model can be increased by pretreatment with these cytokines. transforming growth factor-β is identifiable by immunohistochemistry in the prenecrotic regions of infiltrates in the murine myocardium and decreases when the macrophages and fibroblasts migrate to the necrotic foci. these growth factors may be responsible for recruitment of the immunologic effectors and may directly affect cardiac function. an intriguing feature of cytokine activity remains their possible role in the secondary development of myocyte hypertrophy and interstitial fibrosis, characteristic of dilated cardiomyopathy. among animals with different forms of viral myocarditis associated with similar intensity of initial myocyte necrosis, only those animals with persistent inflammation develop interstitial fibrosis, reflected by fibroblast proliferation and an increase in the extracellular matrix. myocardial fibrosis correlates well with the presence of t lymphocytes and macrophages, which in their activated state release fibrogenic cytokines such as fibroblast growth factor and transforming growth factor-β. matrix metalloproteinases (mmps), and their inhibitors, are thought to play a critical role in the process of myocardial remodeling. some of the cytokines elaborated during the course of viral myocarditis, such as tnf-α, disturb the balance between mmps and their inhibitors by increasing mmp, leading to failure of collagen cross-linking and worsened ventricular function ( fig. . ). this pathophysiology may present opportunities for prevention of the development of dilated cardiomyopathy resulting from myocarditis. extracardial reservoir secondary transfer to the target organ (e.g., heart) viral replication in the target organ viral protein expression lymphocytic myocarditis models in animals have conclusively demonstrated the association of viral infection and myocarditis. this association clearly exists in humans, but the proportion of cases that can be accounted for by viral infection is not known. the myocardial damage in murine models of viral myocarditis occurs in two distinct phases: an early phase of direct viral cytotoxicity in which virusspecific t-lymphocyte-and antibody-mediated cytotoxicity predominate; and a late or chronic phase in which the persistent viral genome, reactive ctls, autoantibodies, cytokines, and microvascular damage mediate myocyte damage and dysfunction. the hypothetical mechanisms of virusinduced autoimmune heart disease are presented in figures . to . . the recognition that immune responses to specific viruses are consequential in the development of myocyte injury has led to exhaustive research to exploit the possibility of designing immunomodulatory and antiviral therapies. the pretreatment of mice with inactivated virus vaccine prevents the manifestations of encephalomyocarditis virus myocarditis. the administration of antiviral therapies reduces the viral load and attenuates the histologic findings of myocarditis. , the antiviral response can be augmented by ifn-α or the exogenous administration of il- . , recombinant murine ifn-γ has also been demonstrated to improve the prognosis of acute murine myocarditis caused by encephalomyocarditis virus by suppressing replication. the murine model has also been the subject of intensive study with clinically applied immunosuppressants, such as corticosteroids, nonsteroidal antiinflammatory agents, , and cyclophosphamide, all of which have demonstrated deleterious effects when given in the acute viremic phase. cyclosporine, when administered in the early viremic phase, worsens myocardial injury but, in the late immune phase, has a beneficial effect. , , similar results have been reported with tacrolimus, and survival improves significantly when immunosuppressants such as cyclosporine, azathioprine, and -deoxyspergualin are used in adjunct to immunomodulators, such as ifn-α. antibodies to tnf-α have been demonstrated to improve survival and reduce myocardial injury. cytokine inhibitors have had promising results in animal models, but human clinical trials have been inconsistent. vesnarinone, a phosphodiesterase iii inhibitor, has demonstrated beneficial hemodynamic effects and inhibits the production of tnf-α and favorably modulates induction of inos. amlodipine has also been shown to increase survival of mice with viral myocarditis by inhibiting expression of inos and production of nitric oxide in vivo and in vitro. the diversity of immunopathogenetic mechanisms and variability in the severity of observed disease in the murine model are only a preview to the potpourri of clinical manifestations of myocarditis in humans. the presentation of unexplained progressive cardiac dysfunction or ventricular arrhythmias should lead to the suspicion of myocarditis, especially when routine cardiac diagnostic studies do not reveal an etiology. the history of an antecedent viral infection or prodrome is often sought but seldom reported and rarely confirmed by convalescent serologies. the presence of mild elevation of creatine kinase mb isoenzyme (ck-mb) or troponin, leukocytosis, or ecg changes may further underscore the possibility of myocarditis. most patients with myocarditis likely remain asymptomatic and never seek medical attention. the high frequency of exposure to cardiotropic viruses and the observation of a fairly high incidence of ecg abnormalities in apparently healthy individuals support this speculation. the incidence of myocarditis in an autopsy series following traumatic deaths in previously healthy individuals has been reported at . %. others have reported incidences ranging from . % to as high as % in unselected autopsy series. , these studies may suggest that at any given time, a significant percentage of the asymptomatic general population has myocarditis. the most common presentation of myocarditis is an acute febrile syndrome associated with pericardial and sys- temic complaints. cardiotropic viruses may cause pericardial inflammation, and patients often present with a syndrome of myopericarditis. chest pain is the most common symptom and is secondary to pericarditis or myocardial injury. a rather dramatic presentation of myocarditis is one indistinguishable from an acute myocardial infarction, complete with chest pain, ecg features suggesting acute ischemic injury, enzymatic evidence of myocardial damage, and echocardiographic or ventriculographic regional wall motion abnormalities, but on endomyocardial biopsy myocarditis is confirmed. [ ] [ ] [ ] most patients presenting with this acute syndrome completely recover, although there are isolated instances where progressive myocyte loss and cardiac failure or sudden arrhythmic death is reported. the segmental wall motion abnormalities result from virus-mediated injury, although local coronary arteritis and vasospasm have been suggested as possible culprits. , symptoms of right and left ventricular failure and even cardiogenic shock are frequently found in patients with biopsy-proven myocarditis, since it is these symptoms that lead to medical attention. however, the true incidence of heart failure in patients with myocarditis is probably much lower. in patients presenting with recent-onset heart failure and biopsy-proven myocarditis, % to % have had an antecedent flu-like illness. neonatal myocarditis is often a fulminant syndrome consisting of fever, tachycardia, tachypnea, cyanosis, and rapid progression to circulatory collapse. mortality rates are the highest in this subpopulation, approaching %. children are known to present with syncope due to heart block. other atrial arrhythmias described with myocarditis include sinoatrial block, atrial standstill, av block, intraatrial conduction abnormalities, atrial tachycardia, flutter, and fibrillation. [ ] [ ] [ ] [ ] [ ] [ ] histologic evidence of possible myocarditis has been described in up to two thirds of patients with lone atrial fibrillation. complete heart block has also been described in certain viral infections, such as epstein-barr virus or mumps, and also with rickettsiae. [ ] [ ] [ ] myocarditis may also manifest as myocardial thickening and fibrosis presenting as diastolic dysfunction or restrictive cardiomyopathy, and asymmetric septal thickening resembling hypertrophic cardiomyopathy. [ ] [ ] [ ] lieberman and coworkers proposed a clinicopathologic description of myocarditis based on the initial manifestations, endomyocardial biopsy, and recovery (fulminant, acute, chronic active, or chronic persistent myocarditis). ventricular arrhythmias are frequently encountered in patients with myocarditis, ranging from innocuous premature ventricular contractions to malignant and incessant ventricular tachycardia, and myocarditis is often incriminated in otherwise unexplained ventricular arrhythmias and sudden death. , myocarditis has been documented as a cause of ventricular repolarization abnormalities in athletes with or without arrhythmias. , ventricular arrhythmias may also be precursors to sudden cardiac death in young athletes with occult myocarditis. in autopsy series, myocarditis accounts for % to % of sudden deaths in young, healthy people. , , , in a population-based retrospective study from turin, italy, an incidence of only . % was reported among , autopsies performed over three decades, but the application of standardized systematic histologic examination and criteria tends to give a higher incidence, in the range of %, among autopsies performed at a general hospital. wesslen and associates reported signs of active, healing, or healed myocarditis in of cases of sudden death in young swedes. among high-performance athletes, sudden death due to undiagnosed myocarditis often stirs media attention. myocarditis has also been anecdotally implicated in sudden infant death syndrome. ventricular arrhythmias are frequently the initial and most prominent presentation of giant cell myocarditis. [ ] [ ] [ ] [ ] , ventricular arrhythmias and sudden death are common in all forms of myocardial failure, but specific immunemediator-induced effects on myocyte electrophysiology could also account for a portion of these arrhythmias. binah summarized a number of the mechanisms recognized by work in his laboratory and in others. as noted above, perforin elaborated by ctls is capable of forming membrane channels that pass charged ions, resulting in action potential shortening and diastolic oscillations. in addition, fas ligand can lengthen the action potential and induce afterpotentials, in part through inhibiting i to and augmenting i cal . the physical findings in acute myocarditis are dependent on the extent of myocardial or pericardial involvement, inciting agent (cardiotropic virus), and other factors. fever occurs occasionally, and in the myocarditis treatment trial (mtt), it was noted in % of patients with myocarditis. sinus tachycardia may frequently accompany the febrile state but is often out of proportion to the fever and is more likely adrenergically mediated, owing to the hemodynamic alterations of the failing heart. significant ventricular dysfunction may also be associated with hypotension, gallops, murmurs of regurgitation, rales, jugular venous distention, hepatomegaly, ascites, pleural effusions, and peripheral edema. pericardial involvement may result in a friction rub. the physical findings are not specific for myocarditis. patients with myocarditis frequently have serologic evidence of an inflammatory state with elevation of nonspecific markers of inflammation, such as erythrocyte sedimentation rate, c-reactive protein, and leukocyte counts. a fourfold increase in virus-specific igg titers in the convalescent period is considered reliable evidence of recent infection and is found in % of patients with myocarditis. , in the mtt, more than half of the patients with biopsy-proven myocarditis had an elevated sedimentation rate. other markers noted to be elevated in myocarditis include tnf-α, icam- , vascular cell adhesion molecule- , interleukins, and soluble fas. , , , unfortunately, these markers are not specific for myocarditis. myocarditis, although associated with myocyte damage and necrosis, results in ck-mb elevation in only % of patients with biopsy-proven myocarditis. more recently, lauer and colleagues reported on ck-mb elevation in only one of fi ve patients with histologic evidence of myocarditis, but cardiac troponin t (ctnt), which is extremely specific for myocardial damage, was elevated in all five. additionally, ctnt was elevated in patients, of whom had immunohistologic evidence of myocarditis. thus, ctnt elevation appears to be highly predictive for myocarditis. in an analysis of stored sera on patients from the mtt, cardiac troponin i (ctni) was elevated in % of patients ( of ) with myocarditis, compared with % ( of ) without myocarditis. in contrast, ck-mb values were elevated in only . % of patients ( of ) with myocarditis. further, the ctni elevations correlated with less than month's duration of heart failure symptoms. antibodies to cardiac antigens can be detected in the serum of patients with myocarditis. , , anti-α-myosin igg antibodies may have promise as a diagnostic tool, and, along with other antibodies, probably play a functional role. , the clinical efficacy of igg immunoadsorption , in dcm supports this notion (see also fig. . ). historically, acute myocarditis was diagnosed with the constellation of clinical symptoms, physical signs, and ecg abnormalities. although no particular feature on the electrocardiogram is pathognomonic of acute myocarditis, sinus tachycardia, repolarization abnormalities, conduction abnormalities, and arrhythmias are common findings. in a series of patients with biopsy-proven myocarditis, morgera and associates noted an abnormal qrs duration in %; abnormal q waves in %; left bundle branch block (lbbb) and right bundle branch block (rbbb) patterns in % and %, respectively; st elevation in %; t-wave inversions in %; and advanced av block in %. in patients presenting earlier in the course of the disease, with symptoms of less than month's duration, % had advanced av block and % had st elevation with t-wave inversions. the latter finding has been noted to portend a poorer prognosis. other predictors of poor outcome include lbbb, rbbb, and other conduction abnormalities, which seem to suggest active, severe, and extensive myocarditis. patients may present with sustained ventricular tachycardia, and continuous ecg monitoring of patients with myocarditis often reveals complex ventricular ectopy and nonsustained ventricular tachycardia. , echocardiography is useful in assessing the extent of left ventricular systolic dysfunction, which may range from mild segmental hypokinesis to severe global hypokinesis or akinesis associated with severe congestive heart failure (chf). patients presenting with chest pain or arrhythmias without chf often have normal echocardiograms. the ventricular dimensions may remain normal or may be only mildly enlarged. there may be an increase in left ventricular sphericity and right ventricular elongation and an increase in wall thickness and left ventricular mass with the interstitial edema and compensatory hypertrophy. , restrictive filling patterns in the left ventricle identifying diastolic dys-function have been reported consistently in biopsy-proven myocarditis. mural thrombi in diffusely hypokinetic ventricles have been reported frequently. hyperrefractile myocardium and other qualitative and quantitative analyses of myocardial texture have been described to assess the degree of active myocardial inflammation. pericardial effusion is a helpful echocardiographic finding, reported in % of patients with myocarditis, though hemodynamic compromise with cardiac tamponade is infrequent. urhausen and associates recently demonstrated that cardiac tissue velocity imaging by ultrasound is more sensitive than magnetic resonance imaging (mri) in some cases in detecting myocarditis with subtle ventricular functional impairment. imaging of leukocyte-mediated inflammation through ultrasound fracture of phagocytosed microbubbles shows promise as a means for detecting many forms of myocardial inflammation, although the method remains to be fully evaluated in humans. cardiac scintigraphy has been proposed as a convenient, noninvasive test with high sensitivity to diagnose active myocarditis. gallium- imaging, which identifies areas of increased inflammation, has been studied in clinical settings and noted to have sensitivity and specificity of % and %, respectively, with a negative predictive value of % for the diagnosis of myocarditis. indium- antimyosin monoclonal antibodies have been extensively studied to identify areas of myocyte damage in acute myocarditis. , this technique has extremely high sensitivity and often detects myocarditis that, on endomyocardial biopsy, is not seen by routine histologic assessment but is detected by immunohistochemistry. dec and coworkers studied patients with dcm with radiolabeled antimyosin antibody and endomyocardial biopsy. thirty-nine patients had abnormal antimyosin scans, but only of had evidence of myocarditis (predictive value of %). however, functional improvement was more likely in antimyosin scan-positive patients irrespective of the biopsy. the left ventricular ejection fraction (lvef) improved significantly in both concordant-positive (scan and biopsy both positive) and discordant-positive (scan positive, biopsy negative) patients, but it did not markedly improve in the negative scan and negative biopsy subset. the investigators proposed that discordant-positive scans represented patients with myocarditis in whom there may have been a sampling error on biopsy, hence the reason for missing the diagnosis. anastasiou-nana's group in athens reported that a combination of minimal or no left ventricular dilatation and a positive indium- antimyosin monoclonal antibody scan is highly specific for myocarditis. other nuclear techniques, such as technetium- m ( m tc)-mibi single photon emission computed tomography (spect) imaging, may also be useful in detecting myocarditis. contrast media-enhanced cardiovascular mri in patients with myocarditis has also been demonstrated to be an excellent tool in visualizing the location, activity, and extent of inflammation. early in myocarditis (day ), the enhancement on mri signals is accentuated and focal, whereas later (day ), this seems to be attenuated and more diffuse. furthermore, the severity of change correlates with prognosis. myocardial phosphorus- magnetic resonance spectroscopy has been utilized in assessing abnormalities in cardiac high-energy phosphate metabolism in patients with dcm and allograft rejection, but its role in the diagnosis of active myocarditis remains to be elucidated. the antemortem diagnosis of myocarditis was made feasible by the development of the endomyocardial biopsy technique. myocardial samples could be obtained via a transvascular approach with minimal discomfort to the patient and a low complication rate. whereas other approaches for acquiring myocardial tissue included percutaneous biopsy and mediastinotomy, , these were fraught with complications, precluding their acceptance into clinical practice. the safe and successful transvascular endomyocardial biopsy first described by sakakibara and konno was readily accepted for surveillance of cardiac allograft rejection in transplant recipients. the use of endomyocardial biopsy for the diagnosis and management of myocarditis was first reported in . subsequently, many reports , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] documented myocarditis in patients presenting with unexplained heart failure or ventricular arrhythmias (table . ). however, there was considerable incongruity in the diagnostic criteria used in these largely anecdotal reports. the dallas criteria were developed in preparation for a large, randomized, multicenter clinical trial of immunosuppressive therapy in myocarditis. these criteria define active myocarditis (see also fig. . a) as "an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of ischemic damage associated with coronary artery disease." furthermore, other causes of inflammation (e.g., connective tissue disorders, infection, drugs) should be excluded. , the dallas criteria also defined borderline myocarditis as an inflammatory infiltrate that is sparse and lacks myocyte injury, and often ( %) on repeat biopsy, borderline myocarditis will histologically progress to active myocarditis. a limitation of endomyocardial biopsy is possible sampling error. the inflammation in myocarditis may be patchy or focal, unlike allograft rejection, which is a relatively diffuse process. although obtaining four samples from the right ventricular septum provides a high sensitivity for detection of allograft rejection in transplant recipients, this may not hold true for myocarditis. in an autopsy study of the right ventricular biopsy technique ( samples taken from the apical septum), only six of patients dying of myocarditis were correctly identified. left ventricular biopsy missed the diagnosis in eight of . in another study using the standard four to six samples, the sensitivity of right ventricular endomyocardial biopsy was reported at %. dec and colleagues reported that employing repeat left and right ventricular biopsies in patients with suspected myocarditis with an initial negative biopsy increases the yield by %. because an ideal study to evaluate sampling error has not been done, the true yield is unknown, but clearly a negative biopsy does not exclude active myocarditis. in the mtt, only % of patients screened had histologic evidence of myocarditis. the european study of epidemiology and treatment of cardiac inflammatory disease (esetcid) demonstrated a % incidence of biopsy-proven myocarditis by expanding the dallas criteria with the use of newer techniques of pcr and in situ hybridization. as discussed earlier, [ ] [ ] [ ] [ ] [ ] [ ] there is a need for validation of new histologic and nonhistologic criteria for diagnosis of this disease to improve upon the dallas histologic criteria. coronary arteriography is usually normal, although in animal models, coronary vasculitis has been reported. the one major exception is kawasaki disease, in which coronary artery aneurysms are frequently seen in association with myocarditis. ventriculograms may demonstrate global or regional ventricular dysfunction, associated valvular regurgitation, and mural thrombi. localized ventricular aneurysms with normal global systolic function have also been reported. the hemodynamic profiles of patients with acute myocarditis are representative of the extent of myocardial and pericardial involvement. in patients with significant ventricular dysfunction, elevated filling pressures with depressed cardiac output and stroke work indices are seen. a restrictive hemodynamic profile can be seen and must be differentiated from that seen with postviral constrictive pericarditis. the true natural history of myocarditis is largely unknown because the great majority of cases is perhaps subclinical and resolves without any significant residual cardiac dysfunction. clinically apparent myocardial dysfunction as seen with acute coxsackievirus b infections also resolves without any adverse sequelae in most cases. it has been estimated that only % of patients with clinically suspected acute myocarditis will proceed to develop dcm, but the true incidence is unknown. the murine myocarditis models frequently develop a pathologic process indistinguishable from that of the human form of idiopathic dcm. the direct link among viral infection, myocarditis, and dcm has not been conclusively proven. isolation of infectious virus from the heart has been achieved in only a few cases of acute fulminant myocarditis in neonates and infants. , given the hypothesis that dcm may develop after viral infection has been eradicated, the presence of virus in the myocardium is neither sufficient nor necessary to link virally mediated myocarditis with dcm. the indirect evidence of viral etiology of dcm relies on ( ) progression of viral myocarditis to dcm in experimental animal models, ( ) apparent progression of myocarditis in some patients to dcm, ( ) increased enteroviral antibody titers in patients with dcm, ( ) presence of viral genomic material in the myocardial tissue of patients with dcm, and ( ) improvement of ventricular function in subjects with dcm receiving immunomodulatory treatments. the major limitations are as follows: the relevance of disease in mice to humans is suspect, most cases of dcm are not preceded by documented myocarditis, and epidemiologic serologic evidence is incomplete. whereas coxsackievirus b igm antibodies are detected with greater frequency in patients with dcm than in normal controls, the frequency is similar to matched community controls and household contacts. , enteroviral genomic sequences are detected in the myocardium of % to % of patients with active myocarditis and in % to % of patients with dcm, but in data derived from most published studies, the average detection frequencies are % for active myocarditis, % for dcm, and not significantly different from % among healthy controls. , in a metaanalysis of the association of enteroviruses with human heart disease, baboonian and treasure concluded that although the causative role of enteroviruses in acute myocarditis, particularly in children, was supported by an overall odds ratio of . [confidence interval (ci), . to . ], and the association of dcm was suggested by an overall odds ratio of . (ci, . to . ), six of studies did not demonstrate an increased presence of viral remnants. the same investigators demonstrated more recently that pcr positivity is not found in minimally affected first-degree relatives of patients with familial dcm, suggesting that in this group, genetic predisposition to viral myocarditis does not underlie the inherited predisposition to development of dcm. in recent studies, other investigators have found strong evidence for a viral link, while others have found no viral vestiges in the myocardium of patients with end-stage heart failure. , regional variation in the etiology of dcm may be responsible in part for the reported differences in pcr positivity. responsiveness of patients with dcm to immunomodulatory interventions provides an interesting line of evidence supporting a viral/immune etiology of dcm. one would expect immune suppression to be an effective treatment in dcm if postviral and other forms of autoimmunity play a causative role in the disease. efficacy of such interventions has been reported in carefully selected patients. , , [ ] [ ] [ ] although the link between myocarditis and dcm is unclear, certain prognostic factors are identifiable. the presence of an abnormal qrs complex on ecg correlates with more severe left ventricular damage and is an independent predictor of survival. left atrial enlargement, atrial fibrillation, and lbbb are also associated with increased mortality. higher baseline lvef is positively associated with survival, whereas intensity of conventional therapy at baseline is negatively associated with survival. the presence of right ventricular dysfunction, as evidenced by abnormal right ventricular systolic shortening on echocardiography, was shown to be the most important predictor of death or need for cardiac transplantation in a group of patients with biopsy-proven myocarditis who were followed longterm. in addition, a net increase in lvef (between initial and final ejection fraction) was associated with improved survival, whereas baseline ejection fraction was not predictive of outcome. the presence and degree of left ventricular regional wall motion abnormalities did not affect the clinical course. light microscopic findings on biopsy have not been found to predict outcome in myocarditis. however, the extent of myocardial inflammation was a predictor of outcome after surgical ventricular remodeling for heart failure. higher baseline serum antibodies to cardiac igg by indirect immunofluorescence was associated with a better lvef and a smaller left ventricular end-diastolic dimension. general supportive measures for patients with myocarditis include a low-sodium diet, discontinuation of ethanol, and fluid restriction, especially in the presence of heart failure. patients with myopericarditis may need analgesics for pain control. recommendations for the limitation of physical activity are based on the murine model of cvb myocarditis, in which forced exercise during the acute phase of illness was associated with higher titers of infectious virus, increased inflammatory and necrotic lesions, and mortality. , , ibuprofen, indomethacin, and salicylates administered to mice after inoculation with cvb also resulted in increased viral titers, increased histologic severity of myocarditis, and increased mortality. this led to the suggestion that even nonsteroidal antiinflammatory drugs should be avoided in patients with active acute myocarditis. the american college of cardiology task force on myopericardial diseases recommends a convalescent period of approximately months after the onset of clinical manifestations before a return to competitive sports. the management of patients with presumed or confirmed myocarditis is primarily directed toward treatment of chf, arrhythmias, and symptoms from pericardial disease. diuretics, vasodilators, and digoxin should be administered to patients with mild-to-moderate systolic dysfunction. inotropic therapy and mechanical support with intraaortic balloon pump or ventricular-assist devices may be required for patients in refractory cardiogenic shock. cardiac transplantation is reserved for those patients who do not improve despite the measures described previously. although there are multiple studies on the use of angiotensin-converting enzyme inhibitors (aceis) in heart failure, the utility of aceis in myocarditis has been studied only in the murine model. early treatment with captopril in a cvb myocarditis model resulted in less inflammatory infiltrate, myocardial necrosis, and calcification. heart weight, heart/body weight ratio, and liver congestion diminished. even with delayed therapy, a reduction in left ventricular mass and liver congestion was evident. the aceis exert a potent vasodilator response, improve pump function, prevent ventricular remodeling, and may have antiarrhythmic properties. hence, all patients with systolic dysfunction, including those with myocarditis, should be placed on maximally tolerated doses of aceis. the use of beta-blockers in patients with mild-tomoderate heart failure due to dcm has been reported to be beneficial, but once again, no trials in humans with myocarditis have been performed. metoprolol-treated mice in an acute cvb murine myocarditis model have increased viral replication, myocyte necrosis, and -day mortality rates. carteolol, a nonselective beta-blocker, has been studied in a chronic myocarditis model and found to have beneficial effects with improved histologic scores, reduced heart weight and volume, and liver congestion. it appears that in the acute setting, beta-blockers should be avoided, and in the chronic heart failure stage, the nonselective beta-blockers may be beneficial. antiarrhythmic therapy may be needed for control of ventricular and supraventricular dysrhythmias. although the data from clinical trials of antiarrhythmic therapy in heart failure have not shown a primary mortality benefit, patients with active myocarditis were excluded in these trials. since immunosuppression is probably not helpful in myocarditis and no other specific therapy is available, one might consider treating the arrhythmias in the usual fashion, but there appears to be a rationale for making the diagnosis of myocarditis in patients who do not have profound ventricular dysfunction along with their arrhythmia. first, the majority of patients with myocarditis have a spontaneous resolution. second, current antiarrhythmic therapy of ventricular tachyarrhythmias is exacting, involving electrophysiologic studies and use of potentially toxic drugs or implantable defibrillators. the benefit of making the diagno-sis of myocarditis is that the patient may require only shortterm protection while the underlying process resolves, which can be provided by using amiodarone or other antiarrhythmic drugs under inpatient monitoring. if myocarditis resolves, antiarrhythmic therapy can be withdrawn. patients whose arrhythmias fail to improve despite histologic resolution of myocarditis, or who survived cardiac arrest, may be candidates for aggressive electrophysiologic approaches and implantable defibrillators. temporary and permanent pacemakers may be required in patients presenting with conduction system abnormalities. clinical trials of immunosuppressive therapy were first reported in children with clinical evidence of myocarditis, prior to the introduction of endomyocardial biopsy. in two series, in a total of eight children presenting with acute onset of severe chf, rapid improvement and survival were noted with adrenocorticotropic hormone or hydrocortisone treatment. , mason and associates reported patients with biopsy-proven myocarditis, half of whom improved with azathioprine and prednisone. gagliardi and coworkers followed children with biopsy-proven myocarditis who were treated with cyclosporine and prednisone. at year, of patients still had histologic evidence of myocarditis. no patient died or required transplantation. however, there was no control group. the data supporting an immunologic basis of myocarditis resulted in multiple treatment trials using immunosuppressants (table . ). the average proportion of patients showing improvement with a variety of immunosuppressants was %. a large number of the trials predated the development of the dallas criteria; thus, the histologic definition of myocarditis was not uniform. immunosuppressive regimens were arbitrary, and the lack of control groups made interpretation of these trials arduous. it was unclear whether immunosuppression was beneficial in those patients with myocarditis, as they can improve spontaneously. further, the infectious complications of immunosuppression were frequently seen and occasionally reported. , the conflicting results from these nonrandomized observations led to the mtt. in a multicenter, prospective, randomized design, the mtt enrolled patients with heart failure of recent onset (< years), left ventricular dysfunction (lvef < %), and biopsy-proven myocarditis (per the dallas criteria). the study screened patients; ( %) had endomyocardial biopsy evidence of myocarditis, and patients had a qualifying lvef of less than % and agreed to enrollment. patients were randomized to three treatment arms: prednisone and cyclosporine, prednisone and azathioprine, and no immunosuppressant treatment. all patients received conventional therapy for heart failure. the prednisone and azathioprine group was subsequently eliminated owing to low patient recruitment in the trial. patients were treated for weeks, and the primary end point was comparison of the mean increase in lvef at weeks. secondary analysis of other markers of left ventricular function, survival, and several immune parameters was performed. at both and weeks, no difference in lvef was observed in immunosuppressive-treated patients compared with untreated patients. at and years, there was no difference in survival or need for cardiac transplantation between groups (fig. . ) . on multivariate analysis, better baseline lvef, less intensive conventional therapy, and shorter illness duration were independent predictors of improvement in lvef during follow-up. analysis of immunologic variables (cardiac igg, circulating igg, natural killer and macrophage activity, helper t-cell level) suggested an association between better outcome and a more robust immune response. a higher level of cardiac igg was associated with a higher lvef and a smaller left ventricular size. the mortality rate for the entire trial was % at year and % at . years. the results of the mtt were important for diagnostic management because the authors recommended that in patients with unexplained chf, the performance of endomyocardial biopsy for the sole purpose of instituting immunosuppressive therapy was not warranted. nonetheless, certain subgroups may benefit from immunosuppressant therapy, including those with gcm, hypersensitivity myocarditis, or cardiac sarcoidosis. using a multicenter database, cooper and colleagues reviewed patients with gcm. the rate of death or cardiac transplanta-tion was %. median survival was . months from symptom onset to death or transplantation. the median survival in patients treated with corticosteroids was . months versus . months in untreated patients. however, patients treated with corticosteroids and azathioprine had an average survival of . months. cyclosporine in combination with corticosteroids, corticosteroids and azathioprine, and corticosteroids, azathioprine, and orthoclone okt survived an average of . months. the uncontrolled nature of this report decreases the reliability of its conclusions. patients with myocarditis associated with a known immune-mediated disease, such as systemic lupus erythematosus, may benefit from immunosuppressive therapy. other potential indications for a trial of immunosuppressant therapy include failure of myocarditis to resolve, progressive left ventricular dysfunction despite conventional therapy, continued active myocarditis on biopsy, or fulminant myocarditis that does not improve within to hours of full hemodynamic support, including mechanical assistance, and persistent ventricular tachycardia or fibrillation. smaller studies have used differing immunosuppressant regimens. kühl and schultheiss treated patients with biopsies classified as immunohistologically positive (more then two cells per high-power field and expression of adhesion molecules), negative dallas criteria, and left ventricular dysfunction. patients were treated with conventional therapy for months, followed by gradual tapering of methylprednisolone doses over weeks (following biopsy and lvef response). therapy was associated with an improvement in ejection fraction in % and improved new york heart association (nyha) functional class in %. four patients ( %) had no change in ejection fraction despite improvement in inflammatory infiltrates. however, study conclusions are limited by the absence of a control group. drucker and coworkers retrospectively reviewed children with congestive cardiomyopathy and dallas criteria of borderline or definite myocarditis. twenty-one patients were treated with intravenous igg ( g/kg over hours) and were compared to historical controls. overall survival was not improved, although there was a trend toward improvement in -year survival rates in the treated group. in the intravenous igg group, the left ventricular function was improved and persisted after adjustment for age, biopsy status, and use of aceis and inotropes. in a comparative study of ifn-α, thymomodulin, and conventional therapy in patients with biopsy-proven myocarditis or idiopathic dcm, an improvement in the active treatment groups was reported for ejection fraction (at rest and during exercise), maximal exercise time, functional class, and ecg abnormalities. in patients with chf, nyha class iii or iv, with symptoms of less than months' duration, intravenous igg resulted in an improvement in lvef and a functional improvement to nyha class i or ii at year of follow-up, in all nine patients who survived, regardless of biopsy results. perhaps strategies with alternative immunosuppressive regimens and different diagnostic criteria will be more successful in demonstrating the utility of immunosuppressants. the esetcid , is a prospective multicenter, placebocontrolled, double-blind study intended to address the natural course of myocarditis, myopericarditis, pericarditis, and the treatment regimens include conventional therapy with diuretics, aceis, digoxin, and antiarrhythmics or defibrillators; specific therapy for cmv and enteroviral myocarditis; and prednisolone and azathioprine for myocarditis without detectable virus. the duration of blinded therapy is months, with follow-up for months. the possible utility of more flexible diagnostic criteria for identification of responders to immunosuppressive therapy was recently suggested in a retrospective analysis by frustaci and colleagues. they found that the patients who had improved with immunosuppression had detectible circulating cardiac-specific autoantibodies but no detectible viral genomic material in their myocardium, while nonresponders had the opposite findings. these observations could be explained by successful suppression of an autoimmune response without the liability of suppressing ongoing antiviral immune activity. immunomodulatory therapies should be considered in cases of myocarditis that display ongoing adverse immune and autoimmune activity. immunosuppressive drug therapy, intravenous igg, tnf-α, and immunoadsorption are the forms of immunomodulation discussed above that have been used in humans (fig. . ). immunoadsorption has been applied primarily in dilated cardiomyopathy, but may hold promise in myocarditis, perhaps especially in phase ( fig. . a) of lymphocytic myocarditis. manipulation of cytokines, chemokines, and other factors that regulate proinflammatory and antiinflammatory processes , , , [ ] [ ] [ ] [ ] should receive attention in the development and assessment of new immunomodulatory therapies. myocarditis has emerged as a special indication for device therapy in recent years. circulatory-assist devices are especially attractive in myocarditis because the disease is usually self-limited. as a result, a relatively short period of circulatory assistance may allow time for the myocardium to recover and the injurious infectious, immune, and autoimmune processes to dissipate. this concept has been successfully tested in patients with severe heart failure due to myocarditis. [ ] [ ] [ ] implanted cardioverter-defibrillator devices have been used to treat ventricular tachyarrhythmias commonly associated with myocarditis. while such therapy may be lifesaving, consideration should always be given to antiarrhythmic drug therapy with protracted monitoring so as to avoid device implantation if possible. those patients with myocarditis who have survived cardiac arrest are candidates for implantable defibrillator devices. in a small series (n = ) composed predominantly of female patients ( %), the outcome of patients with active lymphocytic myocarditis confirmed by histologic examination of the explained heart was significantly worse than in controls undergoing transplantation for other diagnoses. this concern has not been validated in the analysis of outcome of , cardiac transplant recipients in the registry of the international society for heart and lung transplantation. one-year actuarial survival in all groups transplanted (idiopathic dcm, myocarditis, peripartum cardiomyopathy, versus other diagnoses) was %. nonetheless, myocarditis may recur in the transplanted heart. prevention prevention of myocarditis is an important developing strategy given the likelihood that a substantial proportion of cases of dcm worldwide are the result of preceding or ongoing myocarditis. several strategies have been considered, including immunization against the most common cardiotropic viruses, , , [ ] [ ] [ ] [ ] functional disablement of the coxsackie-adenovirus sarcolemmal receptor, , and early induction of immune tolerance. while immunization seems to have the greatest potential, scientific, medical, geographic, and political impediments are formidable. the advances in treatment strategies for hiv-infected patients have successfully resulted in prolonged survival times, and noninfectious complications of aids, such as dementia and heart disease, have become increasingly prevalent. early in the history of the aids epidemic, reports emerged of a rapidly fatal dcm affecting hiv-infected patients. , since the early reports, several clinical and echocardiographic series , [ ] [ ] [ ] [ ] [ ] have suggested that a subgroup of hiv-infected patients are predisposed to the development of progressive heart disease. in a prospective echocardiographic survey of hiv-infected adults over a period of years, patients were found to have significant cardiac dysfunction. dilated cardiomyopathy occurred in of and was strongly associated with a cd count of less than /mm and poorer survival. it has been estimated that clinically significant cardiac disease occurs in % to % of hiv-seropositive individuals. an interesting hypothesis to explain the high frequency of dilated heart muscle disease is the presence of myocarditis in hiv-infected patients with left ventricular dysfunction. reilly and colleagues reported in an autopsy series of consecutive aids patients a significantly higher incidence of myocarditis in those with clinically apparent cardiac disease or dcm. there have been other reports of higher prevalence of myocarditis in an endomyocardial biopsy series of hiv-seropositive patients compared with those without risk factors for hiv who were biopsied for suspected myocarditis. human immunodeficiency virus-related myocarditis has unique and atypical immunopathogenic features. it is characterized by increased cd t lymphocytes and sole induction of mhc class i, perhaps as a part of the systemic depletion of cd t cells. the myocarditis may not be readily apparent on histology owing to the accompanying lymphopenia, and special immunohistology and histochemistry techniques may need to be employed. although in situ hybridization techniques have demonstrated hiv- transcripts in cardiac myocytes, interstitial dendritic cells, and endothelial cells, the pathologic significance of this finding is still unclear because patients with evident transcripts may or may not have clinical disease. also, it is not evident that myocyte injury is a result of direct cytotoxicity of the virus, transcripts, cytokines, or other cardiotropic viruses. a large number of hiv-seropositive patients with left ventricular dysfunction also manifest evidence of nonpermissive or latent infection of myocytes with cmv immediate-early (cmv ie- ) genes. although evidence for classic intranuclear inclusions of active lytic cmv infection is rarely found, there is increasing speculation that the latent viral infection may be responsible for enhanced mhc expression and provide a stimulus for ongoing immune injury, as seen with most models of myocarditis. a role for direct cytokine-mediated cardiac injury has also been proposed in hiv-infected populations with myocardial dysfunction. both tnf-α and il- , known to be elevated in hiv infection, directly inhibit cardiac contractility in vitro, and the former has been implicated in causing myocardial dysfunction. increased catecholamines may be responsible for microvascular spasm and chronic ischemic dysfunction. the clinical management of patients with hiv-related myocarditis and cardiomyopathy is targeted toward improving congestive symptoms, afterload reduction, and digitalis for improved neurohormonal axis. a specific role for antiviral therapies is controversial, since medications like zidovudine and ifn-α are themselves recognized as cardiotoxins. zidovudine has been known to result in premature termination of myocyte mitochondrial dna chain replication. despite worldwide eradication of smallpox, the bioterrorism threat arising from the existence of stored variola virus has prompted military and civilian smallpox vaccination programs in the united states. myocarditis emerged as a known, rare complication of smallpox vaccination during the eradication effort in the s and s. its incidence varied with the vaccinia strain used to produce the vaccine, and with the method used to detect myocarditis. the true incidence is not known. current vaccination programs use the original new york city board of health strain of vaccinia (dryvax) and new vaccines. while the occurrence of myocarditis in the united states's current military dryvax vaccination program appears to be higher ( . %, or about one in , ) than historical estimates, its incidence after new vaccines has not been determined. previously vaccinated individuals have a much lower risk of developing myocarditis. full functional and symptomatic recovery occurs in most patients. while involvement of eosinophils has been noted, the mechanisms responsible for postvaccination myocarditis are not known. bacterial infection of the myocardium occurs frequently in association with infective endocarditis, usually in the form of myocardial abscesses adjacent to the valve ring (see chapter ) . myocardial involvement has also been reported in association with a wide range of bacterial pathogens in the absence of endocarditis. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] with most of these agents, myocardial involvement is uncommon and occurs principally in the setting of overwhelming systemic infection. cardiac involvement after streptococcal infection is usually manifested as acute rheumatic fever, which develops to weeks after onset of pharyngitis and has a distinctive histologic appearance (see chapter ) . streptococcal pharyngitis may also be associated with a nonrheumatic form of myocarditis that occurs concurrently with the febrile illness. [ ] [ ] [ ] [ ] the most common clinical manifestations are chest pain and marked st-segment and t-wave abnormalities on the electrocardiogram, which correlate with segmental wall motion abnormalities observed with echocardiography. cardiomegaly and chf are uncommon. histologic examination reveals lymphocytic infiltrates and myocyte necrosis in the absence of aschoff bodies, similar to the findings in viral or idiopathic myocarditis. bacteria are not present in the myocardium, and it is hypothesized that inflammation is caused by streptococcal exotoxins in a manner similar to that in diphtheritic myocarditis. although vaccination has virtually eliminated diphtheria in most western nations, it remains an important public health problem in many underdeveloped countries, and may be the most common etiology of myocarditis worldwide. infection with c. diphtheriae is usually confined to the respiratory mucosa. systemic manifestations are due to secretion of a potent exotoxin. the ecg abnormalities suggesting myocardial involvement are present in a high proportion of patients, but clinical evidence of cardiac dysfunction occurs in only % to % of cases. nevertheless, cardiac involvement is the most common cause of death in fatal infections. disturbances of av conduction, including bundle branch blocks and complete av block, are observed frequently in affected patients and are associated with a mortality rate of % to %. patients may also present with progressive cardiac dilatation and chf. histologic study reveals diffuse mononuclear cell infiltrates associated with myocyte necrosis. corticosteroid therapy does not appear to be effective in the prevention or treatment of diphtheritic myocarditis, although only one prospective trial has been performed. one report suggested that administration of carnitine may decrease the incidence and severity of cardiac involvement. spirochetal myocardial disease lyme disease lyme disease is caused by the spirochetal organism b. burgdorferi, which is transmitted to humans by certain species of deer ticks in endemic areas of north america, europe, and asia. the acute phase of the illness is characterized by fever, myalgia, lymphadenopathy, and a characteristic rash known as erythema chronicum migrans. the organism persists in many tissues, and chronic manifestations include arthritis and a variety of neurologic syndromes. manifestations of cardiac involvement develop in % to % of patients at an average of . weeks (range, days to months) after the acute illness. , , disturbances of av conduction are the most common manifestations, occurring in % of cases, with complete or high-grade block in more than %. the av block is usually supra-hisian, with a narrow complex escape rhythm. temporary transvenous pacing is required frequently, but av block almost always resolves within to days. endomyocardial biopsy may reveal lymphocytic infiltrates with associated myocyte necrosis, and spirochetes may be identified in biopsy specimens. lyme carditis occasionally develops in patients without a preceding rash or other symptoms of acute lyme disease. therapy with a to week course of antibiotics (doxycycline, amoxicillin or cefuroxime) is recommended for patients with lyme carditis. , antibiotic therapy has proved effective in the prevention and treatment of chronic arthritic and neurologic syndromes, but its use in cardiac disease has not been tested prospectively. evidence of diffuse myocardial involvement is common, including evolving stsegment and t-wave abnormalities on the electrocardiogram, reversible abnormalities of left ventricular wall motion, and diffuse myocardial uptake on gallium scan. one fatal case of pancarditis has been reported, but frank heart failure is uncommon. a high incidence of positive serologies for b. burgdorferi was reported in european patients with chronic dcm, and in two patients the organism was cultured from myocardial biopsies. , it has been suggested that unrecognized lyme carditis may be responsible for a small but significant proportion of cases of idiopathic dcm. evidence of severe myocarditis is present at autopsy in a high proportion of fatal cases of leptospirosis and relapsing fever. , nonspecific ecg abnormalities are common in these diseases, but clinical evidence of left ventricular dysfunction is rare. although previously uncommon, the incidence of fungal infections of the heart has increased markedly since the early s. this increased incidence is due to several factors, including the increasing use of antibiotics, immunosuppressive agents for transplantation, and chemotherapy, as well as the increasing application of cardiac surgery and the increasing prevalence of intravenous drug abuse. candida infection the most common fungal organisms causing cardiac infection are candida species. candida endocarditis occurs most frequently after thoracic surgery and in intravenous drug abusers. immunocompromised patients, on the other hand, are more likely to develop candida myocarditis without involvement of the valves or endocardium, usually in the setting of disseminated systemic infection. [ ] [ ] [ ] autopsy studies reveal extensive myocardial involvement in % to % of patients who die of systemic candidiasis. histologically, candida myocarditis consists of focal abscesses (usually microscopic, although gross nodules may be present) interspersed with areas of normal myocardium. clinical manifestations typically include nonspecific ecg abnormalities, disturbances of av conduction, including complete heart block, and tachyarrhythmias. cardiomegaly and chf are rare. myocardial involvement is usually not recognized antemortem. myocardial involvement is present in % of patients with disseminated aspergillosis, and myocardial invasion is almost always present in patients with aspergillus endocarditis. as in other tissues, histology is characterized by microscopic and macroscopic abscess formation. , extensive vascular invasion by fungal hyphae results in thrombosis and coagulation necrosis. although aspergillus endocarditis has been treated successfully, myocarditis is uniformly fatal. cardiac involvement in actinomycosis occurs in only % of cases and usually develops by direct extension from a contiguous focus of pulmonary or mediastinal infection. [ ] [ ] [ ] hematogenous seeding of the myocardium occurs occasionally. myocardial involvement is characterized by necrotizing abscess formation with masses of mycelial bodies and characteristic sulfur granules. in many cases, cardiac symptoms are absent, but patients may present with chest pain characteristic of pericarditis, pericardial tamponade, or chf. myocardial involvement has rarely been reported in immunocompromised patients with disseminated coccidioidomycosis and cryptococcosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cardiac involvement is usually not clinically apparent antemortem, although death due to progressive chf has been reported. cardiac involvement with blastomycosis and histoplasmosis is extremely uncommon and usually results from direct extension from a contiguous intrathoracic focus. rocky mountain spotted fever caused by infection with rickettsia rickettsii is characterized by a diffuse vasculitis, and in fatal cases, death is usually due to vascular collapse. vasculitis of the coronary vessels may also be present, and lymphocytic infiltrates with myocyte necrosis are present in approximately % of fatal cases. , although cardiac dilatation and cardiogenic pulmonary edema occur infrequently, echocardiography demonstrates systolic left ventricular dysfunction in the majority of patients. , clinical evidence of myocarditis has been reported in association with scrub typhus due to rickettsia tsutsugamushi, whereas q fever (coxiella burnetii) usually causes endocarditis in its chronic form. it is estimated that to million people in south and central america are infected with t. cruzi, and chagas' cardiomyopathy resulting from this infection is the most common cause of chf and cardiac death in these endemic areas. , the parasite is transferred to humans by triatomine insects known as reduviid bugs. the clinical course of infection is characterized by an acute phase, an indeterminate or latent phase of variable duration, and a chronic phase. , after inoculation, parasites are disseminated throughout the body, with the highest concentrations appearing in striated and cardiac muscle and autonomic ganglia. a lesion may appear at the point of entry, and an acute illness develops, characterized by fever, myalgia, edema of the face and lower extremities, hepatomegaly, and generalized lymphadenopathy. because of the nonspecific nature of the symptoms, the acute phase of the disease is usually unrecognized. rarely, acute inflammatory myocarditis develops during the acute phase, with ecg abnormalities, cardiomegaly, and chf. histologic examination in these cases demonstrates inflammatory infiltrates adjacent to myocytes containing large numbers of intracellular parasites. these findings suggest that cardiac manifestations during the acute phase of the illness may be due to direct lysis of myocytes by parasites. , the acute illness resolves over a period of weeks to months, and patients enter the indeterminate phase. these patients are asymptomatic, with low-level parasitemia, and antibodies to t. cruzi are present. although the electrocardiogram is normal, echocardiography and left ventricular cineangiography demonstrate focal wall motion abnormalities in a high proportion of cases, most commonly involving the left ventricular apex and posterior wall. endomyocardial biopsy is frequently normal but may reveal hypertrophy, fibrosis, and inflammatory infiltrates in up to % of patients without clinical manifestations. manifestations of chronic chagas' disease develop in % to % of infected patients after a highly variable period, which may be as long as years. , involvement of autonomic ganglia may cause megacolon or megaesophagus, but the heart is the organ most commonly affected. histology is characterized by focal areas of inflammation or fibrosis interspersed with areas of normal myocardium. endomyo-cardial biopsy reveals myocarditis in approximately % of patients. , this process frequently involves the specialized conducting tissue, and therefore disturbances of av conduction, especially rbbb with or without associated left anterior fascicular block, are present in up to % of patients. complete heart block may require permanent transvenous pacing. ventricular arrhythmias are also frequent, and the initial manifestation of the disease may be sudden death due to ventricular tachyarrhythmia or complete heart block. decreased ventricular function is present in almost all patients with chronic chagas' disease, and in its most advanced form, chagas' disease presents as a congestive cardiomyopathy with four-chamber dilatation. a characteristic apical aneurysm is usually present. [ ] [ ] [ ] left ventricular thrombus is frequently observed, and systemic embolization is common. , this advanced form of the disease is usually fatal within a few years. diagnosis of chronic chagas' cardiomyopathy is dependent on detection of circulating antibodies to t. cruzi by one of several serologic methods. parasites are usually not detected in the myocardium, but low-level parasitemia can be demonstrated by hemoculture or xenodiagnosis, using uninfected reduviid bugs allowed to ingest the patient's blood. the pathogenic mechanisms leading to myocardial injury, in some patients occurring many years after the initial infection, are poorly understood. the presence of inflammatory infiltrates in the absence of detectable parasites suggests the possibility of autoimmune injury, as postulated for viral and idiopathic myocarditis. support for this hypothesis includes the demonstration of antibodies to t. cruzi as well as antiidiotypic antibodies that cross-react with myocyte antigens. , histologic studies demonstrate loss of autonomic ganglia, and physiologic studies are suggestive of marked autonomic dysfunction. [ ] [ ] [ ] withdrawal of parasympathetic tone may lead to excess sympathetic stimulation, which can cause cardiomyopathy. histologic studies also demonstrate abnormalities of the microvascular bed, , and in vitro experiments demonstrate altered endothelial cell function and increased platelet-endothelial cell adhesion. , all three reports suggest that progressive focal myocardial disease is the result of ischemia due to obstruction of the microvascular bed. treatment of chronic chagas' cardiomyopathy is supportive, with the use of standard therapy for chf. dynamic cardiomyoplasty has resulted in symptomatic improvement in some patients. the role for left ventricular reduction or the commonly known batista procedure is controversial. antiarrhythmic therapy may be indicated for sustained ventricular tachyarrhythmias, and a permanent pacemaker should be implanted in patients with high-degree av block. two antiparasitic drugs are available for the treatment of american trypanosomiasis. both nifurtimox and benznidazole decrease the level and duration of parasitemia and decrease mortality in patients with acute chagas' disease. low-level parasitemia persists in most treated patients, however, and it is unclear whether therapy in the acute phase decreases the incidence of subsequent progression to chronic chagas' disease. whereas earlier studies with these drugs have not been shown to decrease progression from latent phase to chronic disease or to decrease symptoms or improve cardiac function in patients with chronic disease, , the recent studies with itraconazole and allopurinol have shown partial success with parasitologic cure and normalization of ecg changes in nearly half the patients. in a randomized, placebo-controlled trial of benznidazole, there was successful negative seroconversion of % of patients with early chronic disease as manifested by seropositivity for t. cruzispecific antibodies after treatment for days. immunosuppressive therapy in patients with malignancies or after organ transplantation has been associated with reactivation causing acute chagas' disease. , reactivation of chagas' disease in this setting has usually responded promptly to therapy. [ ] [ ] [ ] african trypanosomiasis african trypanosomiasis is caused by trypanosoma gambiense or t. rhodanese and characteristically presents with progressive somnolence owing to central nervous system involvement. autopsy studies demonstrate a pancarditis involving the mural and valvular endocardium as well as the myocardium in up to % of fatal cases. [ ] [ ] [ ] [ ] the conduction system and autonomic ganglia may also be involved. nonspecific abnormalities are often present on the electrocardiogram, but other clinical manifestations of the frequent cardiac involvement are apparently uncommon. patients with acute infection by t. gondii are usually asymptomatic, but they may have a transient syndrome of fever and lymphadenopathy. the infection usually persists in a latent phase, with cysts deposited predominantly in the brain and myocardium. immunosuppression after chemotherapy, in transplant recipients, and in patients with aids may be associated with disseminated infection characterized by severe encephalitis and myocarditis. [ ] [ ] [ ] [ ] myocarditis after transplantation occurs frequently in seronegative recipients of hearts from seropositive donors. [ ] [ ] [ ] endomyocardial biopsy demonstrates intracellular toxoplasma pseudocysts and a mixed interstitial infiltrate, frequently including eosinophils ( fig. . d ). toxoplasma myocarditis can be successfully prevented by a -week course of pyrimethamine imitated after early transplantation or treated with pyrimethamine and sulfadiazine. cardiac involvement in metazoal infections is uncommon. up to % of patients with echinococcosis have cardiac cysts. [ ] [ ] [ ] these patients may present with pericardial or atypical chest pain, chf owing to inflow or outflow obstruction, ventricular arrhythmias, or pulmonary hypertension owing to diffuse pulmonary embolization of scolices. the diagnosis is usually documented by two-dimensional echocardiography, and surgical excision is indicated, when possible, even in asymptomatic patients. trichinosis, caused by the parasite t. spiralis, is usually a benign syndrome characterized by fever, myositis, and eosinophilia. mild, asymptomatic myocardial involvement is probably common, as suggested by frequent ecg abnormalities and pericardial effusion noted by echocardiography. rarely, a severe myocarditis develops, which is the apparent cause of death in most fatal cases. [ ] [ ] [ ] eosinophils are prominent in the interstitial infiltrate. t. spiralis does not become encysted in the heart, and larvae are seldom identified in the myocardium. myocardial injury is thought to be immune mediated, and therapy with corticosteroids is generally recommended, although prospective trials have not been performed owing to the infrequent occurrence of this syndrome. the mucocutaneous lymph node syndrome or kawasaki disease occurs predominantly in children under the age of years and is most prevalent in japan. , it has been recognized worldwide, and in the united states and the developed world, it has replaced rheumatic fever as the most common cause of acquired heart disease in children. it is widely believed to have an infectious etiology, but no agent has yet been identified. its diagnosis is based on recognition of clinical features of the illness, which include remittent high-spiking fever with distinctive conjunctival injection, anterior uveitis, strawberry tongue with erythema, dryness, fissuring and peeling of the lips and mouth, erythematous truncal rash, redness of palms and soles with periungual desquamation, and cervical lymphadenopathy. the principal cardiovascular manifestation of the disease is a multisystem arteritis with frequent involvement of the coronary arteries. coronary arteritis leads to aneurysm formation and thrombosis. the most common cause of death is myocardial infarction due to aneurysm rupture or coronary occlusion. myocardium obtained by endomyocardial biopsy or at autopsy reveals histologic evidence of myocarditis in a high proportion of patients. [ ] [ ] [ ] [ ] segmental wall motion abnormalities and nonspecific ecg changes are frequently present in the absence of coronary aneurysms. , these findings have been attributed to myocarditis, but they might also reflect ischemia due to small vessel arteritis. congestive heart failure in the absence of infarction is uncommon. intravenous gamma-globulin and high-dose aspirin are effective in the prevention of coronary aneurysms and thrombosis, but their effect on myocarditis is not known. giant cell myocarditis is a rare but frequently fatal disorder. it is defined histologically by extensive but patchy myocyte necrosis with areas of intense multicellular inflammatory infiltration that includes histiocytes, lymphocytes, and the characteristic multinucleated giant cells (fig. . b) . , [ ] [ ] [ ] there has been a great deal of controversy as to whether gcm and cardiac sarcoidosis are distinct pathologic entities because multinucleated giant cells in gcm seldom organize to form granulomas. , , litovsky and associates showed that gcm is characterized by myocytic destruction mediated by cytotoxic t cells, macrophagic giant cells, and eosinophils. in contrast, cardiac sarcoid is an interstitial granulomatous disease without myocytic necrosis. although the etiology of gcm is unknown, it has been associated with a medley of autoimmune disorders and perhaps is immunologically mediated. thymomas, systemic lupus, rheumatoid arthritis, wegener's granulomatosis, ulcerative colitis, chronic hepatitis, myasthenia gravis, myositis, pernicious anemia, takayasu's arteritis, and lymphomas have been associated with gcm. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the clinical presentation of gcm may mimic lymphocytic myocarditis, although arrhythmias and heart failure are usually more severe and rapidly progressive. , frequently, patients with gcm present with conduction system abnormalities, ventricular tachycardia, or even sudden cardiac death. [ ] [ ] [ ] [ ] , , , , giant cell myocarditis has also been reported to present as asymmetric septal hypertrophy. the natural history of gcm is obscure owing to its rare occurrence, but the isolated reports in the literature suggest that it carries a poor prognosis. davidoff and coworkers reported that % of patients with gcm required cardiac transplantation or died during a -year follow-up period compared with the % of patients with lymphocytic myocarditis. cooper and colleagues reported on patients with gcm collected in a worldwide registry. the registry patients had an % rate of death or need for transplantation, which was significantly worse than that for the patients with lymphocytic myocarditis seen in the mtt. the median survival with gcm was . months. the patients treated with immunosuppressive regimens including cyclosporine, azathioprine, and prednisone had an average cardiac survival of . months compared with . months for the untreated patients. the rate of recurrent gcm in the transplanted patients was % (nine of ). the role of immunosuppressive therapy for gcm is unknown, but at least anecdotal and registry reports suggest possible benefit of cyclosporine and prednisone with or without azathioprine. cardiac transplantation remains the last therapeutic resort for these patients, although there is risk of recurrent disease, , , which seems to be associated with abatement of immunosuppressive therapy after transplantation and may represent atypical rejection in the allograft. it usually resolves with intensification of the immunosuppressive regimen. eosinophilic myocarditis loffler first described the association of eosinophils with cardiac disease, and reported "endocarditis parietalis fibroplastica" in association with eosinophilia. the endocardial disease with eosinophilia is well recognized and extensively reviewed elsewhere. , myocardial involvement is rare and frequently fatal; hence, diagnosis is often made postmortem. endomyocardial biopsy is essential to the antemortem diagnosis of eosinophilic myocarditis (fig. . c ). myocarditis is believed to represent a more fulminant and necrotic form of the endocardial disease. eosinophils have the ability to secrete highly toxic cationic proteins into areas of inflammation and to produce harmful oxygen radicals and potent lipid mediators, leading to myocyte necrosis as seen in proximity to degranulating cosinophils. animal experiments have confirmed that exposure of myocytes to eosinophil granule proteins is lethal, and ventricular function in the intact heart is reduced in hypereosinophilic states. eosinophilic myocardial infiltrates have been reported in association with profound eosinophilia caused by an allergic diathesis, parasitic infection, drug hypersensitivity, vasculitis, or churg-strauss syndrome, , , but eosinophilic myocarditis can occur in the absence of profound eosinophilia. further, eosinophilic myocarditis may present as acute myocardial infarction, sudden death, cardiogenic shock, or nonspecific chest pain and dyspnea. the natural history of eosinophilic myocarditis is usually swift and ominous with rapid evolution to refractory heart failure or intractable arrhythmias, leading to death. early biopsy-aided histologic confirmation is fundamental to antemortem diagnosis. clinical improvement may occur with corticosteroid therapy. cardiac sarcoidosis sarcoidosis is a multiorgan, noncaseating granulomatous disorder of unknown etiology. histologically, it may involve the lung, lymph nodes, skin, liver, spleen, parotid glands, and heart. right heart failure owing to pulmonary manifestations of pulmonary hypertension and pulmonary fibrosis is the predominant cardiac finding. asymptomatic cardiac involvement is common, with a quarter of the patients having sarcoid granulomas in the heart at autopsy. characteristically, the noncaseating granulomas infiltrate the ventricular walls and become fibrotic. they may involve the conduction system, although there is no definite predilection for specialized tissues. there may be transmural involvement with fibrous replacement of portions of the myocardium and aneurysm formation. the fibrous transition of granulomas may result in early diastolic dysfunction, but as the disease progresses and with extensive involvement, systolic impairment occurs. whereas cardiac involvement in sarcoidosis commonly occurs as part of the systemic affliction, isolated cardiac sarcoidosis in the absence of systemic disease has been described. the clinical presentation of cardiac sarcoidosis is variable and may depend on the amount of myocardium replaced with granulomas and the amount and location of scar tissue. rhythm abnormalities and conduction disorders predominate, although asymptomatic patients with mildly restrictive filling patterns may elude medical attention. patients with chf may show clinical features of restrictive cardiomyopathy or dcm. papillary dysfunction with mitral regurgitation and pericardial involvement with effusiveconstrictive disease have also been described. radionuclide myocardial imaging with thallium and gallium is helpful in identifying patients with myocardial involvement. magnetic resonance imaging has also been proposed as a diagnostic modality. , histologic diagnosis with endomyocardial biopsy is corroborative, but a negative biopsy does not rule out the possibility, owing to sampling error. the combination of bilateral hilar adenopathy and myocardial disease suggests cardiac sarcoidosis in a young person. corticosteroids are indicated when myocardial involvement, conduction abnormalities, and ventricular arrhythmias are present. patients with scintigraphic uptake of gallium may be more responsive to corticosteroid therapy. perma-nent pacemakers may be needed to treat the conduction abnormalities. implantable defibrillators may be utilized in the prevention of sudden death. heart failure is treated in the conventional manner, whereas heart transplantation is reserved for intractable heart failure. heart-lung transplants are performed infrequently for patients with pulmonary involvement, but there is a significant risk of recurrent disease. peripartum myocarditis/cardiomyopathy virchow and porak first reported the association of pregnancy with dcm in in an autopsy series. peripartum myocarditis/cardiomyopathy occurs in one of every , to , pregnancies. the incidence is higher in africa, and it increases with older age, multiparity, multiple gestations, and prior history of peripartum myocarditis/cardiomyopathy. peripartum cardiomyopathy is currently believed to be a myocarditis of unknown etiology, perhaps an infectious, autoimmune, or idiopathic process. the viral myocarditis hypothesis stems from the observations that pregnant mice are more susceptible to cardiotropic viruses, with increased viral replication, and with the increased hemodynamic burden of pregnancy, the myocardial lesions worsen. recently, it has been postulated that after delivery, the rapid degeneration of the uterus results in fragmentation of tropocollagen by enzymatic degradation. this releases actin, myosin, and their metabolites, and antibodies are formed that then cross-react with the myocardium. an association between tocolytic therapy and cardiomyopathy has also been reported. while the diagnosis of peripartum myocarditis/cardiomyopathy is traditionally made during the last trimester or during the first months postpartum, earlier occurrence has been reported. the presentation is usually of decompensated ventricular systolic failure in the absence of any identifiable cardiac pathology. the lvef normalizes in approximately % of women and is more likely to normalize if the initial lvef is > %. therapy is tailored to the decompensated state with diuretics, digoxin, and vasodilators (aceis are contraindicated in pregnancy). inotropic therapy may be needed for supporting those in cardiogenic shock, along with the use of mechanical circulatory-assist devices. although there are anecdotal reports of benefit of immunosuppressive therapy, the routine use of these agents cannot be recommended; in fact, the only indication would be biopsy-proven fulminant myocarditis. cardiac transplantation is an alternative therapeutic option and may be offered to those with intractable heart failure, but it is preferred that transplantation be delayed. the early outcome after transplantation in these patients is often unfavorable, with increased allograft rejection, and the natural history of peripartum myocarditis/cardiomyopathy suggests that more than half of the patients have spontaneous resolution. there are perhaps two different subgroups. one presents with a rapidly progressive, fulminant course with often nearcomplete resolution of myocardial dysfunction within days and excellent long-term prognosis. the other group has late, insidious onset and presents with progressively worsening heart failure with poor prognosis. it is often difficult to differentiate this from the common variety of dcm. myocarditis is a focal or diffuse inflammation of the myocardium, which has multiple infectious and noninfectious etiologies. autoimmunity, triggered most often by viral infections, is a prominent pathophysiologic mechanism of myocarditis. overt and clinically inapparent myocarditis is an important cause of dilated cardiomyopathy. virus-induced lymphocytic myocarditis progresses through three stages: active viral infection, autoimmunity, and dilated cardiomyopathy. myocarditis is no longer a diagnosis of exclusion; histology, histochemistry, dna and rna detection, tissue and circulating antibody detection, and a variety of imaging techniques can be used together or, in some cases, independently to make the diagnosis and to establish the disease stage. treatment of myocarditis must be tailored to the phase of disease. many new therapies based on knowledge of the molecular pathophysiology of 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role of sampling error insensitivity of right ventricular endomyocardial biopsy in the diagnosis of myocarditis myocardial infarction in kawasaki disease: clinical analyses in cases ventriculographic findings in the convalescent stage in eleven cases with acute myocarditis localized left ventricular aneurysms with normal global function caused by myocarditis diagnosing and treating active myocarditis viral myocarditis. a review similar prevalence of enteroviral genome within the myocardium from patients with idiopathic dilated cardiomyopathy and controls by the polymerase chain reaction enteroviral myocarditis and dilated cardiomyopathy: a review of clinical and experimental studies meta-analysis of the association of enteroviruses with human heart disease absence of viral nucleic acids in early and late dilated cardiomyopathy evidence of viral infection in the myocardium of american and japanese patients with idiopathic dilated cardiomyopathy no evidence for persistent enterovirus 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myocarditis and other myopericardial diseases and mitral valve prolapse ace inhibitors in non-ischaemic heart failure: results from the mega trials beneficial effects of captopril in acute coxsackievirus b murine myocarditis adrenergic beta-blocking agents in congestive heart failure due to idiopathic dilated cardiomyopathy betablocker treatment of dilated cardiomyopathy. beneficial effect of carteolol in mice myocarditis of unknown etiology (fieldler's?) treated with acth; report of a case in a -year-old boy with improvement acute aseptic myocarditis: corticosteroid therapy dilated cardiomyopathy caused by acute myocarditis in pediatric patients: evolution of myocardial damage in a group of potential heart transplant candidates immunosuppressive therapy in experimental and clinical myocarditis lack of objective improvement in ventricular systolic function in patients with myocarditis treated with azathioprine and prednisone treatment of chronic myocarditis with corticosteroids 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human cytomegalovirus immediate-early gene transcripts within cardiac myocytes of patients with hiv-associated cardiomyopathy cellular and mitochondrial toxicity of zidovudine (azt), didanosine (ddi) and zalcitabine (ddc) on cultured human muscle cells smallpox vaccination and myopericarditis: a clinical review incidence and follow-up of inflammatory cardiac complications after smallpox vaccination eosinophiliclymphocytic myocarditis after smallpox vaccination congestive heart failure in the course of typhoid fever myopericarditis as an initial presentation of meningococcemia. unusual manifestation of infection with serotype w myocarditis in legionnaires' disease myocarditis with microabscess formation caused by listeria monocytogenes associated with myocardial infarct typhoid fever with myocarditis serologic evidence for chlamydia trachomatis myocarditis carditis associated with mycoplasma pneumoniae infection streptococcal tonsillitis and acute nonrheumatic myopericarditis atrioventricular block complicating acute streptococcal tonsillitis acute nonrheumatic perimyocarditis complicating streptococcal tonsillitis myocarditis associated with acute nasopharyngitis and acute tonsillitis principles and practice of infectious diseases diphtheric myocarditis cardiac complications of diphtheria diphtheritic myocarditis. a histochemical and electron microscopic study failure of corticosteroid therapy to prevent diphtheritic myocarditis or neuritis the protective effect of carnitine in human diphtheric myocarditis clinical pathologic correlations of lyme disease early lyme disease cardiac involvement in lyme disease: manifestations and management lyme carditis. electrophysiologic and histopathologic study complete heart block as the sole presentation of lyme disease isolation of borrelia burgdorferi from the myocardium of a patient with longstanding cardiomyopathy lyme borreliosis as a cause of myocarditis and heart muscle disease in: mandell gl, ed. principles and practice of infectious diseases cardiac fungal infections: review of autopsy findings in patients candida myocarditis without valvulitis cardiac candidiasis in cancer patients the potentially lethal problem of cardiac candidosis aspergillus pancarditis following bone marrow transplantation for chronic myelogenous leukemia aspergillus myocarditis primary actinomycosis of the pericardium pericardial actinomycosis with cardiac tamponade from a contiguous thoracic lesion cardiac actinomycosis unusual manifestations in a cardiac transplantation patient overwhelming myocarditis due to cryptococcus neoformans in an aids patient cryptococcal myocarditis in acquired immune deficiency syndrome chagas' heart disease in the united states myocardial involvement in rocky mountain spotted fever cardiac manifestations of rocky mountain spotted fever cardiopulmonary dynamics in a severe case of rocky mountain spotted fever myocardial function in rocky mountain spotted fever: echocardiographic assessment m-mode echocardiographic abnormalities in rocky mountain spotted fever the indeterminate form of human chronic chagas' disease: a clinical epidemiological review trypanosoma species (american trypanosomiasis chagas" disease): biology of trypansomes pathophysiological insights into the cardiomyopathy of chagas' disease eosinophil activation in acute and chronic chagasic myocardial lesions and deposition of toxic eosinophil granule proteins on heart myofibers ultrastructural characteristics of different stages of human chagasic myocarditis right ventricular endomyocardial biopsy in chronic chagas' disease electrophysiologic findings in long-term asymptomatic chagasic individuals cardiac morbidity and mortality due to chagas' disease: prospective electrocardiographic study of a brazilian community m-mode and two-dimensional echocardiography in chronic chagas' heart disease. a clinical and pathologic study echocardiographic features of impaired left ventricular diastolic function in chagas's heart disease left ventricular cineangiography in chagas' disease: detection of early myocardial damage the usefulness of the resting electrocardiogram for characterizing acute chagas' heart disease in the rat chagas' heart disease and myocardial infarct. incidence and report of four necropsy cases association of elevated anti-sarcolemma, anti-idiotype antibody levels with the clinical and pathologic expression of chronic chagas myocarditis major trypanosoma cruzi antigenic determinant in chagas' heart disease shares homology with the systemic lupus erythematosus ribosomal p protein epitope increased capacity of the coronary arteries in chronic chagas' heart disease: further support for the neurogenic pathogenesis concept functional evaluation of sympathetic and parasympathetic system in chagas' disease using dynamic exercise plasma norepinephrine in chagas' cardioneuromyopathy: a marker of progressive dysautonomia microvascular changes as a cause of chronic cardiomyopathy in chagas' disease enhanced platelet adherence and aggregation in chagas' disease: a potential pathogenic mechanism for cardiomyopathy treatment of chronic chagas' disease with itraconazole and allopurinol randomised trial of efficacy of benznidazole in treatment of early trypanosoma cruzi infection reactivation of chagas' disease during therapy of acute lymphocytic leukemia kidney transplantation and chagas' disease. a two-year follow-up of a patient with parasitemia chagas disease and kidney transplantation chronic intracellular protozoan infections and kidney transplantation heart transplantation in patients with chagas' disease cardiomyopathy pancarditis affecting the conducting system and all valves in human african trypanosomiasis agents of african american trypanosomiasis (sleeping sickness) isolated toxoplasma myocarditis in acquired immune deficiency syndrome lethal cardiac and cerebral toxoplasmosis in a patient with acute myeloid leukemia after successful allogeneic bone marrow transplantation endomyocardial biopsy in the diagnosis of toxoplasmic myocarditis primary and reactivated toxoplasma infection in patients with cardiac transplants. clinical spectrum and problems in diagnosis in a defined population toxoplasmosis in cardiac transplantation cardiac echinococcosis: clinical picture and complications two-dimensional echocardiographic features of echinococcosis of the heart and great blood vessels. clinical and surgical implications cardiac hydatid cyst with clinical features resembling subaortic stenosis cardiac involvement in trichinosis trichinosis with neurologic and cardiac involvement. review of the literature and report of three cases myocarditis caused by trichinella spiralis cardiac dysfunction in trichinosis kawasaki syndrome: clinical features. pathophysiology, etiology and therapy diagnosis and management of kawasaki disease kawasaki syndrome kawasaki syndrome-cardiovascular manifestations myocarditis in kawasaki syndrome. a minor villain? cardiac biopsy of kawasaki disease pathology of the heart in kawasaki disease gallium- myocardial imaging for the detection of myocarditis in the acute phase of kawasaki disease (mucocutaneous lymph node syndrome): the usefulness of single photon emission computed tomography myocarditis in kawasaki disease high-dose intravenous gammaglobulin for kawasaki disease giant cell myocarditis: evidence for the macrophage origin of the giant cells giant cell myocarditis: an autoimmune disease? sarcoidosis of the heart. a clinicopathologic study of necropsy patients (group ) and review of previously described necropsy patients (group ) isolated myocarditis versus myocardial sarcoidosis. a contribution to the discussion regarding points of resemblance between these and a report of three illustrative cases giant cell myocarditis: an entity distinct from sarcoidosis characterized by multiphasic myocyte destruction by cytotoxic t cells and histiocytic giant cells a case of 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of recurrent giant cell myocarditis in a transplanted heart to intensive immunosuppression giant cell myocarditis: first report of disease recurrence in the transplanted heart giant cell myocarditis-like appearance after transplantation: an atypical manifestation of rejection? endocarditis parietalis fibroplastica mit bluteosinophilie, ein eigenartiges krankheitsbild the pathogenesis of endomyocardial fibrosis: the role of the eosinophil eosinophilia and heart disease acute necrotising eosinophilic myocarditis relation between eosinophilia and endomyocardial disease electron-microscopic and immunohistochemical studies on endomyocardial biopsies from a patient with eosinophilic endomyocardial disease toxic effects of human eosinophil products on isolated rat heart cells in vitro evolution to dilated cardiomyopathy from acute eosinophilic pancarditis in churg-strauss syndrome eosinophilic myocarditis associated with high-dose interleukin- therapy eosinophilic myocarditis manifesting as myocardial infarction: early diagnosis and successful treatmento right heart impairment in sarcoidosis: haemodynamic and echocardiographic study cardiac sarcoid: a clinicopathologic study of unselected patients with systemic sarcoidosis ventricular tachycardia and ventricular aneurysm due to unrecognized sarcoidosis predominant myocardial sarcoidosis comparison of clinical features and prognosis of cardiac sarcoidosis and idiopathic dilated cardiomyopathy myocardial sarcoidosis myocardial involvement in patients with sarcoidosis. an analysis of patients diagnosis of cardiac sarcoidosis aided by mri magnetic resonance imaging as an aid to the diagnosis and treatment evaluation of suspected myocardial sarcoidosis in a fighter pilot sarcoidosis of the heart diagnostic and prognostic value of myocardial scintigraphy with thallium- and gallium- in cardiac sarcoidosis cardiac sarcoidosis with sudden death: treatment with the automatic implantable cardioverter defibrillator sarcoidosis and transplantation peripartum cardiomyopathy: a comprehensive review viral myocarditis during pregnancy: encephalomyocarditis virus infection in mice viral myocarditis and cardiomyopathy peripartum heart failure associated with prolonged tocolytic therapy pregnancy-associated cardiomyopathy: clinical characteristics and a comparison between early and late presentation peripartum myocarditis and cardiomyopathy peripartum cardiomyopathy: clinical, hemodynamic, histologic and prognostic characteristics key: cord- -k h cc authors: Çatlı, tolgahan; atilla, huntürk; miller, eva kathryn title: acute viral rhinitis date: - - journal: all around the nose doi: . / - - - - _ sha: doc_id: cord_uid: k h cc rhinitis refers to any kind of inflammatory condition of the nasal mucosal linings. generally, acute rhinitis is associated with environmental allergies or respiratory viral infections. viral microbes with numerous types and subtypes can infect the respiratory epithelium of the nasal cavity in a repetitive fashion throughout the year, or during a specific period of time such as winter or fall. among all forms of inflammatory diseases of the nasal mucosa, acute viral rhinitis (avr) has unique epidemiological, clinical, and therapeutic characteristics. as the most prevalent type of rhinitis, avr is also the most common form of any infectious disease of the human body. although it is almost always self-limiting, in rare circumstances disease might progress and the clinical scenario could become complicated. common complaints and physical findings related to avr are similar to those seen with other types of rhinitis such as allergic, hormonal, senile, or drug induced. the clinician must interpret these symptoms and findings in the context of other parameters such as “duration, environmental factors, and patient characteristics” to establish an accurate diagnose and appropriate therapeutic management. in this chapter, we aim to discuss the epidemiology, pathogenesis, clinical findings, differential diagnosis, and therapeutic management of avr in light of the recent literature knowledge. it is our hope that this chapter may aid medical professionals who encounter avr in daily practice. avr is the most common form of human respiratory infectious diseases, and the predominant inciting pathogens are composed of numerous types and sub types in nature. acute infection of the upper airways is usually viral, rather than bacterial. human rhinoviruses (hrv) are responsible for up to half of the cases of upper respiratory infections (uri) or "colds" [ ] . other viruses such as coronaviruses, adenoviruses, respiratory syncytial virus (rsv), influenza viruses, and parainfluenza viruses account for a relatively minor proportion of viral colds. these viral agents infect the nasal respiratory epithelium and also other components of the upper/lower respiratory tract after inoculation through respiratory droplets of an infected person. although oral inoculation might be an alternative source of viral transfer, the risk is relatively low. risk of experiencing avr in a single year is much more probable for children than adults. while children experience - colds a year, adults usually experience - colds per year [ ] . rhinoviruses can be cultured by a nasopharygeal swab for up to weeks after inoculation [ ] , and even longer in the immune compromised host. usually, the inflammation is controlled by the host immune response and almost always disease is self-limited. however in some circumstances, the natural course of the disease may be altered and complications may arise such as bacterial sinusitis or otitis, asthma exacerbation, pneumonia, chronic post-viral cough, and cavernous venous thrombosis. nasal respiratory epithelium has four types of cells: basal, goblet, ciliated, and un-ciliated columnar cells, which are separated from the lamina propria by a basal membrane [ ] . although the nasal mucosa is in persistent and repetitive contact with the outer world and suffers various sources of injuries such as infectious and physical, the mucosa is armed with various mechanisms for epithelial repair and remodeling. the usual suspect triggers of avr are the most common respiratory viruses including human rhinoviruses, rsv, influenza viruses, parainfluenza viruses, and adenoviruses [ ] . these viruses infect the nasal epithelial cells, damage tight junctions, disrupt membranes, and induce cell death. the epithelial infection process begins with viral entry into the nasal cell via receptors. the identified receptors for rhinoviruses, the most common cause of avr, include intercellular adhesion molecule- (icam- ) and toll-like receptor (tlr ) [ , ] . viral endocytosis is the next step after receptor binding and is followed by expression and duplication of the viral genetic material within a couple of hours after the initial viral and human cell interaction. rhinovirus-infected cells upregulate some integral molecules such as interleukin- b (il- b) and nuclear factor (nf)-kb and these molecules upregulate icam- , enhancing infectivity and promoting inflammatory infiltration [ ] . after invasion, the virus begins to dominate host cell metabolism in order to replicate itself, and this usually results in host cell destruction. cell death after invasion and/or apoptosis may limit viral replication by augmenting antigen presentation and host immune response. viral inflammation "triggered by the infected cells and augmented by the host immune system" is composed of an activated cascade of numerous biological pathways and results in elimination of the offending agent. this inflammation is also the source of various clinical symptoms experienced during the course of an avr, due to injury of the nasal mucosal linings. during the inflammation of the nasal mucosa, particularly in the first days of clinical rhinitis symptoms, cellular infiltration of the nasal cavity predominately by the polymorphonuclear leukocytes is the characteristic histopathological finding. these ultrastructural events are usually reflected as the typical symptoms and physical findings of the avr such as "excessive mucus production and rhinorrhea, nasal blockage, sneezing, watery eyes, and some degree of nasal and ocular pruritus" [ ] . this symptom and sign complex is defined as "rhinitis symptom and sign complex (rssc)" by doyle et al. [ ] . although many studies in adults experimentally exposed to viruses (hrv, rsv, etc.) showed that even viral replication and shedding is evident, these symptoms and signs are observed in only % of the volunteers [ ] . if rssc is present, it is typically delayed relative to viral exposure and this time period differs between virus types. this delay is about h for rhinoviruses and about days for rsv [ ] . however, the resolution period is usually similar for all types of viruses and typically takes - days. with these rssc, in most of the cases selfdiagnosis is not unusual. however, if the symptoms are of longer duration and/or with elevated body temperature, chills and/or body aches, a "flu" diagnosis is probable and clinician must be aware of the possible serious morbidities. the underlying mechanism of the associated symptoms and physical findings of avr is basically triggered by the inflammatory cascade of the immune system [ ] . for example, the lowered activation threshold of the nasal epithelial neural network that is affected by the inflammatory molecules triggers the sneeze reflex. in a similar manner, nasopharyngeal sensitization occurs and coughing is triggered to facilitate expectoration. concurrently, nasal epithelial intercellular tight junction remodeling and mucin production allow for the clearance of nasal mucosal surface and also produce a functional barrier from the outer environment. altered nasal vascular resistance, mainly driven by venous pooling, instigates the increased nasal airway resistance and decreased nasal airway patency by mucosal swelling and excessive mucus secretions. in daily practice, patients with rssc almost always present to medical professionals (rhinologists, pulmonologists, family physicians, etc.) with a self-diagnosis, but physicians must acquire a detailed medical history and perform meticulous physical examination for a precise diagnosis and adequate treatment. the first step is taking a detailed history, where the patient is asked about any chief complaints and timing of symptoms, duration, and severity. in a case of avr, chief symptoms to consider include "excessive mucus production and rhinorrhea, nasal blockage, sneezing, watery eyes, and some degree of nasal and ocular pruritus," as well as "chills, muscle aches, etc." in avr, these symptoms have an acute beginning and typically resolve after days of a self-limiting course. in an uncomplicated circumstance, blood or other laboratory testing is not necessary and history and physical findings are adequate to make a diagnosis. on physical examination, the patient's general appearance is very typical with a pale face, watery nose/eyes and reddish nose tip. detailed upper aero-digestive tract examination reveals "edematous, pale, and watery inferior turbinates bilaterally with a blocked nasal passage." physicians should assess these anatomic changes when considering this and other possible rhinitis etiologies. although the main tool for differentiation of important rhinitis etiologies is taking a detailed medical history (including family history); physical examination and laboratory testing are also other important complementary tools. in a patient with early acute rssc, generalized symptoms and occasional fever are not uncommon; and physicians need to detail the history of the patient to differentiate other etiologies that do not present with generalized symptom or fever. physical assessment must include systems other than the nasal cavity such as ocular, pulmonary, or systemic findings that may provide some important clues about the nature of the rhinitis. in a patient with rhinitis, accompanying conjunctivitis may be a component of avr or allergic rhinitis. cervical lymphadenopathy is also another physical finding commonly associated with avr and must be considered during the evaluation. clinicians must prepare an algorithm in the evaluation of a patient with rhinitis, because some presentations of rhinitis could be eliminated after certain basic questions such as duration of rssc, accompanying symptoms, and viral history. in a patient with acute symptoms ( week or less) without a viral exposure history, acute exacerbation of allergic rhinitis is possible and clinicians must detail the medical and family histories to determine if viral pathogenesis is likely or not. another clinical entity seen in children with an acute rssc (especially with unilateral symptoms) is a foreign body in the nasal cavity, particularly when there is malodorous discharge. in cases involving longer duration of symptoms, other chronic pathologies such as seasonal/perennial allergic rhinitis or non-allergic rhinitis should be considered. these rhinitis etiologies are summarized in table . , and also may be categorized by acute or chronic rssc. for a disease such as avr which is usually self-diagnosed, limited to a short course and self-treated in a non-professional setting, it is difficult to encourage every patient to use antiviral drugs or even seek medical care. however, in placebocontrolled studies comparing effective antiviral drugs (interferon-alpha [ifn-alpha], zanamivir, etc.) with placebo, there is consistently earlier resolution of symptoms and signs by about day for both rhinoviruses and influenza [ , ] , in the anti-viral arms. antiviral agents primarily decrease viral shedding, but other steps of viral infection may be targeted [ ] . in usual clinical practice, avr receives very traditional treatment strategies such as "hydration with plenty of fluids, herbal and nutritional supplements (vitamin c, selenium, zinc, etc.), homemade soups, honey, and multiingredient (antihistamine, oral/topical decongestant, antiinflammatory, etc.) over-the-counter (otc) drugs." these regimens are reasonable because the basic underlying mechanisms of rssc are targeted with anti-inflammatory molecules and hydration. bed rest, activity restriction, and cigarette cessation also offer some degree of symptom relief. antibiotics are not necessary unless secondary bacterial infection occurs during the viral infection. in the case of suspected bacterial super infection (acute bacterial rhinosinusitis, bacterial lower respiratory tract infection, etc.), these are usually treated as classic bacterial infections for - days with antibacterial prescriptions. emerging data suggests that at least days of antibiotic therapy for community-acquired pneumonia may be sufficient, monitored with serial physical assessments. vaccination is another optimistic strategy in the prevention of avr by creating an immune memory response to the particular virus subtype [ ] . however, the large number of virus subtypes and yearly changing characteristics may dampen the enthusiasm for broad implementation. application of such a proposed vaccine should be first limited to the "at risk" population such as "young, elderly, and immunocompromised" patients. concerning transmission routes of these viruses, education strategies of the population about viral dissemination and hand-washing seem to be the best logical option in the fight of our kind against these micro-enemies. as one of the most common infectious diseases of human kind, avr is a familiar, though sometimes insidious phenomenon and should come to the forefront of each clinician's mind when assessing rhinitis in the hopes of preventing possible complications. human rhinovirus is the most common pathogen responsible for almost % of the cases. rssc is defined with "excessive mucus production and rhinorrhea, nasal blockage, sneezing, watery eyes, and some degree of nasal and ocular pruritus." there is no standard laboratory or radiological assessment tool for avr, but if the clinical course becomes complicated, complementary studies should be performed. particularly in a susceptible individual, avr might develop complications such as bacterial super infections of the upper or lower airways and thus may further have more serious morbidities and even mortal consequences. in the treatment of an avr, some palliative strategies such as "bed rest, activity restriction, cigarette cessation, oral and/or parenteral hydration with anti-inflammatory drugs" can be the initial approach although some anti-viral molecules (inf-α, zanamivir, etc.) are shown to be effective in avr treatment by shortening the duration of rssc. this is neither a first-line therapy nor is it widespread in practice for the management of a cold. vaccination is theoretically a good option for the prevention of any viral disease; however, the large number of virus subtypes and yearly changing charac- teristics dampen our enthusiasm for their usage. finally, the education strategies of the population about viral dissemination and cold transmission, including hand-washing/hygiene techniques and prevention of spread, seem to be the best and most logical option in the fight of our kind against these micro enemies. the diagnosis and management of sinusitis: a practice parameter update nasal epithelial repair and remodeling in physical injury, infection, and inflammatory diseases viral-induced rhinitis liiness in the home. a study of , illnesses in a group of cleveland families sites of rhinovirus recovery after point-inoculation of the upper airway tissue remodelling in upper airways: where is the link with lower airway remodelling? european position paper on rhinosinusitisand nasal polyps rhinovirus infection induces expression of its own receptor intercellular adhesion molecule (icam- ) via increased nf-kappab-mediated transcription nasal cytokine responses to natural colds in asthmatic children an update on the patho-physiology of rhinovirus upper respiratory tract infections diagnosing rhinitis: allergic vs. nonallergic viral and bacterial rhinitis illness and otological changes during upper respiratory virus infection nasal cytokines as mediators of illness during the common cold changes in human nasal mucosa during experimental coronavirus common colds zanamivir for treatment of symptomatic influenza a and b infection in children five to twelve years of age: a randomized controlled trial viral respiratory infections due to rhinoviruses: current knowledge, new developments effect of rimantadine treatment on clinical manifestations and otologic complications in adults experimentally infected with influenza a (h n ) virus th international conference on antiviral research. respiratory viruses key: cord- -mm en os authors: isaiah, amal; pereira, kevin d.; correa, armando g. title: tracheal infections date: - - journal: infectious diseases in pediatric otolaryngology doi: . / - - - - _ sha: doc_id: cord_uid: mm en os infectious processes of the trachea represent a distinct clinical entity with an evolving landscape owing to advances in airway management and vaccination practices. untreated inflammatory processes of the trachea may present in the form of acute airway obstruction, potentially resulting in significant morbidity and even mortality. therefore it is important to recognize the cardinal features of some of the common tracheal infectious processes to differentiate them from non-infectious pathology, as the latter is associated with a more indolent course. as with most other infectious processes of the airway, pathogens causing tracheal infection can be bacterial, viral or fungal in nature. viral etiology represents the most common cause of laryngotracheal infection in a child. bacterial infections of the trachea are responsible for more significant morbidity, including prolonged hospitalization, need for endotracheal intubation and even an occasional tracheostomy. the current chapter describes the clinical features and microbiology of tracheal infections at large, explores the utility of diagnostic tests, and provides an algorithm for management. a plethora of epidemics , changes in immunization practices and endotracheal intubation have resulted in a better understanding of the pathogenesis of tracheal infections. historic descriptions fi rst appeared for croup; with the name derived from anglo-saxon root kropan referring to a child with a barking cough. this was fi rst described in print as early as [ ] . much deliberation ensued concerning the treatment of this condition, with o'dwyer being the fi rst physician credited with treatment of acute croup either by insertion of a modifi ed endotracheal tube [ ] or a tracheostomy [ ] in separate instances. the fi rst reported case of tracheitis was published in by pierre blaud [ ] . an increase in incidence was observed during each infl uenza a virus pandemic-h n during the great spanish fl u ( ), asian fl u caused by h n ( ), hong kong fl u resulting from h n ( ), and more recently the pandemic h n of . autopsies performed during the pandemic showed tracheal denudation, maceration, de-epithelialization and other pathologic changes consistent with tracheitis [ ] . bacterial tracheal infections still maintain a low level of presence in infants and children presenting with symptoms of airway obstruction, requiring icu admission and potentially endotracheal intubation. different diagnostic terms have been used for conditions that affect the larynx and trachea. although it is useful to distinguish between supraglottic and subglottic laryngitis, this distinction is often diffi cult when the child is fi rst seen. laryngotracheitis or croup syndrome is a useful preliminary descriptive diagnosis until more defi nitive information is available [ ] . " croup syndrome " also has been used to emphasize the variety of possible causes and location of laryngotracheal disease. in this chapter, "croup" is used to refer to subglottic laryngitis or laryngotracheitis, presumably viral. " epiglottitis " is an imprecise term often used in place of the better " supraglottitis " as the epiglottis may be minimally involved in some cases in which most of the swelling is in the aryepiglottic folds. preferred terms for tracheal infections are (with the usual terms in parentheses): croup, supraglottitis (epiglottitis), and suppurative tracheitis, laryngotracheitis, laryngotracheobronchitis, or laryngotracheobronchopneumonitis (bacterial tracheitis), depending on the extent of the bacterial superinfection [ ] . tracheal infections have a signifi cantly lower incidence compared to infections of the upper respiratory tract, with - % of all children requiring outpatient evaluation for viral croup within the fi rst years of life. croup also requires hospital admission in about . - . % of all children evaluated for the same in emergency settings [ , ] . viral croup has the highest incidence in the second year of life and is virtually non-existent in the fi rst months. the incidence is slightly higher in male children (odds ratio = . ), and is highest in late fall and early winter [ ] . a time-series analysis performed from a large number of children admitted with a principal diagnosis of croup in ontario suggested a strong component of seasonality, with a biennial mid-autumn peak and annual summer trough [ ] . of interest, the overall number of hospitalizations has continued to decrease in the last years, given the improvement in diagnosis and treatment. marx et al. [ ] from the centers for disease control (cdc) studied the overall burden of croup and showed that the mean annual number of croup hospitalizations is about , (range, , - , /year) in the u.s. ninety-one percent of hospitalizations occur among children < years of age. the authors also reported that minor peaks in croup hospitalizations occurred each year in february, and major peaks occurred in october of odd-numbered years, which coincides with peak parainfl uenza type activity . supraglottitis , in contrast, has no seasonal peak. this disease, almost always caused by haemophilus infl uenzae type b and accompanied by bacteremia, has been virtually eradicated by widespread immunization during infancy. while the peak age frequency for croup is - years, supraglottitis occurs in older children, with a peak between and years. suppurative tracheobronchitis also tends to be a disease of preschool and school-age children [ ] . the reported incidence for bacterial tracheitis in the literature is about . / , [ ] . this estimate was based on the combined experience of four pediatric intensive care units. the incidence of tracheal infections caused by other pathogens such as fungal or mycobacterial origin is exceedingly low. over the last two decades, the availability of nebulized epinephrine as well as injectable corticosteroids have changed the landscape of serious, life-threatening tracheal infections, with the re-emergence of bacterial tracheitis. currently, bacterial tracheitis has three times the risk of respiratory failure associated with it than epiglottitis and viral croup combined [ ] . acute laryngotracheitis, considered to be the most common cause for croup, is almost exclusively caused by viral organisms. both bacteria and viruses may be responsible for infections with collateral components, such as laryngotracheobronchitis, and the more general laryngotracheobronchopneumonitis. in , the fi rst evidence for association between croup and two newly isolated myxoviruses, parainfl uenza virus types and , resulted in separation of two categories of cases-mild, requiring only outpatient follow up, and severe, requiring hospitalization [ ] . parainfl uenza is a rna paramyxovirus that actively replicates in respiratory epithelial cells and is comprised of four major serotypes. parainfl uenza type , more commonly associated with bronchiolitis or bronchopneumonia, can also produce severe croup in an endemic pattern, while type is rarely seen. parainfl uenza and , account for > % of all causes of croup. a large series studied instances of lower respiratory tract infections (lris) wherein approximately % of all isolates were identifi ed as parainfl uenza viruses. of these, parainfl uenza type accounted for about %. conversely, the propensity of the various organisms to produce symptoms of croup reached % for both parainfl uenza and . for parainfl uenza type , the number dropped to about %, whereas all the other microorganisms accounted for about - %. thus, parainfl uenza viruses were the most common cause for all age groups; whereas respiratory syncytial virus (rsv) caused croup in infants and the infl uenza viruses and m. pneumoniae were signifi cant causes of croup only in children older than years of age. summertime croup may be due to enteroviruses, adenovirus, or parainfl uenza type . among other important viral pathogens causing tracheal infections, rsv was studied in isolates from sentinel practices in england and wales from to , during which an increase in mortality, by as much as - %, was observed in comparison with parainfl uenza and infl uenza viruses [ ] . prematurity is associated with an increased risk for mortality, with factors such as a decrease in gestational age, increased perinatal oxygen requirements and discharge within months of the rsv season increasing the likelihood for hospitalization [ ] . among the rare viral causes, measles, by virtue of immunosuppression, leads to a bacterial superinfection that results in a condition termed measles-associated bacterial tracheitis (mabt) , which carries an increased risk for need of artifi cial airway and intensive care admission [ ] . bacterial tracheitis is much less common when compared to that of viral origin. previous reports have shown that the most consistent organism is s . aureus , followed by s . pneumoniae and m. catarrhalis [ ] . due to the universal immunization against h. infl uenzae type b , the incidence has dropped signifi cantly. similarly, immunization against c . diphtheriae has restricted the incidence of diphtheritic tracheitis to unimmunized children only. reports of this are largely limited in modern literature, compared to the beginning of the century when tracheostomy was a routine practice to circumvent acute airway obstruction due to formation of pseudomembranes [ ] . fayon et al. [ ] studied independent risk factors for development of bacterial tracheitis in a large series of children admitted to the picu (n = ), and found that the incidence of bacterial nosocomial tracheitis in that population was about . %. the pathogens isolated in this series were in agreement with other studies of bacterial tracheitis, comprising staphylococcus aureus and gram-negative bacteria, and sometimes, mixed fl ora. in this population, tracheitis was attributed to young age, with smallsized airways in which thick secretions and mucosal infl ammation being blamed for impairment of air fl ow and increased stasis. head trauma, neuromuscular blockade and mechanical ventilation were independent variables that increased the risk of infection, but the last two risk factors may be physiologically collinear, given that most patients who were administered neuromuscular blockade were intubated, and vice versa. given the evolution of design features of modern day endotracheal tubes as well as enhanced monitoring of cuff pressures, reports of laryngotracheitis induced by indwelling endotracheal tubes have largely been limited to historic data [ ] . modern endotracheal tubes use materials that intrinsically inhibit or are coated with substances such as micronized silver to reduce bacterial growth by providing less scaffolding for colonization [ , ] . infectious agents such as mycobacterium tuberculosis and fungi have been previously reported to have caused isolated instances of tracheal infection with a picture of long-term respiratory failure requiring a tracheostomy during the course of treatment that may be prolonged [ ] . chronic aspiration as well as gastroesophageal refl ux (gerd) may accelerate laryngotracheal injury facilitating the development of tracheitis in those children. despite the narrow spectrum of pathogens isolated from the plethora of tracheal infections described, these can have varied presentations, and typically differ in the outpatient vis-à-vis inpatient setting. acute viral croup manifests in the form of a viral prodrome characterized by clear rhinorrhea, lowgrade fever, sore throat (in older children) and cough [ ] . this usually lasts about - h, and typically progresses to hoarseness and the pathognomonic croupy cough that has a bark-like character. rarely, febrile convulsions can occur. nighttime symptoms are usually worse which frequently prompts the parents to seek care in the emergency room. the typical course of progression goes through stridor that is inspiratory in nature but may also be associated with an expiratory component that results from unique features of the larynx in young children [ ] . the infant larynx is narrowest in the subglottic segment, and infl ammation of this area results in a fi xed obstruction that leads to expiratory stridor. children presenting multiple times with acute viral croup may have a masked presentation of subglottic stenosis wherein the already narrowed subglottic larynx is further reduced in diameter due to croup-related infl ammation [ ] . hoarseness results from edema of the true vocal cords, often reducing their mobility. wheezing is infrequently present. with increased severity, suprasternal and intercostal retractions may be present, and tachycardia and tachypnea are relatively common. it is important to note that reduction in the intensity of stridor in a sick-appearing child may be sign of impending respiratory failure as airfl ow may be reduced to the point where stridor may not be present. when an infectious cause is not present in croup, the clinical course is abbreviated, with the noticeable absence of the viral prodrome. this condition is frequently referred as " spasmodic croup ", although episodic croup is a more appropriate term as it is typically triggered by an allergic etiology and often recurs [ ] . pediatric angioedema shares features with croup, but is often associated with facial or neck swelling that is acute in onset [ ] . rarely, an undetected foreign body may masquerade as acute croup. although a viral prodrome may be absent, an unsuspecting physician may be drawn into an acute airway emergency due to commonality of symptoms [ ] . the severity of viral croup may be assessed using one of the many scoring systems available. the most well-known of these, the westley croup score [ ] , utilizes key clinical signs including chest retractions, stridor, cyanosis, level of consciousness and air entry to obtain a composite score that is predictive of the need for intubation. as croup is primarily a clinical diagnosis, the utility of the westley score, as with other stratifi cation systems, such as the alberta clinical practice guideline working group [ ] , may be limited to use in a research scenario. for example, using the westley score , johnson et al. [ ] showed that ≥ % of children present with symptoms of generally mild croup, and less than % were diagnosed with severe croup. peltola et al. [ ] studied the clinical courses of croup caused by parainfl uenza and infl uenza viruses to highlight the differences in morbidity caused by the different viral strains in hospitalized children. in general, there were no signifi cant differences in the patterns of clinical features due to infections with the three parainfl uenza subtypes, except that parainfl uenza was associated with wheezing. however, children with croup due to microbiologically-confi rmed infl uenza virus tended to be hospitalized for longer ( days vs . days for parainfl uenza). in addition, the rates of readmission were higher for infl uenza due to the relapsing course of respiratory distress during the few days following discharge. the requirements for corticosteroids as well as supplemental oxygen also tended to be higher for those caused by infl uenza virus, emphasizing its enhanced virulence. notwithstanding the generally predictable course of viral croup, it is important to differentiate it from other acute disorders of the pediatric airway. rapidity in progression to high fever, odynophagia, anxiety and relative aphonia should always alert the practitioner to supraglottitis (epiglottitis), which is a rare occurrence following introduction of universal immunization against hemophilus infl uenzae type b . posturing in the upright position and extension of the neck with drooling in an anxious-appearing child mandates the need to secure the airway in a controlled setting such as the operating room. care should be taken to not agitate the child for that may precipitate respiratory collapse. if there is history to suggest incomplete immunization, laryngeal diphtheria should be considered. this condition tends to have a slower progression, and has historically been associated with the presence of exudative membranes in the oropharynx. bacterial tracheitis typically is a secondary infection following a primary viral respiratory infection due to a cascade of events resulting from tracheal mucosal injury, impaired phagocytic function and cytopathic effects of the viral infection. this condition usually is recognized after reasonable efforts to treat viral croup have failed. children with bacterial tracheitis are acutely ill, with symptoms to suggest dehydration and organ failure in the presence of other host factors such as immunodefi ciency. often seen in an inpatient setting in children admitted to the icu with respiratory failure, bacterial tracheitis can have a variable presentation in the absence of pathognomonic clinical signs [ ] . the cardinal initial signs of bacterial tracheitis include cough, stridor and a rapidly changing course of illness that progresses to respiratory failure quickly. children affected are usually older (> years). other symptoms on admission may include choking episodes, dyspnea, dysphagia, neck pain, dysphonia and agitation. in one study by bernstein et al. [ ] , children younger than years of age were twice as likely to be intubated, compared to older children. the same study compared changing fi gures for mortality-prior to the s, the mortality rate for bacterial tracheitis approached %, but with advances in mechanical ventilation and airway management, that fi gure has dropped to - % in more recent series. from the majority of studies, it is clear that the diagnosis of acute viral croup is chiefl y based on clinical examination and does not necessitate laboratory testing. that said, if there is suspicion for a concurrent lower respiratory tract infection, white blood cell count with differential, as well as routine postero-anterior/lateral chest and neck radiographs may be indicated. in viral croup, the white count is often at the high end of normal, and may be higher in approximately % of hospitalized children [ ] . administration of corticosteroids may cause leukocyte demargination, which can lead to spuriously elevated counts during the course of treatment. plain fi lm radiography often is utilized to evaluate laryngotracheal edema in croup, but has inconsistent results. the typical picture is that of narrowing of laryngeal air column in the subglottic segment, approximately for ~ - mm below the level of the vocal cords, resulting from mucosal edema [ ] . this has been historically referred to as the steeple sign ( fig. . ), but is observed only in ~ % of instances [ ] . this, coupled with reduced sensitivity for differentiating between viral croup, epiglottitis and bacterial tracheitis undermines the usefulness of routine radiographs for diagnosis. however, some investigators such as mills et al. [ ] , have reported sensitivity and specifi city of > % respectively. the best practice in these circumstances is to consider radiographs in those children in whom the clinical presentation is atypical and whose respiratory status is stable enough to undergo positioning prior to obtaining the fi lms [ ] . alveolar gas exchange is usually not affected by viral croup, unless there is concurrent presence of laryngotracheobronchitis, asthma or pulmonary insuffi ciency [ ] . thus, pulse oximetry and respiratory rate have been shown to have poor correlation with clinical status or hypoxia due to artifacts [ ] . evidently, the uncompromised standard is clinical observation with pulse oximetry as a useful adjunct in instances wherein the lower airway is also affected. in cases where operative control of the airway is required, telescopic tracheobronchoscopy , aided by the ventilating bronchoscope provides the gold standard for assessment of the airway in severe croup, or when alternate pathology, such as supraglottitis, is suspected. in the ambulatory setting, children who present with recurrent croup should be examined for concurrent abnormalities. chun et al. [ ] evaluated children who were previously diagnosed with recurrent episodes of croup. a third of these children were found to have synchronous lesions such as subglottic stenosis, edema and cysts. in the same study, abnormal rigid endoscopic fi ndings were more likely to be seen in children under the age of years, highlighting the need for a higher index of suspicion and lower threshold for performing airway endoscopy in this age group. microbiologic investigations to determine etiology are increasingly being performed due to the availability of molecular and standard virologic methods. these tests are usually not recommended for diagnosis in mild cases of croup, but may be warranted in children hospitalized and/or requiring mechanical ventilation. real-time polymerase chain reaction (rt-pcr) and viral cultures are also indicated with atypical courses of the infection, as described by reports of novel pathogenic strains for viral croup, e.g. coronavirus nl detected in samples isolated from europe [ ] . an improved panel based on an rt-pcr assay has been developed for infl uenza a and b viruses, rsv and parainfl uenza , , and . according to one study, the application of pcr increases the sensitivity of respiratory viral diagnosis, with results being made available within h, thus increasing clinical relevance [ ] . with claimed sensitivity of ~ % and specifi city approaching %, several authors have increasingly validated their cost-effectiveness [ ] . as mentioned earlier, the routine use of these tests in mild croup is unsubstantiated. in children undergoing rigid endoscopy or endotracheal intubation for bacterial tracheitis and other serious airway infections, routine contact bacterial cultures and broncho-alveolar lavage with cultures may be obtained to facilitate culture-directed therapy. jones et al. [ ] fi rst described laryngoscopically-directed cultures in bacterial tracheitis from copious mucopurulent material obtained from the subglottis, which grew s. aureus in most instances (fig. . ) . plain fi lm radiographs in these instances are consistent with progression of an infl ammatory response (fig. . ) . these results have been replicated from a number of other centers [ , , ] . the treatment of tracheal infections has evolved over the course of the twentieth century, from initial descriptions of primitive endotracheal intubation to tracheostomy performed for acute airway distress secondary to laryngeal diphtheria [ - ] . the fi rst recognized form of treatment was the use of mist (humidifi ed aerosol) produced by hot water, historically reported by keeping children close to a running tub with the door closed, leading to accumulation of mist. discovery of therapeutic benefi ts from the use of corticosteroids and racemic epinephrine have revolutionized the manner in which croup is treated, and advanced in mechanical ventilation as well as development of rigid telescopes have improved treatment of tracheal infections of bacterial origin as well. these therapeutic strategies are summarized below. croup kettles were fi rst introduced in the late nineteenth century to provide aerosolized mist to alleviate the symptoms of viral croup [ ] . later, cool mist was observed to have the same degree of therapeutic benefi t as warm mist, and this avoids the risk of burns. there are at least three postulated fig. . endoscopic photographs of ( a ) early bacterial tracheitis, with increase in exudates seen in the subglottis without overt purulence, ( b ) crusting and purulence seen that progressed to ( c ) erythema, pseudomembranes and overall infl amed-appearing tracheal mucosa arrows point to anatomical changes that can be due to ongoing tracheal infl ammation mechanisms, which include (i) a soothing effect on infl amed mucosa, (ii) reduced viscosity of tracheal secretions and (iii) activation of laryngeal mechanoreceptors leading to reduction of turbulence [ ] . however, humidity may also trigger bronchospasm, thus the duration of therapy should be carefully monitored. recent studies have, however, shown that the benefi ts offered by mist treatment may be overemphasized. three separate studies, that did not include untreated controls, determined that the effi cacy of aerosolized mist may not be proportional to the degree of mist saturation, for e.g. the effects of humidity at three different levels ( %, % and %) remained the same [ ] . in yet another study, the effect of nebulized saline was identical to mist. lastly, a recent cochrane review of data concluded that the benefi t of mist therapy remains unproven [ ] . despite the various recommendations for dosages, routes and drugs for use of corticosteroids, a number of large-scale studies have exemplifi ed their therapeutic effi cacy. their mechanism of action is related to the reduction of vascular permeability, resulting in a reduction of laryngeal and tracheal mucosal infl ammation. russell et al. [ ] in a cochrane collaboration reviewed studies and showed that corticosteroids resulted in a rapid improvement of westley score, fewer return visits and/or readmissions, reduction of stay in the emergency room as well as the overall need for concurrent use of epinephrine. the benefi ts are not readily apparent in children with mild croup as the symptoms begin to resolve in about the same time taken for steroids to show treatment benefi t. following adoption of corticosteroids as a standard fi rst line of therapy in acute viral croup, overall hospitalization and the burden of the disease on healthcare systems worldwide began to fall. this was acknowledged following the guidelines formulated by the canadian pediatric society that encouraged the use of intravenous dexamethasone as initial treatment of croup [ ] . among steroids, dexamethasone is used in a dose of . mg · kg − body weight given either orally or by the intramuscular route. as dexamethasone is a potent steroid with a prolonged half-life, repeat doses are often unnecessary. other investigators have shown that orally administered dexamethasone is as effi cacious as parenteral formulations. the choice of route should hence be determined based on cost and availability. yet another study failed to show differences in therapeutic benefi t between three different doses ( . , . and . mg · kg − ) of dexamethasone, so a single dose ( . mg · kg − ; maximum of mg) may be suffi cient in the outpatient setting [ ] . a double-blind, randomized control trial compared three different treatment strategies that included placebo, nebulized budesonide and oral dexamethasone [ ] . in this study, the overall rates of hospitalization were much less in the group treated with dexamethasone ( %), compared with budesonide ( %) and far less compared with placebo ( %). other studies have also advocated for the use of aerosolized budesonide given the rapidity of its action and effectiveness comparable to that of nebulized epinephrine [ , ] . the primary benefi t offered by the use of aerosolized/nebulized epinephrine is the reduced need for intubation. early studies showed immediate clinical benefi t with use of . % racemic epinephrine, and the more recent studies demonstrated the same amount of benefi t for l-epinephrine at a ratio of : used with ml saline [ ] . initial studies represented a major paradigm shift in management of children with severe croup, obviating the need for endotracheal intubation or tracheostomy [ ] . the therapeutic effects of epinephrine are mediated via α-adrenergic receptors that results in constriction of capillary arterioles and reduced infl ammation. unfortunately, although the effects are almost immediate, they only last approximately h, and hence the child should be watched for a reasonable period of time prior to discharge. this therapy may be suitable even in the outpatient setting if the observation period is adequate, although lack of improvement at about an hour following treatment may convert an outpatient encounter to hospitalization [ ] . when conditions such as tetralogy of fallot, tachycardia or ventricular outlet obstruction co-exist, epinephrine should be used cautiously [ ] . with the peak effect occurring between and min, the child should be carefully monitored for the rebound effect-which usually occurs h after treatment. the recommended dose is thus . % ( . ml in . ml of saline) for children < months of age and . ml for infants and children > months. substituting isotonic with hypertonic saline ( %) may enhance the effect by absorbing water from the submucosa. when respiratory failure is impending (cyanosis, severe retractions with lack of airfl ow, and persistent desaturations), endotracheal intubation is indicated until laryngeal edema resolves. this is usually transient and rarely evolves into a need for long-term mechanical ventilation, in children treated with steroids during the course of intubation, the time to extubation is shortened and the need for reintubation also is reduced [ ] . use of the physiologic leak test, by either vocalization around the cuff, or sustained difference in inspiratory and expiratory tidal volumes serves as a guide for extubation in these children. since its description in , use of helium as a carrier for oxygen (heliox) has benefi cial effects in reducing eddy currents that interact with each other and thereby reduce turbulent fl ow. heliox is routinely used in children with post-extubation stridor to reduce the risk of re-intubation. as an appreciable number of children with severe croup progress to respiratory failure needing an artifi cial airway, using heliox can reduce the work of breathing by easing the delivery of oxygen to the lower airway past the site of obstruction. assessments with croup scores and blood gas analyses reaffi rm the benefi cial role of heliox as a useful adjunct to potentially circumvent the need for endotracheal intubation [ ] . a randomized trial showed benefi t comparable to racemic epinephrine in moderate to severe croup [ ] . as the etiology of croup is not bacterial, there is no role for routine use of antibiotics. in the past, inappropriately prescribed antibiotics have been reported to cause superinfections, prolongation of hospital stay as well as general increase in costs [ ] . however, when the diagnosis of bacterial tracheitis is strongly suspected, the management changes wherein culture-directed antibiotic therapy is the gold standard of treatment. empiric therapy with coverage that includes s. aureus , s. pyogenes , s. pneumoniae and h. infl uenzae is indicated. in an era when both hospital-acquired and communityacquired methicillin-resistant s. aureus (mrsa) are prevalent, the combination of vancomycin and a third generation cephalosporin (such as cefotaxime or ceftriaxone) is a reasonable choice until identifi cation and susceptibility of the causative organism is established. in an intubated patient, tracheal aspirates should be obtained. a very useful adjunct to anti-microbial therapy is the use of frequent pulmonary toilet and debridement of tracheal pseudomembranes under bronchoscopic guidance [ ] . in the presence of other co-morbidities such as immunosuppression, mortality increases. reported complications of bacterial tracheitis include pneumomediastinum, sepsis and multi-organ failure, bronchospasm and impaired gas-exchange due to the burden of pseudomembranes with toxic shock syndrome [ ] . severe croup caused by infl uenza viruses as a part of epidemics may require treatment with neuraminidase inhibitors [ ] . with the increasing number of options to treat croup, an algorithm is useful to stratify the burden of the disease and dictate an appropriate protocol. this is shown in fig. despite the relative commonality of laryngotracheal infections, there are no clearly defi ned guidelines by national organizations for their treatment. the clinical picture can be often frightening to the parents who bring the child to the emergency room. fortunately, the vast majority of children show signs of rapid improvement after initiation of treatment using steroids and racemic epinephrine. only a very small proportion of children require hospitalization and an even smaller proportion require intubation and mechanical ventilation. complications are rare exceedingly rare. in contrast, bacterial tracheitis may have varied presentations and often requires endotracheal intubation and debridement in the operating room. mortality is higher but early institution of culture-directed therapy is known to reduce the severity of disease and the incidence of complications. it is important for the astute clinician to recognize the symptoms of croup and maintain a high index of suspicion for conditions that masquerade as croup, such as a foreign body, supraglottitis (epiglottitis), subglottic stenosis and other anatomic abnormalities. remarks on croup and its treatment fifty cases of croup in private practice treated by intubation of the larynx, with a description of the method and of the dangers incident thereto changing epidemiology of life-threatening upper airway infections: the reemergence of bacterial tracheitis pulmonary pathologic fi ndings of fatal pandemic infl uenza a/h n viral infections moffet's pediatric infectious diseases: a problem-oriented approach croup: an -year study in a pediatric practice lower respiratory tract illness in the fi rst two years of life: epidemiologic patterns and costs in a suburban pediatric practice croup hospitalizations in ontario: a -year timeseries analysis pediatric hospitalizations for croup (laryngotracheobronchitis): biennial increases associated with human parainfl uenza virus epidemics bacterial tracheitis: a multicentre perspective association of type hemadsorption (parainfl uenza ) virus and asian infl uenza a virus with infections croup impact of infl uenza and respiratory syncytial virus on mortality in england and wales from rehospitalization for respiratory syncytial virus among premature infants measles-associated bacterial tracheitis bacterial tracheitis caused by corynebacterium diphtheriae nosocomial pneumonia and tracheitis in a pediatric intensive care unit: a prospective study new therapy for postintubation laryngeal edema and tracheitis in children reduced burden of bacterial airway colonization with a novel silver-coated endotracheal tube in a randomized multiple-center feasibility study biofi lm formation in endotracheal tubes. association between pneumonia and the persistence of pathogens tracheitis in pediatric patients viral croup viral croup subglottic stenosis in infants and children corticosteroid treatment of laryngotracheitis v spasmodic croup in children pediatric angioedema: ten years' experience subglottic foreign bodies in pediatric patients nebulized racemic epinephrine by ippb for the treatment of croup: a doubleblind study antibiotic prescribing for canadian preschool children: evidence of overprescribing for viral respiratory infections clinical courses of croup caused by infl uenza and parainfl uenza viruses bacterial tracheitis: a varied entity is bacterial tracheitis changing? a -month experience in a pediatric intensive care unit pillsbury rd hc. severe hospitalized croup: treatment trends and prognosis viral croup: current diagnosis and treatment the usefulness of lateral neck roentgenograms in laryngotracheobronchitis experience of pulse oximetry in children with croup utility of bronchoscopy for recurrent croup croup is associated with the novel coronavirus nl rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by infl uenza a and infl uenza b viruses, respiratory syncytial virus, and parainfl uenza viruses , , , and a sensitive, specifi c, and costeffective multiplex reverse transcriptase-pcr assay for the detection of seven common respiratory viruses in respiratory samples bacterial tracheitis reexamined: is there a less severe manifestation? otolaryngol head neck surg bacterial tracheitis management of the sick infant controlled delivery of high vs low humidity vs mist therapy for croup in emergency departments: a randomized controlled trial humidifi ed air inhalation for treating croup glucocorticoids for croup outpatient treatment of moderate croup with dexamethasone: intramuscular versus oral dosing a comparison of nebulized budesonide, intramuscular dexamethasone, and placebo for moderately severe croup nebulized budesonide is as effective as nebulized adrenaline in moderately severe croup nebulized budesonide for children with mild-tomoderate croup state of the evidence for standard-of-care treatments for croup: are we where we need to be? ten-year experience with ippb in the treatment of acute laryngotracheobronchitis racemic epinephrine in the treatment of laryngotracheitis: can we identify children for outpatient therapy? placebo-controlled trial of prednisolone in children intubated for croup effi cacy of helium-oxygen mixtures in the management of severe viral and post-intubation croup a randomized comparison of helium-oxygen mixture (heliox) and racemic epinephrine for the treatment of moderate to severe croup bacterial tracheitis-an old disease rediscovered bacterial tracheitis in children prevention and control of infl uenza. recommendations of the advisory committee on immunization practices (acip) clinical practice. croup key: cord- -rz id mt authors: braun, serge title: non-viral vector for muscle-mediated gene therapy date: - - journal: muscle gene therapy doi: . / - - - - _ sha: doc_id: cord_uid: rz id mt non-viral gene delivery to skeletal muscle was one of the first applications of gene therapy that went into the clinic, mainly because skeletal muscle is an easily accessible tissue for local gene transfer and non-viral vectors have a relatively safe and low immunogenic track record. however, plasmid dna, naked or complexed to the various chemistries, turn out to be moderately efficient in humans when injected locally and very inefficient (and very toxic in some cases) when injected systemically. a number of clinical applications have been initiated however, based on transgenes that were adapted to good local impact and/or to a wide physiological outcome (i.e., strong humoral and cellular immune responses following the introduction of dna vaccines). neuromuscular diseases seem more challenging for non-viral vectors. nevertheless, the local production of therapeutic proteins that may act distantly from the injected site and/or the hydrodynamic perfusion of safe plasmids remains a viable basis for the non-viral gene therapy of muscle disorders, cachexia, as well as peripheral neuropathies. skeletal muscle can act as an effective platform for the long-term production (and secretion) of therapeutic proteins with systemic distribution and for the introduction of dna vaccines eliciting strong humoral and cellular immune responses (for review see [ , ] ). conversely, the treatment of hereditary neuromuscular diseases is particularly challenging for non-viral vectors. among issues are as follows: ( ) the size of the muscle tissue, which represents half of the total mass of the organism, ( ) the poor accessibility of profound muscles or peripheral nerves, and ( ) the progressive tissue remodeling along the natural history of some muscle diseases with active processes of necrosis/regeneration and fibrosis/lipidosis. s. braun (*) afm-telethon, evry cedex, france e-mail: sbraun@afm-telethon.fr on the other hand, non-viral vectors do bear interesting advantages over recombinant viruses. non-viral vectors are made of plasmid dna, naked or complexed to a variety of versatile molecules such as cationic lipids or polymers. they are ( ) well characterized, and their structure can be fine-tuned [ ] , and ( ) mostly nonimmunogenic provided, they are not carrying protein motifs. this allows repeated administrations for chronic diseases, ( ) comparatively easy to produce at a large scale [ ] , ( ) less limited by size constraints, leaving the potential to deliver widetype genetic material, as large as kb [ ] (this is far beyond the size of coding sequences such as the dystrophin cdna for duchenne muscular dystrophy), and non-viral vectors ( ) can remain functional for a long period of time in skeletal muscles [ ] . episomal plasmid dna can persist for life in rodents and for many years in larger animals if they are delivered into low turnover tissues, including the brain and spinal cord, heart, or muscle (for review see [ ] ). synthetic vectors have been constructed as substitutes to viral vectors for delivering therapeutic genes and many other drugs in humans [ ] . the principle is based on the self-assembly of supramolecular complexes, often through electrostatic interactions between the positively charged vectors and the dna negatively charged phosphate residues [ ] . in these complexes, dna is condensed and compacted and is less exposed to nuclease degradation. among these, cationic lipid-and polymerbased systems have been the most extensively studied [ ] [ ] [ ] . in early studies, dna was encapsulated in neutral or anionic liposomes without changing the structures of the liposomes [ , ] . the ratio between the cationic charge of the liposome and the negative charge of the dna usually controls the size of complexes [ ] , typically in the range of nm to μm quasi-spherical particles with an ordered (often multilamellar) organization. their positive total charge enables them of efficiently interacting with the negative residues of the cell membranes and internalizing into the cell, which occurs mainly through the endocytosis pathway [ , ]. systemic bio-distribution of non-viral vectors is dependent upon their capability of escaping from blood vessels in the target tissue. vectors must be small enough (less than nm) to cross through vascular endothelial cells and gain access to surrounding tissues [ ] . furthermore, they should also be designed so that they can be ignored in contrast to viral vectors, non-viral gene transfer is not elicited to a large extent by active intake processes. therefore, a sophisticated vector may be needed to facilitate the cellular uptake and appropriate intracellular processing of the transgene. significant developments in artificial complexes combined different functions for improved gene transfer. many cationic liposomes are normally accompanied by a neutral lipid such as dioleoylphosphatidylethanolamine (dope) or cholesterol. dope is frequently useful because it can fuse with other lipids when exposed to a low ph, as in endosomes, which triggers the release of the associated dna into the cytosol [ ] . other popular modifications use ligand binding to peg. various targeting approaches have been investigated, including incorporation of peptides, antibodies, and sugar into the lipid vesicles to facilitate tissue targeting (for review see [ ] ). however, the association of all of these components results in complex structures that require thorough formulation and galenic studies. after cell entry, intracellular barriers may impair successful gene delivery. vectors need to escape from the endosomal or lysosomal membrane to avoid degradation of the plasmid dna [ ] . endosomal release of dna by cationic polyplexbased vectors may be based on the physical disruption of the negatively charged endosomal membrane after direct interaction with the cationic complex [ ] , or a "proton-sponge" phenomenon [ ] resulting in osmotic swelling and endosomal membrane rupture, followed by the release of the polyplexes into the cytoplasm. addition of a fusogenic helper lipid such as dope facilitates the formation of a destabilizing hexagonal phase with the endosome membrane and enhances gene expression by promoting the release of dna from the endosomal compartment ( fig. . and [ ] ). it should be mentioned the majority of cytoplasmic plasmids fail to reach the nucleus due to cytoplasmic nucleases. in contrast to short nucleic acids (such as oligonucleotides) which diffuse freely, the plasmid dna imports to nucleus by an active transport process via the nuclear pore complex and receptor proteins that include importin α, β, and ran [ ] . nuclear localization signals or designed peptides can be linked to the plasmid dna to facilitate nuclear import (for review see [ , ] ). a number of therapeutic concepts have been explored in humans using more or less refined non-viral gene delivery systems in the view of therapies for genetic disorders and of immunologic disorders. as of today, despite a number of very sophisticated chemistries, non-viral vectors were not completely satisfactory in transferring genes to muscle tissues following systemic administration. many complexes show excellent transfection activity in cell culture, but most do not perform well in the presence of serum, and only a few are active in vivo [ ] . they remain at least logs of magnitude less effective than viral vectors. therapeutic doses require high concentrations of complexes. besides the relatively large size of many synthetic vectors (often above nm), the main obstacles in the use of synthetic complexes via systemic delivery are their aggregation, instability, toxicity, and , including negatively charged naked plasmid dna (or polynucleotides) delivered either directly or combined with physical methods (ultrasound, electroporation) or complexed with various chemical entities such as cationic lipids or polymers. (b) uptake pathways involve either fusion with the muscle cell membrane-, receptor-, clathrin-, caveolae-, or pinocytosis-dependent endocytosis. this is followed by endosome formation, escape from endosome, degradation, nuclear import of the plasmid dna/polynucleotide, and transgene expression propensity to be captured by the mononuclear phagocyte system, leading to their rapid clearance by phagocytic cells in the liver, spleen, lungs, and bone marrow. these particles readily aggregate as their concentration increases. toxicity is often linked to the colloidal instability of synthetic vectors resulting from interactions with molecules in biological fluids, leading to large aggregates. these aggregates, which are generally ineffective gene delivery agents, can be absorbed onto the surface of circulating red blood cells, or embolized in microvasculatures, preventing them from reaching the intended target cells. this opsonization process can also activate the complement system, one of the innate immune mechanisms against "foreign" particles within the bloodstream, which in turn activates the phagocytosis and initiates an inflammatory response [ , , ] . skeletal muscles possess poorly permeable, tight endothelial (maybe less in the case of chronically inflamed tissues) layers and a highly regulated microcirculation [ ] . the implication is that one would not expect particulate systems to be distributed easily from the blood circulation to skeletal muscles. thus, the prospects for non-viral particulate vector widespread distribution from the systemic circulation are limited at present. only one systemic delivery attempt was initiated in a neuromuscular disease indication. this was in hereditary inclusion body myopathy in a single patient intravenously perfused with a lipoplex in a compassionate trial. the patient showed signs of increase of sialic acid-related proteins and stabilization in the decline of muscle strength [ ] . the administration of vectors directly to the target tissue avoids most of the obstacles encountered by systemic delivery. however this approach remains hampered by the diffusion limitations and immune cell clearance in the interstitial region of the target organ. indeed, transgene expression following direct intramuscular needle delivery of complexes is often localized in regions that are close to the injection site. this implies that the dispersion of colloidal particles within muscle is a critical issue, and there is a need for basic studies of the effect of formulation on dispersion within solid tissues such as skeletal muscle. nevertheless this poor efficiency remains compatible with applications that require only low levels of the therapeutic proteins, such as genetic vaccines, cancer, or peripheral limb ischemia (table . ). interestingly, retrograde transport seemed to be obtained as some gene expression was found in the peripheral and central nervous system following intramuscular administration [ ] . delivery of therapeutic genes to peripheral neurons upon a peripheral and minimally invasive intramuscular administration of polymeric nanoparticles was shown to be feasible in animal models [ ] . naked dna can be manufactured in a very cost-effective manner and is a very stable material that can be stored at room temperature for long periods of time following lyophilization. it is composed of a bacterial plasmid that contains the cdna of the therapeutic gene under the transcriptional control of various eukaryotic [ ] (continued) regulatory elements and a bacterial origin of replication to allow production in bacteria. a strong promoter may be required for optimal expression in mammalian cells. for this, some promoters derived from viruses such as cytomegalovirus (cmv) or simian virus (sv ) have been used. however, virally derived promoters, such as the cmv promoter, may not be suitable for applications to chronic diseases, as illustrated by the negative impact of inflammatory cytokines (interferon-γ or tumor necrosis factor-α) [ ] . thus, muscle-specific alternatives to the cmv promoter have been proposed, such as the desmin promoter/enhancer, which controls expression of the cytoskeletal protein desmin [ ] or the creatine kinase promoter [ ] . even in vaccines, the vaccinating immune responses obtained were shown to be of a comparable magnitude to those in mice immunized with dna vaccines containing nonspecific promoters. for clinical efficacy and safety of chronic disease applications, it may be necessary to maintain appropriate levels of a gene product in order to prevent toxicity and to be able to modulate or resume transgene expression in response to disease evolution or immune problems. artificial systems for the control of genes are based on two elements: a chimeric transcription factor responding to a small inducer or even electric field and an artificial promoter composed of multiple binding sites for the transcription factor followed by a minimal promoter. inducible gene expression systems use endogenous elements that respond to exogenous signals or stress, such as cytokines, heat, metal ions, and hypoxia. however, neither muscle-specific nor inducible promoters in the absence of induction are devoid of leaky activity [ ] . if hypomethylated bacterial cpg sequences are maintained on the plasmid dna backbone or promoter elements, a t helper (th ) immune response (but only for a short period and with no induction of anti-dna antibodies) can be generated which may however be advantageous in view of genetic vaccination, alone or in priming-boost regimens with viral vectors [ ] . following the serendipitous demonstration of transgene expression in skeletal muscle injected with naked dna by wolff [ ] , plasmid dna has been used extensively in a variety of indications [ ] . uptake and expression of numerous transgenes have been demonstrated in various species following intramuscular administration of naked dna. expression peaks at around days, followed by a slow decrease and a prolonged steady state (years), in case of non-immunogenic transgene. the very long-term expression is probably linked to the postmitotic state of skeletal muscles and the persistence of administered genetic material as an extrachromosomal episomal elements [ ] . the efficiency of plasmid gene transfer into skeletal muscle (and other tissues) by direct injection is low (~ % of cell nuclei) and remains confined at the injection site (along the needle track) across species [ ] , and it further decreases with the plasmid size. nevertheless, naked plasmid dna administration was used in animal models to provide a systemic source of therapeutic protein, for genetic vaccination against pathogens and tumor cells or for therapeutic angiogenesis. in the later case, local gene delivery to focal lesions in the peripheral vasculature, for the production of highly active hormones, is ideally suited to the use of intramuscular or percutaneous vector delivery. in humans, intramuscular injections of naked plasmid encoding angiogenic factors (such as vegf or hgf) were used in small numbers of patients with critical limb ischemia and did demonstrate promising clinical efficacy for the treatment of peripheral arterial disease. ischemic pain and ischemic ulcers in the affected limb were relieved or markedly improved in further trials ( [ ] and table . ). importantly, all those plasmid-based preclinical and clinical trials resulted in a very good safety record ([ ] and table . ). a meta-analysis of clinical trials ( patients total) of local administration of pro-angiogenic growth factors (vegf, fgf, hgf, del- , hif- alpha) using plasmid or viral gene transfer by intra-arterial or intramuscular injections showed that, despite promising results in single studies, no clear benefit could be identified in peripheral artery disease patients, irrespective of disease severity [ ] . locally injected naked dna is being evaluated in muscle regeneration approaches such as myostatin propeptide gene gun delivery [ ] and for genetic motoneuron disorders. in the later case, smn induction in a mouse spinal muscular atrophy model was observed following intramuscular injection of a tetanus toxin c fragment plasmid [ ] . artificially or spontaneous regenerating muscle fibers display a higher, but still limited, efficiency of transfection [ ] . physical methods (electric or ultrasound pulses, ballistic gene gun), which either create transient pores in the cell membrane or increase passive diffusion, were shown to improve up to -fold gene transfer to skeletal muscles [ ] . the pulse parameters and the type of material used (i.e., needle versus externally applied plate electrodes) are of critical importance [ ] . selective electro-sonoporation in a defined area using microbubble contrast agents showed increased plasmid-vegf delivery in skeletal muscle allowing therapeutic angiogenesis in chronically ischemic skeletal muscles with undetectable tissue damage [ ]. a slightly higher risk of random integration of plasmid dna into genomic dna may also be seen [ ] . still limited penetration of the genetic material in the tissue is obtained (in the range of ~ cm). widespread delivery to large or deep muscles remains challenging. muscle damage and inflammation [ ] are induced by these methods which peak at around days and resolve at weeks postinjection with both th and th immune responses potentially occurring [ ] . therefore, this strategy may not be suitable in already inflamed tissue such as dmd muscles. high levels of gene expression in the limb and diaphragm muscles have been achieved by the rapid injection of naked dna in large volumes via locoregional hydrodynamic intravascular delivery with both blood inflow and outflow blocked surgically or using external tourniquets [ , ] . the endothelium in muscle is continuous and non-fenestrated, showing low permeability to macromolecules, including plasmid dna. the hydrodynamic pressure induces extravasation of the injected dna, probably by expanding the endothelium and thereby making pores accessible for dna entry. the mechanism of plasmid dna uptake by the muscle cells is still not clear and may involve both low-affinity receptor-mediated and nonspecific processes [ , ] . the procedure safety is supported by a large body of data collected in mice, rats, dogs, and nonhuman primates. the edema caused by the injected fluid is resolved within h and even the minimal signs of observed muscle toxicity clear within weeks postinjection [ , ] . the hind limb perfusion procedure is a rather quick and simple technique, which may be applied to chronic diseased muscles [ ] or other chronic diseases such as anemia [ ] . based on successful preclinical studies using the mdx mouse and golden retriever muscular dystrophy (grmd) dog models of duchenne muscular dystrophy, and the positive (expression -though very low-, and safety) outcome of a phase i trial of intramuscular injection of myodys ® , a full-length dystrophin plasmid, in duchenne patients (the first completed gene transfer clinical trial in neuromuscular diseases) [ ], the ground was set for a human clinical trial using myodys ® into the forearm of duchenne patients. a dose escalation study of single-limb perfusion with . % saline was carried out in nine adults with muscular dystrophies under intravenous analgesia. the study led by fan et al. demonstrated feasibility and safety up to % of limb volume in the upper extremities of the young adults with muscular dystrophy. perfusion at % limb volume was associated with short-lived physiological changes in peripheral nerves without clinical correlates in one subject [ ] . this study used lower cuff pressures than in our nonhuman primate studies ( - mm hg vs. - mm hg in nonhuman primates) [ , ] . from our studies in the mdx mouse and grmd dog models of duchenne dystrophy, and in nonhuman primates, the minimal volume needed for efficient naked dna limb perfusion is % of the limb volume [ ] . whereas arterial limb perfusion did not turn out to be safe in grmd dogs (personal data not shown), up to ten consecutive naked dna limb perfusions every other day appeared very safe in both dystrophic mice and dogs. even though head-to-head comparison would be necessary, our studies suggested that gene transfer was higher in diseased muscles than in wild-type animals. we also noticed that the highest transfection efficiencies were found in nonhuman primates; up to % of limb muscles expressed reporter genes following a single-limb perfusion [ ] . therefore, limb perfusion of a naked dna remains a valid approach to treat limb dystrophic muscles as an alternative to viral vectors in seropositive patients or in indications that require large transgenes with regional gene transfer [ ] . ex vivo approaches using gene-corrected stem cells with non-viral vectors are also being explored. human artificial chromosome (hac) vectors have the capacity to carry large genomic loci and to replicate and segregate autonomously without integration into the host genome. hac vectors containing the entire human dystrophin gene (dys-hac) with its native regulatory elements allow dystrophin expression at levels similar to native dystrophin isoform expression levels. since they can be stably maintained as episomal elements in host cells, the dys-hac could be introduced into several types of patient stem or progenitor cells for ex vivo therapy, e.g., induced pluripotent stem cells, mesoangioblasts, ac , and mesenchymal stem cells [ ] . one of the main issues, however, is the translatability of stem cell therapy in muscle disorders [ , ] . the development of successful non-viral gene delivery systems to skeletal muscle is highly dependent on the proportion of muscle (or their innervating motoneuron) cells that need to be transfected. more than years of research and testing in animal models and in human trials gear us toward two types of muscle-directed non-viral gene transfer applications: . direct injection. this represents a far simpler but poorly efficient approach. provided highly active gene products are used, non-viral gene therapy becomes increasingly amenable to infectious, cancerous, and peripheral ischemia diseases. vectors could be both naked dna and synthetic complexes. . intravascular delivery. simple intravenous perfusion of non-viral vectors is as of today far less practicable. regional hydrodynamic delivery of naked dna offers several advantages over viral vectors which hold potential for muscle diseases, including limb-girdle muscular dystrophies and peripheral neuropathies. nevertheless, muscle gene therapy using systemic administration of non-viral vectors retains major hurdles that need to be overcome before any human applications. disclosure author declares having no potential competing financial interests. the mechanism of naked dna uptake and expression non-viral gene delivery in skeletal muscle: a protein factory synthetic vectors for gene delivery: an overview of their evolution depending on routes of administration solid lipid nanoparticles for applications in gene therapy: a review of the state of the art in vitro and in vivo delivery of intact bac dna-comparison of different methods cationic liposomes as non-viral carriers of gene medicines: resolved issues, open questions, and future promises muscular gene transfer using nonviral vectors nonviral gene therapy lipofection: a highly efficient, lipid-mediated dna-transfection procedure mechanism of oligonucleotide release from cationic liposomes cellular and molecular barriers to gene transfer by a cationic lipid nuclear import of plasmid dna in digitoninpermeabilized cells requires both cytoplasmic factors and specific dna sequences improvement of exogenous dna nuclear importation by nuclear localization signal-bearing vectors: a promising way for non-viral gene therapy? bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids cationic transfection lipids key issues in non-viral gene delivery microcirculation in skeletal muscle hereditary inclusion body myopathy: single patient response to intravenous dosing of gne gene lipoplex efficient gene transfer from innervated muscle into rat peripheral and central nervous systems using a non-viral haemagglutinating virus of japan (hvj)-liposome method bdnf gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression efficient vaccination by intradermal or intramuscular inoculation of plasmid dna expressing hepatitis b surface antigen under desmin promoter/enhancer control long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and cmv promoter expression levels in vivo electrotransfer into skeletal muscle for protein expression adjuvant properties of cpg oligonucleotides in primates direct gene transfer into mouse muscle in vivo long-term persistence of plasmid dna and foreign gene expression in mouse muscle direct gene transfer into nonhuman primate myofibers in vivo delivery of dna into muscle for treating systemic diseases: advantages and challenges high-pressure transvenous perfusion of the upper extremity in human muscular dystrophy: a safety study with . % saline evaluation of hydrodynamic limb vein injections in nonhuman primates dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid dna functional efficacy of dystrophin expression from plasmids delivered to mdx mice by hydrodynamic limb vein injection gene-based therapies of neuromuscular disorders: an update and the pivotal role of patient organizations in their discovery and implementation dna vaccines to attack cancer 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(hiv- )-specific t cells capable of proliferation in healthy subjects by using a prime-boost regimen of dna-and modified vaccinia virus ankara-vectored vaccines expressing hiv- gag coupled to cd + t-cell epitopes safety and immunogenicity of cytotoxic t-lymphocyte poly-epitope, dna plasmid (ep hiv- ) vaccine in healthy, human immunodeficiency virus type (hiv- )-uninfected adults vaccine research center study team ( ) phase safety and immunogenicity evaluation of a multiclade hiv- dna candidate vaccine clinical experience with plasmid dna-and modified vaccinia virus ankara-vectored human immunodeficiency virus type clade a vaccine focusing on t-cell induction a randomized, placebo-controlled phase i trial of dna prime, recombinant fowlpox virus boost prophylactic vaccine for hiv- first human trial of a dna-based vaccine for treatment of human immunodeficiency virus type infection: safety and host response ev : a phase i trial to compare the safety and immunogenicity of hiv dna-c prime-nyvac-c boost to nyvac-c alone excellent safety and tolerability of the human immunodeficiency virus type pga /js plasmid dna priming vector vaccine in hiv type uninfected adults a human immunodeficiency virus (hiv- ) clade a vaccine in clinical trials: stimulation of hiv-specific t-cell responses by dna and recombinant modified vaccinia virus ankara (mva) vaccines in humans safety and immunogenicity of a gag-pol candidate hiv- dna vaccine administered by a needle-free device in hiv- -seronegative subjects cross-subtype antibody and cellular immune responses induced by a polyvalent dna prime-protein boost hiv- vaccine in healthy human volunteers clinical phase testing of the safety and immunogenicity of an epitopebased dna vaccine in human immunodeficiency virus type -infected subjects receiving highly active antiretroviral therapy protective efficacy of a recombinant dna vaccine against hepatitis b in male homosexuals: results at months induction or expansion of t-cell responses by a hepatitis b dna vaccine administered to chronic hbv carriers strong hcv ns / a, ns b, ns a, ns b-specific cellular immune responses induced in rhesus macaques by a novel hcv genotype a/ b consensus dna vaccine a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial development of a preventive vaccine for ebola virus infection in primates toxicological safety evaluation of dna plasmid vaccines against hiv- , ebola, severe acute respiratory syndrome, or west nile virus is similar despite differing plasmid backbones or gene-inserts the threat of avian influenza a (h n ). part iv: development of vaccines safety, efficacy, and immunogenicity of vgx- , a therapeutic synthetic dna vaccine targeting human papillomavirus and e and e proteins for cervical intraepithelial neoplasia / : a randomised, double-blind, placebo-controlled phase b trial safety of asp , a cytomegalovirus dna vaccine, in recipients undergoing allogeneic hematopoietic cell transplantation: an open-label phase trial clinical development of a cytomegalovirus dna vaccine: from product concept to pivotal phase trial safety and immunogenicity of a bivalent cytomegalovirus dna vaccine in healthy adult subjects safety, tolerability and humoral immune responses after intramuscular administration of a malaria dna vaccine to healthy adult volunteers clinical trial in healthy malaria-naive adults to evaluate the safety, tolerability, immunogenicity and efficacy of mustdo , a five-gene, sporozoite/hepatic stage plasmodium falciparum dna vaccine combined with escalating dose human gm-csf dna immunization with dna coding for gp results in cd t-cell independent antitumor immunity inability to immunize patients with metastatic melanoma using plasmid dna encoding the gp melanoma-melanocyte antigen gene electrotransfer of plasmid antiangiogenic metargidin peptide (amep) in disseminated melanoma: safety and efficacy results of a phase i first-in-man study phase i study of a plasmid dna vaccine encoding mart- in patients with resected melanoma at risk for relapse safety and immunogenicity of tyrosinase dna vaccines in patients with melanoma generation of mammaglobin-a-specific cd t cells and identification of candidate cd epitopes for breast cancer vaccine strategies immunogenicity of a plasmid dna vaccine encoding chimeric idiotype in patients with b-cell lymphoma her /neu dna vaccination for breast tumors a phase i trial of dna vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer taking electroporationbased delivery of dna vaccination into humans: a generic clinical protocol dna vaccines: an active immunization strategy for prostate cancer development of pro-angiogenic engineered transcription factors for the treatment of cardiovascular disease therapeutic angiogenesis with intramuscular nv fgf improves amputation-free survival in patients with critical limb ischemia constitutive expression of phvegf after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia effect of fibroblast growth factor nv fgf on amputation and death: a randomised placebo-controlled trial of gene therapy in critical limb ischaemia naked plasmid dna encoding fibroblast growth factor type for the treatment of end-stage unreconstructible lower extremity ischemia: preliminary results of a phase i trial clinical safety and preliminary efficacy of plasmid pudk-hgf expressing human hepatocyte growth factor (hgf) in patients with critical limb ischemia plasma vascular endothelial growth factor (vegf) levels after intramuscular and intramyocardial gene transfer of vegf- plasmid dna vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial basic fibroblast growth factor in patients with intermittent claudication: results of a phase i trial results of a double-blind, placebo-controlled study to assess the safety of intramuscular injection of hepatocyte growth factor plasmid to improve limb perfusion in patients with critical limb ischemia non-viral vectors for gene therapy: clinical trials in cardiovascular disease intramuscular vascular endothelial growth factor gene therapy in patients with chronic critical leg ischemia results from a phase ii multicenter, double-blind placebo-controlled study of del- (vlts- ) for intermittent claudication in subjects with peripheral arterial disease design of the del- for therapeutic angiogenesis trial (delta- ), a phase ii multicenter, double-blind, placebo-controlled trial of vlts- in subjects with intermittent claudication secondary to peripheral arterial disease treatment of thromboangiitis obliterans (buerger's disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results long-term follow-up evaluation of results from clinical trial using hepatocyte growth factor gene to treat severe peripheral arterial disease induction of antigen-specific tolerance in multiple sclerosis after immunization with dna encoding myelin basic protein in a randomized, placebo-controlled phase / trial double-blind, placebo-controlled study of hgf gene therapy in diabetic neuropathy vascular endothelial growth factor gene transfer for diabetic polyneuropathy: a randomized, double-blinded trial improvement in chronic ischemic neuropathy after intramuscular phvegf gene transfer in patients with critical limb ischemia key: cord- -stcrozdw authors: nan title: abstracts of papers presented at the th meeting of the deutsche gesellschaft für hygiene und mikrobiologie, virology section, göttingen, .– . . date: - - journal: zentralbl bakteriol mikrobiol hyg a doi: . /s - ( ) - sha: doc_id: cord_uid: stcrozdw nan schaefer, a., zibirre, r., kabus, p., kohne, jutta, and koch. g. na +, k + -atpase activity was studied in a plasma membrane rich fraction isolated from control and poliovirus infected he la cells and compared to membrane potential and amino acid uptake in parallel cultures of intact cells. na + , k + -at pase activity in membranes from infected hela cells increased relative to control with a maximum at minutes post infection (+ %) and decreased again ( rnin.: - %). similar but slighter changes were observed in the membrane potential dependent tetraphenylphosphonium (t pp+ ) uptake, indicating a correlation between membrane porential in intact cells and our measurment of plasma membrane na + , k +-at pase. at approximately h post infection we observed a decrease in the uptake of amino acid (methionine, leucine) in infected cells relative to controls. these results suggest that the decline in amino acid upt ake is not mediated by virus-induced changes in the na + , k + -atpase activity or membrane potential. mpi f. biochemic, abt. virologic, sequence homology in different strains of fmdv and other picoma viruses marquardt, o. restriction enzyme generated subgenomic fragments of cloned cdna prepared from rna of the strain k of fmdv were compared quantitatively for sequence complementarity with radioactive rna from strains cobb and a s or with rna from mengo or polio virus in hybridization experiments by use of the southern technique. nucleic acid sequences neighbouring or including the ' end of viral genomes are demonstrated to be ofo homologous in fmdv. in contrast, sequences coding for the capsid proteins vpl and vp were remarkably heterologous (less than ) in fmdv. sequences coding for non-structural proteins showed - homology. thus no highly conserved coding sequence was detectable in fmdv by this technique. no hybridization could .be detected between k specific dna and polio rna, while weak hybridization was observed with mengo rna at areas including the ' end and with a part of the gene for precursor protein p . abr, molekularbiologie, physiol-chern. inst., grindelallee , d- hamburg modification of poliovirus capsid proteins scharli, claudia and koch, g. poliovirus type , strain mohoney contains a protein kinase activity. a highly purified (two cycles of csci gradient centrifugation) poliovirus preparation is able to transfer the y-phosphoryl-group of [ p]_atp to acid precipitable material. when the reaction product is anal yzed by sds page, all structural proteins of polio are phoshorylated. most of the label is incorporated in the minor capsid protein vpo and to a lesser extend in vp , vp and vp . hepatitis a virus (hav) was isolated directly from human stool in diploid human fibroplasrs, viral antigen was expressed only after days of incubation of the infected cultures. in contrast, hav first adapted to growth in human hepatocellular carcinoma cells caused antigen expression in fibroplast cultures already days after infection. during further passage the time of first appearance of antigen in infected fibroblasts decreased to about days in both passage series (i. e. cultures inoculated with virus recovered directly from human stool or after adaption to human hepatoma cells). antigen was predominantly cell-associated and was shown by immunofluorescence to be located exclusively in the cytoplasm. -biophysical properties of hav particles extracted from infected cells were comparable to those described for hav extracted from human stool. those findings are of importance for preparation of large amounts of hav for vaccine. production. max von pettenkofer-inst. u. inst. f. biochemie d. univ. d- miinchen cloning of hepatitis a virus genomes helm, k. von der, winnacker, e. l., and deinhardt, f. subgenomic fragments of the genomic rna of hepatitis a virus (hav) were cloned via edna into pst site of pbr . by restriction enzyme pattern analysis and hybridization of the cloned hav dna with selected fragments of the ha v rna it was determined that the major part of the cloned hav dna fragments are located at the ' end of the genome but a few clones are distributed over the entire genome. we have now identified subtype specific sites in hbv-restriction map s. the maps were aligned to the eco ri site. only subtype ay revealed one subtype specific site of each xbai ( bp), h ineli ( bp) and bam hi ( bp). they are situ ated outside the s-gene. employing rhis system the subty pes of three hbv-ona sequences that ha ve been published so far , could be determined . thereupon the first triplets of a hbv (ad)-dna sequence of the s-gene could be com pared with pub lished data . nucleotide exchanges in triplets and did not cau se an amino-acid (aa)-exchange, bu t cha nges in the triplets and do cause such exchange. thus, in subtype ay, aa is thr, and aa is thr, wh ile in subtype ad, aa is ser and aa is pro. these exchanges may produce different conf ormation of th e peptides. dept. m ed. mic robio l., univ. - gottingen characterization of the hepatitis b virus (hbv) associated proteinkinase gerlich, w. h. purified hbv-core preparations were shown to contain a pr ot eink inase which phosphory lates the major core protein (albin and robinson, ) . in this study it was found th at th is enzyme copurifies with the light (d = . ) but not with the heavy subfraction (d = . ) of the core-particles. the enzyme has a high affinity for atp and it transfers approx. one ph osphat e per particle within minutes. the p-phosphate introd uced in vitro cannot be remove d from core-particles by digestion with alkaline ph osph at ase. after lysis of the cores with sos the p can be cleaved from rhe protein. t his suggests th at th e enzyme and its ph osphate acceptor site are located with in the pa rticle. after acid hydrolysis the incor porated !p co-migrates with ph osph oserin but not with phosphor hreonin or phos pho tryosin. m ax von petrenk ofer-inst., univ. - miin chen a phosphokinase activity associated with the hepatitis b virus (hbv) core helm, k. von der, roggendorf, m., and siegert, w. prepar ation s of core antigen positive (hbcag) fractions obta ined from hbv positive hu man liver tissue are associated with a ph osphokinase wh ich copurifies with the hbcag in cscl density-and sucrose velocity grad ient centrifugations. the acceptor protein for this kina se is the d hbcag protein; casein as an exogenous acceptor is also phospho rylate d to a minor extend. the ph osphorylated amino acid is serine or threonine and not tyrosi ne, as it is frequently the case with tumor virus specific prot ein kinases. dept. med . microbiol., univ. - gott ingen effect of glycosidases on the proteins of hepatitis b surface antigen (hbsag) stibbe, w. and gerlich, w. h . the protein composition of purified hbsag was stud ied by sos·gc e ectrophoresis and staining with silver. in additio n to p and gp of hbsag two furt her proteins, gp and gp , were found consistently in preparations from different blood donors. the glycoprotein nature of gp and gp was shown by the increase of electrophoretic mobility after treatment with endoglycosidase h or neuraminidase. immune precipitation with anti-hbs in ripa-buffer confirmed the specifity of the glycoproteins, the change in apparent mol. weight after treatment with neuraminidase was larger for gp ( ) than for gp ( ) or gp ( ). the data suggest that gp and gp are multiply glycosylated proteins with n-glycosidic linked carbohydrate side chains of the mixed type. inst, f. virologie, univ., hb s antigen screening was done in the blood samples of , pregnant women. blood samples were found to be hb s antigen positive. women were asymptomic carriers, woman suffered from chronic hepatitis. children have been born until now. the examination of cord blood showed hb s antigen in samples; the venous blood samples taken thereupon demonstrated hb s antigen only in two samples. the hb s antigen negative children were treated with hbig. the follow-up examination of the treated children showed no signs of infection up to now. -one child born to an hb e antigen positive mother was already infected at the time of birth. another child born to an anti hb e positive mother became hbs antigen positive at the age of months. unfortunately, this child had not been treated with hbig at the time of birth. willers, h., ponnath, h., sipos, s., and moller, r. sera of , pregnant women living in the hannover area were investigated for the presence of hbsag. healthy hbsag carriers were found: ( . / ) among , women of german origin and ( . / ) among foreign-borne women from southern european and non-european countries. out of the hbsag carriers ( , german women and / foreign-borne women) were recognized to be hbeag-positive. -the risk of perinatal transmission of hepatitis b virus in infants of hbeag-positive mothers is suggested to be % and of hbeag-negative mothers / . -thus in the hannover area out of , infants born to german mothers and out of , infants born ro foreign mothers become hbsag carriers due to perinatal hepatitis b virus-infection. max von pettenkofer insr., univ. d- munich a radioimmunoassay for anti-hbc igm using higher concentration for the dilution of serum ( mg/ml) and hbcag ( mg/ml) was compared with an elisa test ]. din. microb iol. ( ) , . onl y of ( / ) hbsag positive blood don ors were positive ( in elisa) and no correl ation with the total anti -hbc titer could be found . after fraction ation of a serum with discrepant results a positi ve result was found in the elisa only in the igg fractions. in a collaborative stu dy with a/torfer et al. (presented also at lisbo n, easl ( ) , who show ed a fold reduction in hepatitis b titer by means of !i-plluv treatme nt. the effectiveness of the cold sterilization procedure as regards reducing virus infectivity is considera bly greater than that of pasteurization ( h dc) for which shikata et al. ( ) sho wed a fold reduction of hep atitis b virus infectivity. it has also been found that factor ix conce ntrate and stab ilized serum (biseko®) der ived from pooled. cold-sterilization hu man plasma transmitted neither hepatitis b nor hepatit is no n-a, non-b in chimpa nzees. max von pettenk ofer-insr., - miinchen comparison of different evaluation systems for determination of viral antibodie s of the igg and igm class in the enzymeimmunoassay r o g g endorf, m. , zachoval, r ., zoulek, g., a nd deinharot, f. t he enzymeimmunoassays used to demonstrate antibodies of the igg and igm class against viral antigens reveal a high sensitivity. at onset of acute viral infections igm antibodies can be demonstrated up to serum dilutions of - • due to th is high sensitivity, the determin ation of antibody tite rs is not accurate and reproducible, because titer end points are determ ined as that dilution of sera giving an . . sample/o .o. negative control equal or greater than . . -for the dete rmination of antibody con centr ations an evaluation method is proposed which correlates, the measured . . of sera at one dilut ion step to the . . of a reference serum which is defined arbitrarily to contain anti body units. using an elisa for detection of antibodies to adenovirus, a significant increase in antibod y units of acute phase sera over that of convalescent pha se sera is observed. low day to da y varia tions are seen in tests car ried out on different days. in an elisa which is designed to detect anti bodies to tick-born e encephalitis virus of th e igm class, the day to day variatio n using antibody units was significantly lower th an using pin ratios. inst. f. virolog ie, jusrus-liebi g-un iv., - gieben; max-planck-insr, f. m olekulare genet ik, d -looo berlin structure and function of the core protein of alphaviruses boege, ulrike and witt man n-liebold, brigitte the com plete primary struc tures of the core proteins of sindbis and semliki forest virus have been established by pr ote inchemical methods. both proteins contain clusters of basic amino acids and proline in th e n-terminal part, suggesting th at its function is to bind to the nucle ic acid. the c-termi nal pa rts have ext ensive iden tical sequence regions, pr ob abl y p roviding the recognition sites for pr otein-protein interactions . both proteins contain within these highl y conserved portion s the sequence gly-asp-ser-gly wh ich is typical for serine proteases and most likely is related to the cata lytic function of the core prot ein as a protease wh en cleaving its own peptide chai n from the nascent protein precursor. experimental studies using peptides which contain certai n sequence regions will help to elucidate the relationships of struc ture and functions. an ac ute and a persistent infectio n with rab ies virus (h ep flury) was established in th e cn s-derive d cell ine -cc-i (ng - ). these cells possess specific membrane recept ors to many hormon es and neurotran smitters including opiate receptors. in both cases we found increases in th e dissociation constant (kn) for the agonist h-etorp hine as estimated by scatcha rd plot ana lysis. h owe ver, in both cases th ere was no change in th e nu mb er of op iat e receptors per cell compa red to uninfected cells. these studies complete our previou s published observations of the impai rme nt o f receptor func tions in rabies virus infe cted cells ( ) . ( ) kosch el, k. an d m. halb ach: j. gen. virol. ( ) , - . insr, f. virologic u. immunobi olo gie d. univ., - wiirzburg effect of measles (sspe) antiserum on viral surface proteins and hormone receptor activity in c /sspe persistently infected cells barrett, p. n. and koschel , k. it has been reported ( ) th at measles antis erum ca n modulate the expression of certain viru s prot eins in acu tely measles infected cells. we have exam ined th e effect of measles (sspe) antiserum on the exp ressio n of vira l surface proteins in c isspe infected cells. we have shown an over % redu ction in the am ount of viral antigen present on the memb ranes of these cells following incuba tion with sspe antiserum. this was demon-strated both by immunofluorescence and rad ioimmunoassay. however this loss of antigen had no effect on memb rane receptor linked camp synthesis. this is discussed with respect to the effect of virus antigen insertion in cell membranes on specialised membrane bound functions. ( ) we have analyzed the effect of phosphorylation and dephosphorylation on the structure and dna binding of d -t antigen. on non-denaturing pore-gradient gels the purified protein migrated with an apparent size of , daltons, in vitro phosphorylation by the protein kinase associated with the purified protein resulted in a shift of most of the protein to a size of , daltons, and it was this form that contained most of the phosphate incorporated. this aggregation was completely reversible by treatment of phosphorylated d -t antigen with alkaline phosphatase. partial tryptic digestion indicated that phosphorylation of sites in the nvrerminal part of the protein is responsible for the observed aggregation. -as show n by protein blotting onto nitrocellulose filters predominantly the , dalton form bound to sv dna. however, onl y a fraction of the in vitro phosphorylated protein did bind to dna, suggesting that aggregation alone is not sufficient for dna bind ing. subclasses of sv large t antigen were separated by zone velocity sedimentation. three major forms, which sedirnented at about - s, - s and - , have been shown to differ biologically and biochemically ( ) . each subclass was tested for specific binding to restriction fragments of sv dna using an immunoprecipitation assay. -all three forms of t antigen bound specifically to a restriction fragment containing the sv origin of replication. however, the - s form bound more origin fragment per unit t antigen than the other forms . the - s form bound equally well to origin dna in the presence and absence of excess cellul ar dna , whereas the binding of the - s form was reduced in the presence of cellular dna . all forms of t antigen from sv -transformed sv cells bound much less origin dna than that from infected cells. ( ) fanning, nowak , burger: j. virol ( ) wart scrapings from several small skin regions of an epidermodysplasia verruciformis patient were tested for papilomavirus-specific sequences by means of a p-labelled hpv dna. uncleaved wart dna contained uniforme full length hpv genomes. cleavage with bam hi revealed six different patterns and a surprising heterogeneity even within small biopsies. at least two virus types showed only limited cross-hybridization with hpv a. one of these closely resembles hpv b (bam hi fragments , and , ; eco ri fragments , and , ). hind ii fragments a and c of his virus perfectly hybridize with hpv a; b, d and anneal only partially and f, g show no detectable hybridization. the dnas of four subtypes were partially characterized and mapped. only dna of the hpv b-like virus was detected in a probe from the center of a carcinoma at the patient's forehead. this dna persists extrachromosomally with more than genome equivalents per cell. inst, f. virologie, zentrum hygiene, univ., hermann-herder-str. , d- freiburg gene expression of papillomaviruses in hamster tumors freese, u. k.,schulte, p., and pfister, h. bpv includes fibrosarcomas in hamsters. the tumors contain large amounts of complete virus genomes which persist extrachromosomally but there is no evidence for capsid protein synthesis or virus particle production. we used this system to study early viral gene expression. rna from the tumors contained a single virus-specific rna with about nucleotides which was shown to be polyadenylated by affinity chromatography on poly-uesepharose. the transcribed dna region of bpv was mapped within the two hpa ii fragments, which are next to the bam hi cleavage site within the . x d bam hiieco ri fragment. cross-hybridization studies under low stringency revealed some sequence relationship of hpv and hpv dna to the transcribed region of bpv . inst. of genetics, univ., d- cologne the adenovirus type -mouse cell system: permissivity and analysis of viral dna in tumor cells starzinski-powitz, a., schulz, m., and doerfler, w. interactions between viruses and eucaryotic cells can be studied in a genetically well defined system like the mouse system. we have investigated whether ad dna is able to replicate in primary mouse kidney cells or in the mouse cell line l . it was shown by southern blot experiments that ad dna was not able to replicate in l cells, w hereas in prima ry mouse kidne y cells of (balb/c x cs /b ) fl origin, viral dna repl icated. moreover , we subcutaneously injected ad into mice of various genetic origins. in ab out mice injected, one rumor emerged in a balblc mouse almost months after injection. restriction pattern anal ysis of the rumor or dna indicated that abou t - copies of ad dn a were integrated and covalently bound to cellular dna. with th e techniques available no deletion or rearrangement of viral dna could be found. t he sites of jun ction betw een viral and cellular dna will now be cloned and sequenced. inst, of genetics, univ., - co logne virus-cell dna recombinants in human cells lytically infected by ad neumann, r., weyer, u., and doerfler, w. there is ample evidence for the notion that virus-cell dna recombinants are formed in human cells productively infected with adenovirus type (ad ). these high molecular weight forms were detected at - h postinfection and were generated at high frequency, a limited set of rather specific recombinants was produced (neumann and doerfler, j. virol. ( ) suggesting th at recomb ination exhibited a cert ain specificity. we now succeeded in molecularly cloning dna frag ments excised from the high mole cular weight dn a of ad -infected hum an cells by th e restri ction endonuclease ecori in agtwes . ab or in acharon b. some of these clon ed frag ments had sequence ho mology to both viral and cellular d na. th is result provided proof fo r the occurrence of virus-cell dna recombi na nts . inst, of genetics, univ., - cologne unmethylated dna sequences in the promoter regions of integrated adenovirus genes correlate with gene expression kruczek, . and doerfler, w. an inverse correlation was established between the levels of dn a met hylation at '-ccg g- ' sites in specific segments of integrated adenovirus dna and the extent to wh ich the se segments were expressed (sutt er and doerfler, pnas ( ) . similar correlatio ns were reported in other viral and non-viral systems. more recently, the results of in vitro experiments prov ided direct evidence for the notion th at dna methylation at specific sites led to gene inactivation (vardimon et al., pnas, in press ). -in the present stud y a detai led methylation map at '-cc gg- ' (h paiilm spi) sites in the expressed early and the silent late genes of ad dn a was determined in three adl -tr ansformed hamster cell lines. the early region s of inte grated ad dna were unmethylated; in particular their promot er regions were unrnethylated ar the hpall sites in all three lines investigated. t he late regio ns including the promot er sites were completely meth ylated. gahlmann, r., deuring, r., stabel, s., winterhoff, u. vardimon, ., and doerfler, w. the sites of junction between viral and cellular dna were sequenced to investigate two problems: . are the sites of insertion of viral dna specific? . what type of recombinatorial events occur in viral dna integration? we have studied junction sites from the ad -transformed hamster line hes, and from the ad -induced hamster tumor lines claci and clac . the virus-cell dna junctions were cloned in the dna of bacteriophage gt· j.b. appropriate restriction fragments were sequenced by the maxam-gilbert technique. there was no direct sequence identity apparent at the viral and cellular junction sequences. deletions at the termini of the viral genome were seen involving (he ), (clac ) or (clacl) nucleotides. peculiar patch type homologies between the cellular and viral sequence adjacent to the site of junction and also in remote areas were observed. these patches may have an important function in integrational recombination. buttner, w., veres-malnar,s., and block, j. as a first step to understand the relationship between structure and function of the adenovirus type dna binding protein (ad dbp) we have begun to determine the primary structure of the dbp gene. the isolation and characterization of tupaia adenovirus (tav) has been reported. in order to construct a genetic map of the tav genome the use of temperature-sensitive mutants (ts) of tav was necessary. -a variety of ts mutants of t av, which were generated by treatment of t av virions ( x pfu) with hydroxylamine, were isolated and preliminary characterized. six of these mutants its: , , , , , and ) did not replicate in tupaia embryonic kidney cells at . dc, but did replicate well at °c. the characterization of these mutants was carried out using complementation tests, host range studies and dna anal ysis using different restriction enzymes. according to complementation analysis four groups were determined : group i = ts , group ii = ts , group iii = ts , and group iv = ts , , and . the host rage study revealed that ts and also had properties of host range mutants. these mutants did not replicate in tupaia bab y fibroblasts in contrast to wild-type virus. genome analysis of these mutants revealed that the mutated region is located between . to . map units. in addition, in ts and mutants a deletion from . kb to . kb (map unit . to ) was detected. interaction of viral substructures with serum and serumcomponents resp. during the final stage in the course of many viral infections complete virus particles as well as viral substructures enter the intercellular space, e. g. hbsag, hbcag in hepatitis b virus infections . the present study deals on the interaction of the potentially infectious adenovirus cores with dna-specific antibodies and immunoglobulin solutions which had been absorbed by dna-antigens. cores were prepared by sarcosyl treatment of purified adenovirus typ . by electron microscopy immunocomplexes could be demonstrated which are composed of several individual core units. by buoyant density centrifugation in metrizamide gradients a drastic rise in the density of cores could be shown too. with immunoglobulin solutions, absorbed with native as well as with denatured dna, so far no complexes could be assessed. a cell line designated sbl-h and several cell clones were established from skin tumours of the sporadic leukosis form. the cells were proved to be free of bovine leukemia (blv), and some other bovine viruses. by indirect immunofluorescence macroschizonts of a theileria species were seen in the cytoplasm. the cells originated from a blvantibody negative animal and carried a female karyotype with some morphological aberrations. evidence for a possible t cell origin of sbl-h cells was obtained. after inoculation of x loa cells into 'nude mice' transplantable sbl-h tumours developed. by incorporarion of h-uridine and electron microscopy, the production of retrovirus particles by the cultured cells was detected . in the simultaneous detection test a highmolecular weight rna co-migrating with felv s rna was demonstrated. no antigenic or genetic relationship between th e skin tumour virus isolate and blv or other major mammalian retrov iruses has been found. -(supported by stiftung volkswagenwerk ). abt. f. pathologie, gesellschafl: f. strahlen-u. umweldorschung, d- neuherberg insectpathogenic baculoviruses: studies of the activation of endogenous c-type retroviruses in mammalian cell cultures schmidt, j. and erfle, v. activation of endogenous c-type retroviruses by baculoviruses was studied in "in vitro" cell culture systems of four mammalian species: mouse, rat, monkey and man. cells were treated with baculoviruses (from larvae and insect cell cultures), baculovirus-dna, c-rype retrovirus-activating chemicals and chemical insecticides alone and in combination. the activation of retroviral genomes was tested by the determination of reverse transcriptase activiry in concentrated cell culture supernatants and by the demonstration of the intracellular localisation of retrovirus structural protein p applying the indirect immunoperoxydase technique. -c-type retroviruses were activated in mouse cells only by the halogenated pyrimidine analogue iododeoxyuridine. in baculovirus-treated cell cultures no c-type retrovirus activation was detectable. in simultaneous treatments of the cells with baculoviruses and chemicals no potentiating effects could be detected. virions of baculoviruses in mammalian cell cultures showed unaltered morphology and upon reisolation their infectivity in homologous insect cell cultures was lowered by approximately one log. no influence upon growth or morphology of the cells could be observed. the gene products of replication-defective oncornaviruses are high molecular weight proteins which comprise a gag related and one gene product. they are probably not cleaved because the p protease gene is lacking. in vitro, however, the gag-specific peptide sequences are cleaved off upon addition of the purified viral pis protease; in the case of the replication-defective, transforming avian sarcoma virus prc ii the cleaved nongag part has a ryrosine-phosphorylaring kinase activity similar to that described for the rsv src-gene product pp src . processing of pr gp , the precursor to the viral glycoproteins of rous sarcoma virus bosch, v., schwarz, r. t., ziemiecki, a., and friis, r. r. the viral glycoproteins of rous sarcoma virus gp and gp are synthesized via a precursor polyprotein pr ". this precursor is already glycosylated and contains the polypeptides of both gp and gp . we have studied the nature and site of processing of pr '~to mature disulfide-linked gp and gp (vgp). we could show that in addition to proteolytic cleavage, processing involves conversion of the high-mannose oligosac-charides found in pr ' " to the complex, sialidated oligosaccharides found in vgp. experiments pertaining to the site of processing indicate that processing does not occur extracellularly as has been proposed by others iklemenz and diggelmann, j. virol. ( ) - . we have determined that the small amount of mature vgp found in infected cell lysates is localized chiefly within the cell, not at the cell surface. we favour the view that further glycosylation and proteolytic cleavage occur concomitantly on smooth membranes within the cell and that subsequent to this, export in virus is rapid. paul-ehrlich-inst., d- frankfurt; lnst. f. virologie, univ., d· giefen, a protein of a molecular weight of about . d has been found to be phosphorylated h after the onset of cell transformation by rous sarcoma virus (rsv). it is assumed to be a physiological target of the pp '" kinase, since, apart from being phosphorylated in the transformed cell, it can be phosphorylated in vitro by the pp "· kinase in tyrosine ( , ) . -using different chromatographic procedures (chromatography on deae-sephacel, poly (a)-sepharose, blue sepharose, and hydroxylapatite, isoelectric focusing and gel filtration ) the , d protein could not be separated from cytosolic malic dehydrogenase activity (c-m dh ). antiserum against the , d protein inhibited c-mdh. the transformation-specific protein "v-myc" of the acute avian leukemia virus mc donner, p., greiser-wilke, irene, and moelling, karin avian acute leukemia viruses transform cells through a virus-coded oncogene. in the case of the rnyelocytornatosis virus, mc , which transforms fibroblasts as well as bone marrow cells in vitro , this oncogene is fused to a viral structural component, p . the fusion protein, v-rnyc, was characterized by using monoclonal antibodies against p . mc transformed quail fibroblasts which do not produce any virus, mc -q , were analyzed b y immunofluorescence. a nuclear fluorescence was observed which was not detected in normal cells or virus-producing cells. the monoclonal antibodies were used to purify the v-myc protein by immuno-affinity chromatography. the purification achieved by this single-step purification was , fold . the eluted protein was precipitable by anti-sera and will be further characterized for its biological properties. med. poliklinik, univ., d- miinchen; abr, f. pathologie d. gsf, neuherberg biochemical characterization of antigens in human leukemic sera that crossreact with sisv p and baevp leib, c., schetters, h., erfle, v., and hehlmann, r. antigens crossreacting with the core proteins p of baboon endogenous virus (baev) and/or of simian sarcoma associated virus (sisv) have been isolated from human leukemic sera by immunoaffinity chromatography. the antigens have an apparent molecular weight of , in sds-polyacrylamide gel electrophoresis. peptide maps performed with the antigens from two different leukemic sera show that the two antigens are identical. peptide analyses of sisv p and of baev p and simultaneous mapping of p proteins mixed with human antigens show that out of major peptides of sisv p and out of major peptides of baev p have identical mobility with peptides of the human antigens. human serum albumin, transferrin, fibrinogen, igg and igm share clearly less peptides of identical mobility with the human antigens. the isoglycoproteins gp and gp were purified from f-mulv particles (propagated in eveline cells) by solubilization (freezing and thawing), ion exchange chromatography (phosphocellulose) and preparative sds-page. prior to amino acid and nh -terminal amino acid sequence analysis, the purified glycoproteins were subjected to high performance liquid chromatography (hplc) to remove contaminants. -the nh -terminal amino acid sequences ( residues) of gp and gp were found to be different (in positions) but highly related. f-mulv gp shows f homology to gp but lacks the potential glycosylation site (sequon) at position in both f-mulv gp and r-mulv gp . et al., ) . in our laborato ry breast can cers, normal breast tissues, benign breast lesions and other carcin om as from south german patients were tested for crossrea ctivit y w ith mmtv-gp . the tests were done by indirect immunoperoxidase staining on paraffin-sections using antise ra against gp prov ided by dr. spiegelman, n. y. specificity of positive reactions wa s controlled by preabsorption with purified mmtv-gp . breast cancers ( / ) and benign breast lesions ( / ) gave positive reactions, whereas normal bre ast tissue and other carcinomas were negative. -the test might be an additional useful diagnostic too l for the earl y detection of micrometastases, for the assignment of metastases from unknown pr imary tumors and in doubtful cases of breast cancer. it can possibly serve as an add itional criterion for the classification of mastopath ies. viruses wer e found in , patients. nearly all illnesses were caused by mumps-and entero-viruses , other viru ses were found in less than % of all cases. the mumpsmeningitides were ascertained constantly over all these year s. in in , in and an increased incide nce of meningitides caused by echo-virus type wa s seen. -there was no seaso na l dep endence on the occurrence of mumps virus meningitides. meningitides, caused by ent ero-viruses was found mor e often in the autumn. male patients fell ill twice as ofte n as female patients. mumps-meningitides were rarel y found in the first year of life. in contras t, enterovirus meningitides could be dete cted during the first year and caused men ingitides to age of . virus meningitides among adults were rarel y found. we suspected that a number of peripheral facial pareses (p.f.p.) considered "idiopathic" might, in fact, have a viral aetiology . patients of the cologne university e.n.t. clinic with so-called idiopathic p.f.p. were examined under the aspect of a viral aetiology. in cases conditions for virological investigations were optimal (paired sera, first serum within the first week after onset of disease). in of these cases a viral infection could be proven (varicella zoster virus: , herpes simplex virus: , coxsackie b : ). in of vzv cases minimal zoster lesions were observed, facial and thoracic. among the oth er patients (with late sera) no viral infection could be proven. in conclusion, by means of alert clinical and virological examinat ions, a considerable fract ion of idiopathic p.f.p. could be associated with a virus infection. neonates were screened from the delivery to the time of discharge for rotavirus infections. daily faecal specimens were examined by an enzyme-linked immunosorbent assay (elisa) and a subgroup of positive specimens were also tested by a negative staining electromicro scopic method. - babies ( . / ) were found to excrete rotavirus. of them were asymptomatic infected and showed mild gastrointestinal symptoms. with one exception viruses were not detected in babies less than h old, but . % of them excreted virus during the second day of life and after days % of the neonates were positive for rotavirus. -excretion persisted for to days. according to our study most babies ( / ) excreted roravirus for days. -a great number of the stools ( %) from the first da y which were tested by elisa were found to have nonspecific activity in the absence of rotaviral antigen. theref ore such stool specimens should only be examined by electron microscopy. the single radial hemolysis test (srh-test = hemolysis-in-gel test) is a suitable technique for detection of rubella and influenza antibodies in a large number of sera. in our stud y we looked for the effect of att achment of the viral antigens to the erythrocytes by different coupling reagent s. chrom ic chloride, cyanogen chloride, glutaraldehyd and tetraazotized - -dianisid ine (tod) were used for the sensitization of the erythrocytes. -in the rubella srh-test no improvement on regard to sensitivity of the test was seen. moreover it is not possible to detect igm specific antibodies after different kinds of attachment in the srh-test. -in the influenza srh-test with allantoic fluid of eggs infected with h n virus it was possible to increase the sensitivity with tod, chromic chloride and potassium periodate. if tween-ether treated hemagglutinin was used only after sensitization with tod, chromic chloride and periodate hemolytic zones were detectable . univ.-kinderkiinik, mathildenstr. , d- freiburg rna-electropherotyping of human rotaviruses . and pastor, s. rotaviruses contain a double-stranded ribonucleic acid genome consisting of segments. this can easily be extracted from crude stool suspension (method by rodger et al., j. clin. microbial. ) . out of rota antigen positive samples contained sufficient rna to produce a satisfying pattern in the sds-acrylamide-electrophoresis. we demonstrate six electropherotypes with differences in the relative mobility of segments , , , , , , , . of these diarrhea producing strains at least two different electropherotypes were found during every outbreak of rotavirus gastroenteritis. our findings are in good agreement with the results reported by rodger ), espejo et al. ( ), and kalica et al. ( , . the so-called m-type of emc virus is capable of inducing diabetes mellitus in mice by a selective b cell damage. the m-emc strain used in our laboratory has partially lost this capacity. we attempted to reisolate a diabetogenic variant and to elucidate the causes of the change. seven serial heart passages in mice did not enrich such a variant. cloning of the virus stock yielded one clone diabetogenic in two of ten animals ( clones tested so far), -in a different substrain of m-ecm virus (the highly diabetogenic dvariant, obtained from dr. petersen) we found both diabetogenic and non-diabetogenic clones. incidence and severity of diabetes has been shown to be age-related. t his safety study was to demonstrate whether or not granulosis virus (gv) of laspeyresia pomonella can rep licate in vertebrates. after feeding gv to nmri-mice, no virus induced antibodies could be detected within eighty days by radioimmunoassay (ria) and no vertical virus transmission was observed. gv did not replicate in mice. human sera, as well as sera from horses, cattle, sheep and swine reacted with gv in the ria. by characterization the positive reacting human immunoglobulin c ass(es), igg showed the strongest positive reaction, less positive reactions were shown by ige and igm and no reaction by iga and igd. by concentrating the immunoglobulins of the negative reacting sera to pg igg/ml, all sera could be recognized as positive. thus a non-immunological reaction has been suggested. infection of human fibroblasts with cytomegalovirus induces typical cytopathic alterations. cell rounding within - h postinfection (p.i.) is followed by an increase in cell size, appearance of cytoplasmic and nuclear inclusions and a morphological change to an epitheloid cell shape by h p.i. in order to elucidate the participation of the cytoskeleton in this alteration of cell morphology experiments were initiated in serum-starved human fibroblasts to visualize changes in actin arrangement by indirect immunofluorescence. a early as h p.i. cytoplasmic microfilamenrs had shortened and were rearranged to a more irregular pattern. at h p.i. actin fibers were absent from rounded cells. the same was observed in epitheloid cells at h p.i. cultivation of infected cells with phosphonoacetic acid resulted in a partial preservation of the normal actin fiber distribution. in contrast, infected cells did not exhibit major changes in actin synthesis as estimated from the specific radioactivity of cytoplasmic actin isolated from h-leucine pulse labelled infected cells by dnaase i-sepharose affinity chromatography and sdspolyacrylamide electrophoresis with fluorography. chicken erythrocytes were coated with glycine-extracted cmv antigen and negative control antigen by treatment with formaldehyde and crcl s and were used for detection of cmv specific serum antibodies in pha tests. their sensitivity was proven to be in good agreement and their specificity superior to results seen in cft. igm-specific cmv antibody detection was performed either after a simple and rapid deae-cellulose exchange chromatography of serum samples or by igg immunoprecipitation combined with me-reduction controls. the main advantage of the modified cmv-phat was seen in the stability of sensitized erythrocytes, which can be lyophilized. lanvers, a., mertens, th., and eggers, h. j. there are reports that herpes simplex virus (hsv) could be isolated from the genitourinary tract of up to ofo of males without manifest herpes. in order to confirm these results we first wanted to establish a method for typing possible hsv isolates. we used published methods: a plaque test in chick embryo cells, a neutralisation test, and the analysis of the early hsv-proteins (sds-page). all methods yielded identical results. we then tried to isolate hsv from materials of asymptomatic males ( urethral swabs, seminal fluids, fresh tissue probes). all materials were immediately inoculated into tube cultures of two cell lines shown to be highly hsv-susceptible. additionally, the tissue probes were co-cultivated with permissive cells. we also induced cell fusion (peg) in such cocultures. despite all efforts we did not isolate any virus from all these materials. for an experimental approach to answer the question as to which viral genes may control pathogenesis after peripheral infection of inbred mice with hsv, we have isolated a variant strain (ang-path) that proved highly neuropathogenic both upon i.p. -or intravaginal infection from the apathogenic strain hsv- ang. two alterations of the ang genome have been detected by restriction enzyme analysis: the loss of the amplifiable b.p. nucleotide sequence typical for ang and a b.p, deletion approximately at the position mapped for the structural viral glyco-protein d. despite the induction of interferon and nk-cells both variants multiply to a similar extent, probably in the same target cells of the peritoneum. the spread from the peritoneum, spleen, liver, thymus and local lymph nodes to the ens seems, however, to be controlled by the action of gene products coded for in only one of the variants. div, of expo virology, insr, for med. microbiology, univ., d- mainz knoblich, a., friedrich, d., goertz,] ., and falke, d. herpesviruses are known to cause infections in men and animals. strong differences in resistance to herpes simplex virus (hsv- ) are noted among mice of various strains. anti-hsv-activity of t-iymphocytes, macrophages and nk cells has been demonstrated in the last years. our interest was focused on neutralizing antibodies in primary hsv- abstracts of the th meeting of the oghm infections of mice. antibodies become detectable by day after infection and reach a plateau level at day , the day we chose to test the sera. comparison of antibody titers in strains of mice revealed titers always to be higher in female mice, whereas no clear influence of either h- -haplotype or background genome could be detected. sexual steroids produced in ovaries and testes were identified to exert influence on antibody formation by castration experiments. treatment of mice with silica once between day before and day after infection resulted in a strong increase of antibody titers both in females and males at the same time abolishing the difference in antibody titers between the sexes. silica could enhance antibody levels also after immunisation of mice with a formol-inactivated hsv-vaccine. bestatin is a small peptide known-selectively to stimulate dna metabolism in t-lymphocytes and to enhance hsv-antibody -titers maximally when given at day after infection . after pretreatment of mice with silica the antibody augmenting effect is already achieved at day after infection . in secondary hsv-l infection bestatin acts best day after infection, too. insr. f. med. virologie d. univ., - heidelberg induction and characterization of herpes simplex virus reisolates, isolated after intertypic superinfection of latent infected tupaias darai, g. and scholz, j. the susceptibility of juvenile and adult tupaias to herpes simplex virus type and had been reported. the intertypic recombination of herpes simplex virus using temperature-sensitive mutants of hsv-l and and superinfection with wild-type hsv-l and was studied. it was found that animals survived an infection of x to x l() pfu of rs mutants of hsv-l and /or which were inoculated intravenously (l for hsv-l = - • and for hsv- = - • ) . the inoculated animals were protected against a superinfection of hsv-l or ( . x pfu/animals). the state of viral latency in surviving animals was investigated. it was found that infectious virus was recovered from ganglia of those animals which had initially been infected with wild-type hsv-l or and /or superinfected with wild-type viruses. in contrast, the infectious viruses were recovered only from spleens of those animals which had initially been infected with ts mutants of hsv-l or and superinfected with wild-type hsv- and /or . it was found that recovered viruses from the spleen of the animals lost their pathogenicity and their natural tissue tropism . significant changes in the genome of the recovered viruses from the spleen were detected. recombination between ts mutants of hsv-l and and challenged wild-type viruses was observed. thus, the pathogenicity and genomic properties of recovered viruses were altered . this observation is the first description of generation of inrerrypic recombinants of hsv-l and in in vivo. oiv. of expo virology, inst. for med. microbiology, univ., - mainz the functions of the hsv-coded dpyk-complex enzyme labenz, j., brauer, d., moller, w. e. g., and falke, d. analysing the phosphorylating capacity of the hsv-coded dpyk of hsv-l and by glycerol gradient centrifugation we detected each three peaks differing in molecular weights using atp, aop or amp as phosphate donors. also by page peaks with differ-ing rj-values could be seen. indeed, p_amp and p_adp were used for phosphorylation of dthd. the amp-dependent activiry could be purified boo-fold. two antisera against hsv-coded pdyk neutralized all three activities. the tk-mutant mdk (b ) did not induce in'i'kr cells the respective activities, only the cellular tk's were detected and identified by their rj-values. further experiments have shown that only the hsv-l-dpyk has thymidylate kinase activity, but not that of hsv- . the hsv-l thymidylate kinase activity could be neutralized by a tk-antiserum. -the ph-optimum, sensitivity to mg'", fe'", zn'", co" and mri'" ions differed. the susceptibility to thiol reagents was different, the amp-dependent activity proved to be susceptible to phenanthroline. also the k m and vrnax-values were detected. a diagram summarizes the biochemical function of the hsv-coded dpyk-complex. finally there is some indication that the enzyme phosphorylates ado by using dtmp or atp as a phosphate donor. the importance of the enzyme complex in hsv-infected cells is discussed. dkfz, inst. f. virusforschung, m neuenheimer feld , deletion of n uc eotide-sequences in cloned hsv- fragments during propagation in the rec a e. coli, hb gray, c. p., jellinghaus, u., and kaerner, h. c. passage of hsv at high mol results in the generation of defective genomes which are of full length, consisting of a relatively short region of the wild type genome that is repeated. they are packaged into mature virus particles and thus leave the cell in a state that is capable of entering a new host. such defective molecules are of interest as they represent a simpler model in which to study replication, recombination and packaging of hsv. -such a defective molecule arising from the serial passage of hsvtype l-ang has been mapped, and restriction fragments have been cloned into pbr using the rec a-e.coh, hb , as host. -all the resulting clones were found to be unstable and to contain deletions, one of which has been characterised in more detail. the isolation and characterization of tupaia herpesviruses (thy to ) has been reported. the analysis of dna of these viruses showed the absence of submolar dna fragments, when the dna was cleaved with different restriction enzymes, as well as of a stem loop structure, when analysed by electron microscopy. with respect to this genomic structure it was of interest to study he recombination events between thv to . recombinants were generated between these viruses using a co-infection technique in vitro on tupaia fibroblasts. recombinants were selected after the stocks of new progency virus were treated with specific antiserum against each parental virus. different recombinants were isolated, plaque-purified and characterized. results for one recombinant thv-r- between thv- and were as follows: (i) the in vitro host range and the in vivo pathogenicity in juvenile tupaia was altered compared to parental viruses. phosphorylation of proteins is a posttranslational modification which is regulatory for the activity of several enzymes. most animal viruses code for phosphoproreins and the degree of their phosphorylation is thought to be a controlling factor during viral macromolecular synthesis. recent studies on protein kinases from a number of tumor viruses have raised the possibility that the phosphorylation of cell proteins is involved in the processes leading to cell transformation. -incubation of purified tree shrew (tupaia) herpesvirus (thv) particles with y. p atp resulted in the incorporation of p labelled phosphate into proteins. a nonionic detergent such as np- was necessary for the detection of protein kinase activity. the incorporation of phosphate was proportional to the quantity of th viral proteins, indicating that the viral proteins can serve as substrates for the viral enzyme. a_ p datp or a_ p atp did not function as phosphate donors. a divalent cation such as mg + or mn'" is essential for the enzymatic activity. a product analysis revealed that six viral polypeptides are phosphorylated. the predominant sites of phosphorylation are the (f-oh groups of serine and threonine. in a herpesvirus was isolated from young goats with a severe generalized infection in california. this isolate has been characterized in some detail and named caprine herpesvirus . recently a herpesvirus was isolated in switzerland from goats with a similar infection. we report a further characterization of both isolates and propose their classification as bovid herpesvirus (bhv- ). -bhv- multiplies rapidly and shows a broad hostcell range. crossneutralization onl y could be observed with bhv- (ibr/ipv-virus), in a one way reaction. in gel immunoelectrophoresis the serologic relationship envolved the major antigenic components of bhv- . we have evaluated two methods for the analysis of antibodies directed against ebvspecified proteins: . indirect immunoprecipitation (ip) and . radioimmunoassay of electroblots of sds page separated ebv-specified proteins (riab). using ip we have identified proteins against which antibodies are made during infection, some of these proteins have also been found using the latter technique. many of the proteins are only reactive with ea'vca+ sera and not ractive with ea-vca + sera and may therefore be candidates for the ea specifying proteins. it seems likely that the failure to identify all proteins using the riab technique is probably due to the strong denaturating conditions used during the sds page step. only those antibodies directed towards the primary sequence of the protein will react with the blotted proteins, whereas with ip analysis the native proteins are available for interaction with the sera. previousl y we demonstrated that the synthesis of ebv-induced proteins in superinfected raji cells (raji ) could be divided into phases: primary, secondary and tertiary (bay-liss and wolf, j. gen. virol., , in press) . recent experiments show that incubation of raji si in the presence of canavanine and the absence of arginine allows only a limited expression of the viral genome. if the cells are released from a canavanine block (applied from to h post infection) then between and h later several new proteins are synthesized, however, if m-rna synthesis is inhibited (with actinomycin d) after removal of the canavanine then these new proteins fail to appear. amongst these proteins are members of the ebv-specified early antigen complex (ea). these results indicate that an arginine-containing protein is synthesized soon after infection and that this protein is required in an active form in order to synthesize m-rna for the secondary group of proteins. amongst these proteins are the major components of the early antigen (ea) complex. max von pettenkofer inst., univ., d- miinchen ; ent-klinik, univ., d- wiirzburg; dept. tumor virology, chinese acad. med. sci., beijing, china seibl, r., richter, w., zeng, y., gu,s.-y., and wolf, h. antibody titers of iga antivirus capsid antigen (vca) can be used for screening early tumor cases, confirming histological diagnoses and longtime surveillance of therapy. however, in high risk areas (s. china, incidence of npc: / ) % of the population have iga anti vca antibody indicating a need for methods which allow monitoring of additional parameters for deciding on the need for therapy. the same need exists in case of long term ( - years) survivors of npc with constant iga anti vca titers. -we have developed a system to collect cell specimens by application of buffer to the tumor site and collection of cells directly on to nitrocellulose filters. these cells can be examined cytologically or for ebv dna in nucleic acid hybridization. for the latter tests a modification of the grunstein hogness colony hybridization test and cloned ebv dna have been used. in reconstruction experiments virus-producing cells could be detected. inst. f. klin. virologie, univ., d- erlangen-niimberg keil, g. and fleckenstein, b. after infection of permissive cell cultures with overlapping restriction-fragments of viral dna derived from different strains of herpesvirus saimiri (h. saimiri) a number of recombinants could be isolated. the analysis of the viral proteins of the wt-strains , omi and s c led to the identification of four proteins that differed within these strains with respect to molecular weight. we are able to localize these proteins in the internal region of the m-dna by comparing the protein patterns of recombinant and parent strains. the exact localization was so far not possible because the recombination events occured mainly in regions near the ends of the l-dna. -furthermore recombinants were constructed to identify the genomic region responsible for oncogenicity. the wt-strains of h. saimiri are oncogenic while attenuated strain -att, that was obtained from strain has lost this property. this may be due to a deletion of , md at the left end of the l-dna. recombinants between this attenuated strain and wt-strains were constructed. the test for oncogenicity in vivo is in progress. the t-iymphoid cells can contain up to genome copies per cell as episomes. we have begun to study translation and transcription in transformed cells in comparison to lytically infected omk-cells. about new proteins can be detected in lyrically infected cells having apparent molecular weights between - kd. -at early and late stages of infection the right part of the genome is preferentially transcribed and each dna-fragment encodes a series of specific rnas. -in contrast, we do not find any virus-specific proteins in the transformed cells after labeling with ss-methionine and subsequent immunoprecipitation. preliminary results may suggest that the only virus-specific rna found in the transformed cells are small rnas with molecular weights of about . kb. -a more detailed analysis has to be performed in order to confirm these hybridization data. max von pettenkofer inst., d- mi.inchen characterization of herpesvirus saimiri glycoproteins modrow, s. and wolf, h. for the identification of herpesvirus saimiri glycoproteins we seperated h. saimiri induced cell proteins on sds-polyacrylamide gels and transfered the polypeptides by elec-trophoretic blotting ( h, , ma/cm ) to nitrocellulose paper using carbon electrodes and buffer soaked sponge to cover the gel/filter layer. lectins, which are known to bind very specifically to certain sugar residues (concanavalin a to d-glucose and d-mannose derivates, soy bean agglutinin to n-acetyl-d-galactosamine and d-galactose, dolichos biflorus agglutinin to n-acetyl-d-galactosamine, ulex europaeus agglutinin to l-fucose) were iodinated using the nen-iactoperoxidase system. the glycoproteins bound to the nitrocellulose sheets were detected by incubation with the z j-labelled lectins. by this, eight viral glycoproteins could be identified, two of them were synthesized in the presence of canavanine, and characterized according to the type of glycosylation. lymphocystis disease (ld) is a virus disease of marine fish with an almost world-wide geographical distribution. this disease is characterised by papilloma-like tumour lesions. lymphocystis disease virus (fdlv) has been tentatively classified as belonging to the family of iridoviridae. this report describes the anatomy of fdlv: the fdlv virions were isolated from a total of fish with ld lesions, caught near the doggerbank, including flounders, dabs, and plaice, which were analysed individually. the purity of the virus preparations was examined by a negative-staining technique and the virion diameters were determined: flounder . ± . nm, ldv-plaice . ± . nm, and ldv-dab . ± nm. dnas of these different ldv isolates were cleaved with different restriction endonucleases and the resulting dna fragments were separated electrophoretically on agarose slab gels. the fragment patterns demonstrate that ldv dna of flonders and of plaice are indistinguishable, but clearly different from those of dab. the determination of the molecular weights of fdlv dnas using contour length measurements by electron microscopy resulted in a value ranging from to x daltons. in contrast the molecular weight estimations by restriction enzyme analysis resulted in lower values ranging from to x daltons. this discrepancy is probably due to a restricted, permutated structure of the ldv genome similar to the genome of frog virus as reported by granoff. the structure of fldv constituents and its interaction with host cells remains obscure. in an effort to clarify the viral components and their functions we have studied the proteins and purified fldv and searched for virion-associated enzymes. -at least distinct viral polypeptides were detected by polyacrylamide gel electrophoresis under denaturing conditions. the polypeptide patterns are remarkably specific for a given fish species (flounder, plaice, and dab) although some heterogeneity was found when proteins of individual fish of the same species were analysed. -it was found that a nucleoside triphosphate phosphohydrolase activity is closely associated with fldv particles. this activity hydrolyses atp with a high preference. the reaction requires a non-ionic detergent and a divalent cation, such as mg +. reaction rates and substrate specifities were determined. the products of the reaction are nucleosides disphophares and inorganic phosphate. characterization of a poxvirus isolated from white rhinoceros (ceratotherium s. simum) pilaski,] ., schaller, k., olberding, p., and finke, hannelore in september an outbreak of poxdisease occurred in white rhinoceroses (ceratotherium s. simum) in the munster zoo. at the same time a similar outbreak was observed in elephants (elephas maximus, loxodonta africana) of the zoo in frankfurt (airline distance about km). in both cases orthopoxvirus strains could be isolated which were similar but not identical in their biological properties (small efflorescences on the cam with hemorrhagic center, inclusion bodies of type a v+, high pathogenicity for rabbit skin, characteristic skin lesions in adult mice) and their dna restriction patterns (xho i, eco r i, hin d iii). both virus strains were incorporated into the group of "cowpoxlike viruses" (baxby and ghaboosi, ) . the results indicate that both outbreaks had occurred independently from each other. characterization of a k protein specifically associated with released extracellular vaccinia particles hiller g. and aulbach, h. infectious vaccinia virus can be isolated from infected cells after experimentally induced lysis (intracellular virus) or from the growth medium of infected cultures (extracellular virus). we have characterized a k protein only present on extracellular particles by its amino acid composition and its behaviour on isoelectric focusing. in addition we have used a k-specific antiserum to detect its distribution within infected cells. - k protein is a late viral protein appearing - h p.i. it is predominantly found associated with the cellular golgi complex but never with structures representing pox virus "factories". later in infection k protein is incorporated into single viral particles preferentially found in the cell periphery. upon electron microscopy approximately % of morphologically mature virions inside the cell are enwrapped by a double-membranate vesicular structure. thus k viral protein is probably a component of this vesicular structure and only vesiculated virions can be released by the cell before lysis occurs. epidemiology of influenza in lower saxony willers, h. and hopken, w. the influenza surveillance in lower saxony is mainly based on laboratory investigations especially on the attempts to isolate influenza viruses throughout the year. -in the winter - and in the winter - the two influenza subtypes h n and hini circulated at the same time. the h n subtype affected during the last years persons of all age-groups whereas the hini subtype affected only persons younger than years. -in the winter - influenza b was found in an epidemiological extent. the disease caused outbreaks mostly in schools and kindergartens but also affected adults. -in - from mid-january to mid-february scattered outbreaks were caused by the a subtype hinl, which particularly affected schoolchildren contrarily to where the hini subtype mostly infected young adults. influenza of the h n subtype circulated from january to march. inst. f. med. mikrobiologie. abr, virologie d. tu, biedersteiner str. , antibodies against influenza c virus in the population of germany, kenia and australia pfeil-putzien, c. and meier-ewert, h. over one hundred human sera from each of the three countries germany, kenia and australia were tested for antibodies against influenza c virus, using the conventional hemagglutination inhibition test (hi). the rate of positive sera, showing a hi titer amounted to ofo for germany, ofo for kenia and ofo for australia. in the age group up to years, ofo of german and ofo of kenian sera had already antibody titers against influenza c virus. the australian sera were tested for the age group of - years and showed ofo seropositivity. the results show that influenza c virus is circulating to a higher extend in the populations of countries with subtropical climate, as compared to the more temperate middle european zones. inst, f. virologie, justus-liebig-univ., d- giessen the proteolytic activation of influenza hemagglutinin, structure of the cleavage site and the mechanism of cleavage garten, w. and bosch, f. x. the hemagglutinin precursor ha is posttranslationally cleaved by proteases to the complex h i , ' in vitro cleavage of ha by trypsin and trysin-like proteases yields infectious virus. cleavage by thermo lysin or chymotrypsin yields non-infectious virus. we have analyzed the cleavage sites of hi, h and hlo hemagglutinins. the amino acid sequences at the cleavage site, i. e. c-terminus of hal and the n-terminus of ha are identical when virus is activated in vivo and in vitro. under both conditions, an arginine residue connecting hal and ha in the precursor is eliminated. the elimination of argmme results in a shift of the isoelectric point of the hemagglutinin as demonstrated by isoelectric focusing. non-activating enzymes cleave only one peptide bond in the ha -nterminal region of activated ha and thus do not affect the isoelectric point. we have also analyzed the cleavage site of the hemagglutinin of fowl plaque virus (h ). a connecting peptide containing several basic amino acids is eliminated. -the data show that activation of the influenza hemagglutinin involves the action of a cellular protease with trypsin-like specificity followed by the action of an exopeptidase of the carboxy-peptidase b-type. the latter enzyme activity is associated with purified virus and can be analyzed by an assay employing peptides bearing h-arginine at the c-terminus. the carbohydrates of the glycoproteins of influenza a strains containing hemagglutinin and neuraminidase of all serotypes known to date have been compared by analysis of glycopeptides labeled with radioactive sugars. analysis of incompletely glycosylated glycoproteins synthesized in the presence of glycosylation inhibitors allowed the determination of the number of oligosaccharide side chains on ha • with all strains, the neuraminidase contains side chains of both the complex type i and the mannose-rich type ii. there are distinct quantitative and qualitative differences between the strains in the distribution of type i and type ii side chains on the hemagglutinin fragments hal and ha • the majority of the hemagglutinin oligosaccharides is located on hal' these side chains are usually of type . only the hem agglutinins of serotype h have, in addition, a substantial amount of type ii side chains on hal' most strains have on ha a single side chain which is usually of type i. with serotype hs this side chain is free of fucose, and with serotype h it appears to be missing completely. serotypes h and h have, in addition to the type i, a type ii side chain on ha • these observations strengthen the concept that the primary structure of the polypeptide chain is an important determinant for the carbohydrate moiety of the hemagglutinin. inst. f. virologie, ]ustus-liebig-univ., d- giessen acylation of viral glycoproteins schmidt, m. f. g. covalent binding of fatty acids to viral glycoproteins was first detected with sindbis virus and vesicular stomatitis virus ( , ) . studies on acylation in other enveloped viruses revealed that covalent addition of fatty acid to spike glycoproteins is a more general feature. while in sindbis and in semliki forest virus both species of spike glycoprotein (el and e ) carry fatty acid chains, acylation with the other viruses studied (corona-, influenza a-and paramyxovirus family) seems restricted to those glycoproteins that are known to carry fusion activity. this new type of modification of viral glycoproteins occurs in a wide variety of host cells including those of human, bovine, mouse, hamster, avian and insect origin ( ) . -with the aid of controlled digestion of h-palmitic acid labelled virus particles and by the analysis of cyanogen bromide fragments of fatty acid labelled glycoprotein the fatty acid bind ing site in influenza hemagglutinin (hat), vsv g-protein and sindbis virus e and e could be located to the membrane spanning portion of the respective proteins ( ). heinrich-pette-institut f. exp. virologie u. immunologie an d. univ., martinistr. , d- hamburg mannweiler, k., bohn, w., rutter, g., and hohenberg, h. in replica-immunocytochemical (ric) -and ultrathin section (us) preparations the ultrastructures of the specific alterations and of virus antigens, which appear at the plasma membrane of he la cell coverslip cultures after infection with an adapted measles virus strain were investigated. as immunomarker protein a-coated gold particles were used ( ) . this method is sufficiently sensitive to enable labeling of even small altered areas of the plasma membrane ( - nm cb). due to the high atomic number contrast in the tem and the small size of the marker (-<:: nm) the ultrastructure of characteristic alterations morphologically still remains visible with ease in a three-dimensional aspect. data obtained by ric and us preparations after labeling with antimeasles immune serum or with monoclonal antibodies against ha ( ) are demonstrated, compared and discussed. bohn, w., rutter, g., and mannweiler, k. by use of the mouse hybridoma technique, monoclonal antibodies were obtained with specificity for the ha ( k), p ( k) and m ( k) polypeptides of measles virus. balblc mice were immunized with native measles virus and measles virus treated with detergents and heat. clones obtained after immunization of mice with native measles virus showed specificity for the ha polypeptide only. after immunization with measles virus, treated with / sodium sarkosyl sulfate (sss) at °c a clone was obtained producing antibodies to the m polypeptide. heating of measles virus in the presence of / sds under reducing conditions elicited a selective immune response to the p and np polypeptides. thus, clones producing antibodies to the p polypeptides were isolated. contains a s rna, but it does not show any infectivity even after trypsin treatment. an activation of the / cl virus can be obtained by i) cocultivation of the cl-e- cells with standard cells (e. g. bhk- ) and ii) serial passaging of the / cl virus in several cell lines (e. g. bsc- ). infectious / cl virus can be detected in the cell supernatant after a period of - days and - days respectively. this virus cannot be propagated in chicken eggs, but it can replicate in serial cell cultures without trypsin treatment; moreover trypsin treatment does not influence the viral replicaiton. in comparison / virus released from an in-vitro generated persistent infection (bsc- cells infected with egggrown / standard virus) can be propagated in chicken eggs. this virus termed / pi virus also can grow on serial cell passages without trypsin treatment, whereas the serial replication of / standard virus on cell cultures depends on the trypsin-activation. inst, f. virologie u. immunbiologie, versbacher str. , d- wiirzburg monzel, p. and koschel, k. the paramyxvviruses measles (sspe) virus and canine distemper virus (cdv) cause an impairment of the catecholamine induced p-adrenergic receptor dependent c-amp generation in persistently infected c rat glioma cells. in cdv persistently infected c cells the number of receptors is greatly reduced. hirata and axelrod have shown that the number of ii-adrenergic receptors could be regulated by methylation of phosphatidyl ethanolamine (pe) resulting in lecithin synthesis ( ) . we have therefore studied the methylation of pe in persistently infected cells by the incorporation of ( h) methyl groups from ( h-methyl)-methionine into pe. in both infected systems, c /sspe and c /cdv, we observed a total loss of catecholamine stimulated p-adrenergic receptor dependent methylation whereas the p-receptor independent methylation of phospholipids is unchanged. inst. f. med. virologie d. univ., d- heidelberg; t robert-koch-institut, d- berlin ; inst. f. virusforschung, dkfz, d- heidelberg kurz, w., gelderblom, h.t, flogel, r. m. , and darai, g. a paramyxovirus was isolated from a kidney biopsy of a tupaia (three shrew) and termed (tpv). the detailed host range study revealed that only tupaia embryonic fibroblasts and tupaia kidney cells are the cells of choice for the efficient propagation of tpv. tpv can be plaque-assayed on tupaia embryonic fibroblasts and this cell line was used for the continued propagation of tpv. electron microscopy of purified tpv revealed the presence of typical paramyxovirus particles. -the hemagglutination test was performed with erythrocytes with a variety of different species. it was found that guinea pig erythrocytes were agglutinated with tpv. the buoyant density of purified virions was determined in sucrose gradient and found to be . g x ml", -the biological chara cterization of tpv which was perfo rmed by host range study in vivo revealed that tpv is highly pathogenic for new born mice and hamsters. -the characterization of viral rna and proteins of th is new member of paramyxo viridae is now in progress. coronaviruses contain two glycoprotein species £ ( k) and £ ( k) which are both synthesized in the r£r of the infected host cell. glycosylation of £ is initiated at the cotranslational level and it can be inhibited by -deoxyglucose and tunicamycin indicating the presence of n-glycosidic carbohydra te protein linkages. particles formed in the presen ce of these inh ibit ors are non infectious and lack detect able amounts of £ . -cell fractionation experiments show that glycosylation of glycoprotein £ occur s posttranslationally in smooth memb ranes. the carb ohydrate protein linkages in £ are susceptible to mild alkaline reductive conditions and n-acetylgalactosamine was determined to be the reduc ing sugar of the released oligo saccharides . this together with the finding that glycosylation of £ is not sensitive to inhibito rs of n-glycosylation suggests that glycoprotein in £ of coronaviruses is the first structural virus glycoprotein containing o-glycosidic side chains exclusively. insr, £. virologie, jusrus-liebig-univ., d- giessen target cells of infectious bursal disease virus (ibdv) of chickens moller, h. and becht, h. infectious bursal disease virus (ibdv), the causative agent of a highl y contagious disease of young chickens result ing in severe necrotic lesions in the bur sa of fabricius (gumboro disease), is a non-enveloped icosahedral particle with a diameter of about nm. its genome con sists of segments of double-stranded rna with mol ecular weight s of . x · and . x · daltons. th e virion is composed of structural pol ypeptides with mole cular weights of kd, kd. kd, kd and kd. the kd pol ypeptide, one of the two main structural proteins, is derived from the kd pol ypeptide, perh aps by proteolytic modification ( ) . with immunofluorescence and "i nfectious center assays" we were able to show th at . after infection of isolated lymphoid cells in vitro only ofo of bur sa cells, % of thymus cells and ofo of spleen cells produce infective virus (i, e. plaques in infectious center assays) alth ough the donor chickens were in the most susceptible age of to weeks. . the number of virus producing cells is not cor related with the appe arance of slgm or sigg. . virus yields seem to be influenced by the cell cycle: the number of chick emb ryo fibro blasts producing plaques in "infectious center assays" is increased after synchro nisa tion of the cells before infection. . cells that produce infective virus show an increased uptake of h-thymidine. . isolated lymphoid cells from thymus or spleen as well as blood lymphocytes can be stimulated by mitogens to produce higher virus yields. infection of borna virus without any influence of the immunoresponse. the antigens first appeared in the nucleus of neurons and sometimes fibroblasts, then filled the cytoplasm, most brilliantly days post infection. thereafter they disappeared from the cytoplasm, but remained persistently only in the nucleus in point shape. no morphological changes were seen in the infected cells during days post infection. we can say that the virus does not kill the cell. for the destruction of nerve cells in in vivo conditions it can be pointed to the importance of the immunological events, which might cause the clinical pictures. abr, molekularbiologie, physiol.-chem. lnst., grindelallee , d- hamburg ; heinrich pette-inst., martinistr. , d- hamburg large scale production of biologically active vsv in eat-cells mack, d., kruppa, j., and breindl, m.i ehrlich ascites tumor cells maintained in mice were used to prepare milligram quantities of biologically active vsv. at the th day after passage when the cell number reached approx. x cells/mouse, mice were infected by intraperitoneal injection of appropriate concentrations of vsv. ascites fluid was harvested after h. virus production was exponential for at least h and continued for at least h p.i. -approximately - mg of viral protein/mouse and x pfulmouse were routinely obtained. the specific infectivity of vsv isolated from eat-cells reached nearly . x pfui,ug protein. the endogenous transcriptase activity of vsv produced in eat, bhk, and hela cells showed no significant differences. large amounts of biologically active vsv may be produced rapidly and much less costly with the described procedure than using tissue culture cells. it should be possible to adapt the procedure for the production of other viruses. lonza a.g., ch- basel, schweiz the quantitative determination of bardac- , formaldehyde, glyoxal and glutardialdehyde in disinfectants weinreis, p. and goller, s. several institutes of hygiene have been provided with two disinfected formulations, a and b, with the intention to analyse these mixtures, containing different amounts of quaternary ammonium compounds, formaldehyde, glyoxal and glutardialdehyde, -the quantitative analysis of the components of the two mixtures depends on well known volumetric and spectrophotometric methods. -the results of this analysis have shown that it is possible to use the described procedure for routine check ups of disinfectants without using microbiological tests. proc . nat. acad. sci virolog y ( ) previous studies have shown, that mouse-neurovirulent recombinants can be obtained from mixtures of avirulent influenza a-viruses provided one of the parents had previously been adapted to that host. this study shows that prior adaptation of parental strains is not necessary and that generation of neurovirulent recombinants is frequent. the gene constellation for neurovirulence was predictible for recombinants derived from a particular pair of parental strains in influenza c virus -infected cells a virus-specific protein of a molecular weight of , dalton can be detected when glycosylation is inhibited by tunicamycin. since this protein can not be found in untreated control cells, it probably constitutes the unglycosylated precursor of the viral glycoprotein gp since the petide pattern is identical for the doublet bands, it remains to be established whether this reflects a differential glycosylation or dissimilar proteolytic cleavage sites. the coding capacity of the viral genome rna-segment no abstracts of the th meeting of the oghm augenklinik d. justus-liebig-univ., - giessen; inst. f. neuropathologie u. virologie, freie univ., - berlin retino-cerebral manifestation of experimental borna disease in rhesus monkeys krey, h.t, roggendorf, w. , and ludwig, h. inflammation of the uveal tract, the retina meninges and brain represent uveo-meningoencephalitic syndromes of unknown origin. one of these, the vogt-koyanagi-harada syndrome is primarily manifested by inflammation of the retinal pigment epithelium, the uveal tract and meninges. severe visual and neurological impairment can occur. in our experimental studies rhesus monkeys were experimentally infected with borna disease virus. after a - week incubation period a progressive retino-cerebral syndrome was observed. focal inflammatory lesions in the retinal pigmentepithelium and the uveal tract were accompanied by encephalitic and meningeal infiltrates. infectious virus could be demonstrated in the retina and in the brain. experimental borna disease can serve as an appropriate model for uveo-meningo-encephalitic syndromes in men. borna disease (bo) virus induced encephalitis in horses, sheep, rodents and primates shows similarities in many aspects with other so-called slow virus diseases. the eeg has shown to be a specific tool in studying encephalitides of different types in man. this is a report on the eeg of the bo virus specific encephalitis. twenty-eight rabbits inoculated by different routes and with different virus preparations were screened for eeg changes. the basic frequency was measured optically and an analysis of the eeg was performed. the following conclusions were made: . a significant slowing down of the basic frequency was observed in the eegs of bo virus infected rabbits. . spikes and spike-waves were present rather regularly and correlated with epileptic seizures at the end of the disease when they appeared rhythmically in intervalls of twenty seconds. rademakercomplexes appeared from the third week on. virological and serological data collected from all animals demonstrated a strong correlation of bo virus specific reactions with the eeg alterations. the patterns of eeg changes are reminiscent of those in sspe. eeg features in this kind of slow virus diseases may have rather similar characteristics which could suggest that they may underlie common pathophysiological mechanism. we established the primary cultures of neural retina and pigment epithelium of rabbit and of the brain of chicken embryo to study the sensitivity of each kind of cells to the key: cord- -wdnnjlcw authors: jandrić, petar title: postdigital research in the time of covid- date: - - journal: nan doi: . /s - - - sha: doc_id: cord_uid: wdnnjlcw nan patents' (crowe ) . worldwide closures of schools and universities have pushed millions of students and teachers online, bringing decades of experience in the field under the public eye (bates ) . commentators compare chinese and western responses to the crisis, often under bombastic titles such as 'coronavirus and the clash of civilizations' (maçães ) . political scientists discuss whether the pandemic is an argument for total dismissal of capitalism or just a passing aberration in its functioning (roberts ) . economists advise us to prepare the new recession (elliott ) . sociologists see worldwide border closures as an anti-globalization experiment (peters et al. ) , and philosophers go back to questions pertaining to human nature. worldwide governments are responding in radically different ways-the government of montenegro has closed down the whole country before it registered the first patient within its borders (world health organization b), while the uk has opted for a laissez faire approach which is hoped to result in herd immunity (dunn and kahn ) . from official news to social networks, everyone and anyone has something to contribute to these debates, creating an infodemic which will be analysed long after covid- is gone. as i write these words on march from self-isolation in my flat in zagreb, croatia, the future of the pandemic is unclear. we have no idea what percentage of the global population will be affected by the virus, whether the virus will mutate, how many people the virus might kill, and what might happen with our politics and economy after the pandemic is gone. at this point, we need to develop immediate measures to protect ourselves individually and collectively-weed out reliable information, self-isolate, reduce panic, develop educated guesses and emergency plans. however, these urgent measures cannot arrive from thin air, and it is just as important to step back and take a birds-eye, longue durée view at the pandemic. while doctors, nurses, politicians, food suppliers, and many other brave people self-sacrifice to support our daily survival, this editorial argues that academics have a unique opportunity, and a moral duty, to immediately start conducting in-depth studies of current events. viruses are nanoscale infectious agents (one nanometre is a billionth of one meter). viruses do not have their own cellular structure and cannot naturally reproduce without a host cell. yet viruses do have genes, which evolve by natural selection, and when they enter the host cell, viruses reproduce through self-assembly. looking at different aspects of their existence, viruses can be understood both as an inanimate matter and as a form of life, and 'the question about the origin of viruses and life itself remains for the most part a philosophical debate and largely dealt with theoretical arguments rather than molecular data, especially because viral genomic repertoires are limited and patchy' (nasir et al. : - ) . according to antonio Šiber ( ) , the border between non-life and life lies somewhere at the point when inanimate organic matter becomes soaked with information which enables self-replication and evolution. while it is easy to agree with Šiber's definition, this point is hard to determine and far from agreed upon. viruses are within our bodies and in our environment. over the centuries, viral pandemics such as the spanish flu have been major biological, social, and cultural events. viral behaviour (and some would say viruses) can also be found beyond the organic world. recent examples include viral internet memes and videos, viral marketing, and computer viruses (computer programs which enter host programs, modify them, and replicate themselves). while computer viruses are clearly even further from life than biological viruses, 'some scientists have begun to ask if computer viruses are not a form of artificial life-a self-replicating organism. simply because computer viruses do not exist as organic molecules may not be sufficient reason to dismiss the classification of this form of "vandalware" as a form of life.' (spafford : ) . this argument sits well with a growing number of posthumanist critiques, which suggest that what we should accept as life is largely 'a normative not a descriptive category' (fuller and jandrić : ) . covid- is an organic virus which has caused various sorts of organic and nonorganic viral behaviours in all spheres of (human) biology, culture, and society. the interplay of these behaviours can be approached through the lens of michael peters' viral modernity, which is 'a concept that is based upon the nature of viruses, the ancient and critical role they play in evolution and culture, and the basic application to understanding the role of information and forms of bioinformation in the social world' (peters et al. ). viral diseases have always been intrinsic to human existence. every age has its own viral modernity, and the covid- pandemic is merely the first global exercise of viral modernity in our 'hard to define; messy; unpredictable; digital and analog; technological and non-technological; biological and informational' postdigital reality (jandrić et al. : ) . these days, we can speak of viral education (exemplified in a current global switch to online education), viral post-truth (exemplified in a global covid- infodemic), viral open science (exemplified in exponential growth of open science and associated publications) (see peters et al. ) , and so on. writing these words from home isolation in the midst of the covid- pandemic, it is hard not to overstate the viral nature of, and viral perspective to, our postdigital reality. while the exact relationships between viral modernity and postdigital reality will need to be soberly examined after the heat of the moment is gone, there is no doubt that the covid- pandemic is an extreme postdigital 'rupture and continuation event' (jandrić et al. : ) , and that this event will significantly influence the way we see and experience the world in the foreseeable future. writing these words at the beginning of the global community outbreak phase of the covid- pandemic, i am painfully aware of their ephemeral nature. hopefully, the pandemic could soon wind down; yet it is just as possible that we might be heading towards a large-scale disaster or towards anything in the between. and yet, most of us cannot do much at this stage. being in self-isolation, my 'research' of covid- consists of cooking nice meals, cleaning my flat, endless consummation of the infodemic, frantic exchange of emails with friends all over the globe, and feeble attempts to make sense of what happens. unsurprisingly, that involves a lot of trivia and a bit of humour. reading semi-serious, semi-bitter, semi-hopeful 'predictions' of a possible baby-boom nine months after introduction of a curfew in italy, austria, and spain, my first instinct was to think of all those academics now sitting at home. those of us who teach are now dealing with the complexities of online education, and many of us will also try and catch up with writing that one paper that has always hovered at the bottom of our to-do lists. in the sea of covid- -related speculations, the only prediction i would put my money on is an increased number of paper submissions to academic journals in the months to come. researchers in some areas of medical sciences, biology, economy, logistics, and others, can help people directly affected by the pandemic through the development of diagnostic tools, medicines, and vaccinations; analysing counter-recession measures; increasing efficiency of shipping food and medicine; and the like. however, what happens to people who have not been infected by covid- but have lost their jobs, cannot pay their mortgages, or have become homeless due to economic slowdown? what happens to the most vulnerable members of the society -children, elderly, disabled, those with mental issues? how many indirect victims will the covid- pandemic create? in our context of advanced global capitalism, what should be done to spread the burden of the pandemic at least a bit more equally? and which consequences will the covid- pandemic have in regards to the environment, surveillance, worlwide rise of fascism, democracy? postdigital viral modernity is equally about biology, culture, and society; in the long run, humanity cannot defend itself from covid- and create a better future without engaging all strata of the society. therefore, it is crucial that academic researchers working in the humanities and social sciences immediately join the struggle against the pandemic. in the postdigital context of viral modernity, decades of training and experience in any academic field can contribute to making sense of the crisis. postdigital researchers should read, research, and write about all imaginable aspects of covid- !-even if that research, at present, does not seem to offer much help in getting us through and over the pandemic. the covid- pandemic has brought a huge social experiment into our homes, streets, cities, countries, and globally. outcomes of this social experiment will follow the whole humankind, probably fairly unequally, far into the future. as i write these words, nurses and doctors undertake huge health risks to support our wellbeing. supermarket tellers undertake similar health risks, but receive much less praise, to bring supplies to people who are not (or will not be) allowed to leave their houses. teachers work nights and weekends to develop learning materials and support their online students. people working in many other occupations, pensioners, children, and many others, need to stay at home, watch the news, and follow instructions. none of these roles is less important than the other. while we obviously need food, healthcare, and education, the virus can be contained only through discipline and solidarity of all strata of the society. the humanities and social sciences are already making significant contributions in areas such as informing citizens, prevention of panic, big data analysis, open science, and others. for instance, uk's wellcome trust statement, 'sharing research data and findings relevant to the novel coronavirus (covid- ) outbreak' (wellcome trust ) has enabled unprecedented levels of sharing covid- -related information which have already significantly contributed to development of diagnostic tools, medical procedures, and vaccines (see peters et al. ). while we struggle against immediate threats, however, we should also keep in mind the broader picture. other areas of the humanities and social sciences, which may now seem unrelated to our immediate struggle against the pandemic, are not less crucial for long-term flourishing of the human race. we, postdigital scholars working in the humanities and social sciences, should not take our home isolations and quarantines as unexpected vacations or opportunties to catch up with old projects. instead, we should look into the strengths of our disciplinary knowledges and research methods to try and create opportunities to contribute to humanity's collective struggle against the covid- pandemic and point towards more sustainable futures. some of our current insights will be hasted, and will serve as mere first-hand testimonies for later (and more balanced) research. some of our insights will be picked up only in the next pandemic. some of our insights will be plainly wrong, and consequently retracted. in our current infodemic, the most of our current produce will probably simply remain overlooked and unread. yet some of our insights may raise awareness of important issues, add more nuance to our thinking, and perhaps even influence the course of the pandemic. it is impossible to know which piece of research will end up in the garbage bin of history, which piece of research will make a difference, and when that difference may surface. anne frank's diary did absolutely nothing to stop the second world war, and poor anne had not lived long enough to even see it published. yet seventy years later, anne frank's diary still makes a huge service to humanity by providing a constant reminder of the perils of fascism. wearing my academic researcher hat, i am not ashamed of naivety of this paper-it honestly represents my current thoughts and feelings about the covid- pandemic on march . these thoughts are likely to be overridden by new developments, but they will nevertheless serve as a testimony of this historical moment. wearing my academic editor hat, i am not afraid of publishing papers that might be proven wrong or even retracted-messy and unpredictable postdigital challenges pertaining to viral modernity require messy and unpredictable attempts at answering. wearing my daddy hat, i am admittedly a bit ashamed of withdrawing into the world of research while my son lives through some of the most challenging times in his -year-old life. yet beneath all these hats, there is a head; in this head, there is a mind; and in this mind, there is a tiny, persistent voice that whispers: knowledge and solidarity are the key to long-term survival and flourishing of the human race. i invite all postdigital scholars to take this voice seriously, get out of our comfort zones, and explore all imaginable aspects of this large social experiment that the covid- pandemic has lain down in front of us. in the midst of the pandemic, many of these efforts may seem useless. yet paraphrasing john fitzgerald kennedy ( ) , those who dare to fail miserably are also those who might change the course of history. advice to those about to teach online because of the corona-virus coronavirus epicenter has shifted from china to europe we're opening everything': scientists share coronavirus data in unprecedented way to contain, treat disease. cbc news world health organization declares covid- a 'pandemic even while canceling mass gatherings, the u.k. is still aiming for deliberate 'herd immunity'. fortune, march prepare for the coronavirus global recession. the guardian, march the postdigital human: making the history of the future the chinese doctor who tried to warn others about coronavirus postdigital science and education day of affirmation address coronavirus and the clash of civilizations viral evolution: primordial cellular origins and late adaptation to parasitism viral modernity? epidemics, infodemics, and the 'bioinformational' paradigm. educational philosophy and theory it was the virus that did it mass quarantine effective against coronavirus in china. statista treći element computer viruses as artificial life press release: sharing research data and findings relevant to the novel coronavirus coronavirus disease (covid- ) outbreak key: cord- - wy rpqv authors: denman, a.m. title: viral etiology of polymyositis/dermatomyositis date: - - journal: polymyositis and dermatomyositis doi: . /b - - - - . - sha: doc_id: cord_uid: wy rpqv nan a.m. denman . . . for we all of us, grave or light, get our thoughts entangled in metaphors and act fatally on the strength of them. george eliot, middlemarch any attempt to ascribe polymyositis and dermatomyositis (pm/dm) to a viral etiology must take into account what is already known about the pathogenesis of this group of disorders. first, it is a rare disease, and there are few pointers to a conventional infec tious etiology. outbreaks of the disease have not been described, and the evidence for a seasonal onset is weak. thus, theories based on a viral etiology must invoke a highly un usual host response to ubiquitous agents, the existence of uncommon viruses with the abil ity to provoke the disorder, or the possibility that virus infections operate synergistically with other factors. second, the immunopathologic features of pm/dm strongly indicate the importance of immune mechanisms in the pathogenesis of most forms of the diseases [ ] . thus, advocates of a viral etiology are confronted with the same problems facing anyone seeking to implicate virus infections in autoimmune diseases in general. finally, pm/dm often accompanies a variety of other inflammatory connective tissue diseases, and these associations must also be satisfactorily explained by any theory based on viral infec tion. increasing understanding of the immunopathologic consequences of viral infections means that such points can be countered con- the author would like to thank mrs. kathy jameson for expert help in preparing the manuscript. attempted isolation electron-microscopic studies viral probes antiviral antibody titers establishing clones from infiltrating t lymphocytes and screening their antigen specificities exploring the antigen specificities of associated autoantibodies in vitro models of virus-infected muscle cells in vivo models of experimental polymyositis ceptually. however, one must concede that the evidence to date for a viral etiology is fragmentary. the available approaches for exploring this possibility are set out in table . . in recent years there have been several claims implicating specific virus infections in isolated patients with pm/dm. these reports have of ten been based on direct isolation of the agent or the ultrastructural appearances of the inflamed muscle. coxsackieviruses have at tracted particular attention because of the relative ease with which these agents induce inflammatory disease in cardiac and striated muscle of mice. in addition, coxsackieviruses have been linked with myocarditis in human infants on epidemiologic grounds [ ] . struc tures resembling picornaviruses were de tected by electron microscopy in muscle biopsy from two patients who died with subacute dermatomyositis [ ] . similar particles were seen in the diaphragmatic and intercos tal muscles of an -year-old girl who suc cumbed to a chronic myopathy [ ] . these authors also claimed that muscle suspensions induced cytopathic effects in culture of pri mary human amnion cells. furthermore, the isolate reacted with antibody to coxsackievirus a . other viruses have been the subject of similar reports. mckinlay and mitchell [ ] described eight children with transient myositis associated with elevated serum levels of creatine (ck) following acute virus infec tions. adenovirus type ii was isolated from one patient and a parainfluenza virus from a second patient. pm complicated the clinical course of a patient with undoubted hepatitis caused by the hepatitis b agent since the diag nosis was confirmed by muscle biopsy and marked elevations of the serum ck level [ ] . hepatitis b surface antigen (hb s ag) was de tected by immunofluorescence studies of the intranuclear and intracytoplasmic inclusions in the biopsy specimen of the affected muscle. mikol et al. [ ] have also described inclusion bodies in a muscle biopsy specimen obtained from a patient with long-standing pm; these workers also claimed to have cultured adeno virus type ii from the biopsy. in addition to the foregoing reports there have been several reports based almost exclu sively on ultrastructural appearances (sum marized by schiraldi and iandolo [ ] . very few of these reports have been confirmed by isolating the virus suspected on ultrastruc-tural grounds. nor indeed have there been many attempts to confirm the viral nature of the inclusion bodies by immuno-electron mi croscopic techniques or other specific stain ing methods. indeed, there are dangers in ascribing pm/dm to a viral etiology on the basis of ultrastructural changes in isolation. katsuragi et al. [ ] have emphasized this point by their description of histochemical studies of a mus cle biopsy specimen from an elderly man with pm. some unidentified objects in the biopsy specimens were initially considered to be in clusion bodies resembling picornaviruses but were later shown to have the chemical charac teristics of glycogen. the most striking im pression from these reports is their scattered nature, implicating several different agents. there is a dearth of reports showing that agents can be consistently isolated and pas saged in vitro from patients with pm/dm. nor have sequential studies of immune re sponses been appended indicating that pa tients with pm/dm attributed to a given agent mounted a cell-mediated or humoral immune response to that agent at the time of alleged infection. there are diagnostic difficulties over some reports. myalgia has often been reported in association with acute respiratory tract infec tions, and the symptoms may be accompa nied by electromyographic (emg) abnormal ities [ ] . an epidemic of acute myositis has also been described in children in association with influenza b virus infection [ ] . tran sient elevation of the ck level was noted in these children, and influenza b virus was iso lated from of the patients studied. how ever, the patients made a complete recovery in to days without specific treatment, and it is clear that postviral myositis of this nature is unrelated to persistent pm as generally en countered. interestingly, the symptoms were almost exclusively confined to the gastrocnemius and soleus muscles. acute myositis has also been described after rubella vaccination, although the accompanying clinical and patho logic features suggested that the muscles were involved as part of a more generalized arthus reaction to vaccination [ ] . the muscle dis orders most commonly provoked by viral in fections have usually taken the form of tran sient myalgia, in contrast to the more sustained character of idiopathic pm/dm. furthermore, the muscle disorders provoked by most spe cific infections have also been focal rather than generalized and, therefore, clearly re lated to localized infection [ ] . muscle symptoms after influenza-like illnesses have also been described in older patients [ ] , including objective muscle weakness and minimal emg changes. however, the viral etiology of the antecedent illness was not con firmed, biopsy proof was not obtained, and there was no consistent rise in the serum ck level or the levels of other muscle enzymes. muscle problems in patients with immu nodeficiency deserve separate comment. muscle inflammation attributable to echovirus infection presents a striking picture in patients with hypogammaglobulinemia [ ] . the muscle inflammation is often unilateral, usually localized, and rarely involves the clas sic sites of muscle involvement in idiopathic pm/dm. the virus has usually been isolated from the cerebrospinal fluid rather than from involved muscle [ ] . local abnormalities are revealed by muscle biopsies and consist of perivascular mononuclear infiltrates and muscle fiber atrophy, occasionally accompa nied by extensive interstitial fibrosis. how ever, the disease picture in these unusual cir cumstances has not been shown to have a wider relevance. there are additional reasons for doubting the relevance of most reports of this nature to the pathogenesis of pm/dm as commonly encountered. ultrastructural changes of a kind likely to be caused by virus infection have not been reported in any systematic study of biopsy specimens from patients with pm/dm diagnosed by accepted criteria. fur thermore, the epidemiologic evidence does not support the concept that exposure to common viruses is linked to the pathogenesis of pm/dm. thus, influenza virus causes tran sient myalgia and has also been isolated from the muscle of a patient with myoglobulinuric pm [ ] . yet massive immunization with swine influenza vaccine during a -week period in was not followed by any in crease in the incidence of pm/dm [ ] . most attempts to identify viruses in clini cal material obtained from patients with pm/ dm have depended on the presence of viruses that can either be recovered as fully infectious particles or recognized ultrastructurally. there is always the possibility that defective replication or latent infection might render the host cell the target for an immune re sponse and yet be undetectable by traditional methods. the powerful techniques now avail able for detecting viral nucleic acid sequences offer one obvious means of probing putatively infected muscle samples. this approach has been applied to the study of heart muscle from patients with myocarditis [ ] . coxsackievirus b has been implicated serologically in the etiology of myocarditis, but virus has not been convincingly detected in dis eased muscle by immunofluorescence or iso lated by conventional culture techniques. these authors used a complementary dna (cdna) copy of coxsackievirus b genomic rna as a hybridization probe for identifying virus-specific sequences in myocardial biop sies. they claim to have obtained positive hybridization signals in nine of samples from patients with myocarditis and related disorders. if confirmed and extended, these experiments will obviously be relevant to sim ilar attempts to identify viral sequences in biopsy specimens from patients with pm/dm. one can also speculate that latent infection might be more readily detectable if the in fected host cells could be cultured for periods of sufficient duration. improving techniques for culturing differentiated muscle cells and for inducing the growth and expression of latent viruses makes this an important area for future research. antibody screening has been frequently used as indirect means of obtaining evidence for a viral etiology in pm/dm. antibody titers to coxsackievirus b have received particular at tention because of the many experimental models in which this group of viruses induces myocarditis. this line of research has pro duced some suggestive but not conclusive evi dence for coxsackievirus infection in patients with cardiomyopathies. cambridge et al. [ ] encountered high neutralization titers to coxsackievirus b more commonly in patients with congestive cardiomyopathy than in con trols. moreover, this occurred more com monly in patients with a short history than in patients with a febrile illness at the onset of their symptoms. interestingly, endomyocardial biopsies did not produce evidence of direct viral infection. although myositis and myo carditis occur most commonly as clinically distinguishable entities, cardiac involvement is often an important feature of idiopathic pm/dm. thus, in one detailed study haupt and hutchins [ ] found evidence of active myocarditis in of patients with pm or related diseases. thus it is reasonable to sur vey antiviral titers to this group of viruses in patients with pm/dm in isolation. in a lim ited study of this problem travers et al. [ ] noted a marked rise of antibody titer to a specific serotype of coxsackievirus b in four patients with pm/dm without any rise in titers to other viruses. detailed surveys of this kind have not been reported in pm/dm. simi larly raised antibody titers to these viruses have been detected by complement fixation techniques in a preliminary study of children with juvenile pm/dm [ ] . the most informative studies of this kind have been those that have sought evidence for an igm response to defined coxsackievirus strains, using enzyme-linked immunosorbent assay (elisa) techniques, since this approach is more likely to detect serologic indication of recent infection. el-hagrassy et al. [ ] used elisa techniques to detect coxsackievirus b-specific igm responses in % of patients with acute myocarditis or pericarditis. in other areas of clinical investigation improved elisa assays have also proved more discrimi nating. king et al. [ ] detected igm responses to a single coxsackievirus b strain in % of children aged to years with insulin-dependent diabetes mellitus; their findings also emphasize the important point that it may be easier to identify recent infec tions by defined viral strains in children than in adults since the latter can be expected to have had cumulative exposure to the entire range of these ubiquitous viruses. surveys of antiviral antibody titers of pm/dm have not provided any other useful clues to agents that might be implicated in the pathogenesis of the disorder. occasionally, high antibody titers to specific agents have been described in isolated cases. thus, john and fink [ ] noted a sustained rise in anti body titers to echovirus type in one patient with pm. however, surveys of antiviral anti body titers to common viruses pose several problems in interpretation. first, antibodies if viruses induce pm/dm, the simplest mech anism by which they could induce this disease is by replicating in muscle cells, thereby serv ing as a direct target for an immune attack. furthermore, ephemeral or latent infection of muscle cells could initiate an immune response subsequently sustained by other mechanisms. in a search for myotropic vi ruses, klavinskis et al. [ ] found that two strains of influenza a virus lyrically infected human synctial myotubes. in addition, one strain was found to infect unicellular precur sor myoblasts. these authors showed that viral proteins were synthesized in the infected cells and they also identified viral antigens on the surface of the infected cells by immunofluorescence and immuno-electron microscopy ( fig. . ). there is little information about will be present in the majority of the popula tion. rising antibody titers or the detection of igm antibody against strains recently detected in the community might be helpful, but such surveys are difficult to organize, particularly given the rarity of the disease. positive finding in cases drawn from widely disparate geo graphical areas would need extensive control studies in those same areas. second, antibody titers to endemic viruses do not necessarily imply that those agents are pathogenetically related to the disease. this point is particu larly troublesome if the virus, such as mem bers of the herpes group, persists life-long in the human host; rising antibody titers may simply reflect nonspecific reactivation. finally, atypical infections with unusual clinical con sequences may be encountered in patients with generalized immunodeficiency or impaired immunity specifically to the infecting agent. this immunodeficiency could be reflected in inappropriately low antibody titers; the com plications of echovirus infections in hypogammaglobulinaemic patients vividly illustrate this problem. the extent to which other viruses implicated in the pathogenesis of pm/dm grow in mus cle cells. nor should interactions between viruses and muscle cells be analyzed exclu sively in terms of immune-mediated damage. there is also the possibility that pm/dm in duced by a putative infectious agent could result from a mixture of direct cytopathic changes and immune damage. for example, hemolytic paramyxoviruses induce perme ability changes in infected cells in addition to their ability to mediate cell fusion. patel and pasternak [ ] have described membrane permeability changes in cell lines infected by influenza virus at low ph which persisted when the ph was shifted to the physiologic range. persistent pm/dm is often character ized by degenerative changes in muscle fibers the suspected association between coxsackievirus infections and muscle disorders of man has prompted a particular interest in animal models of myocarditis and pm induced by this group of viruses. moreover, it has proved relatively easy to induce inflammatory disorders of muscle with these agents. coxsackievirus-induced myocarditis has been studied particularly intensively. the factors determining the outcome of such infections are set out in table . . to some extent the outcome of infections by coxsackievirus b is governed by the genes peculiar to the infecting strain. indeed, strains belonging to the same group differ in their ability to grow in host tissues and in their resulting virulence. virulent and avirulent strains may appear identical by criteria such as antigens detected by conventional polyclonal antisera, biophysi cal properties, and genomic structure. further more, even virulent coxsackievirus strains indistinguishable by conventional criteria induce a variety of clinical syndromes, including myocarditis. for example, adoles cent cd-i mice inoculated with one variant of coxsackievirus b developed readily observable myocarditis, whereas a second variant was virtually innocuous [ ] . these variants were indistinguishable in terms of their ability to grow in cell lines in vitro or heart muscle cells in vivo, to stimulate defective particles that might interfere with viral replication, or to stimulate host immune factors such as interferon. however, more stringent analysis may reveal basic differences between virulent and avirulent strains. some of the factors determining the outcome of coxsackievirus b infections are governed by the genes of the infecting strain. prabhakar et al. [ ] showed that monoclonal antibodies can detect differences between strains of virus considered antigenically homogeneous by conventional analysis. these authors used monoclonal antibodies to detect antigeni cally distinct variants of coxsackievirus b . they also found that the rate of antigenic mutation is extremely high in this strain, resembling that long recognized to occur in influenza viruses. further analysis of antigenic variants of coxsackievirus b shows that clinical isolates display a mixture of highly conserved, moderately conserved, and poorly conserved epitopes [ ] . their studies suggested that a high rate of error in rna replication is responsible for these frequent antigenic changes. moreover, this appeared to be independent of any selection pressure. receptor availability? interferon induction in interstitial cells? express la antigens? antibody-all relevant epitopes recognized? cell mediated immune responses: antiviral autoimmune access to myocardium interferon production see text for discussion and references. despite this marked antigenic diversity, all the isolates were neutralized by conventional polyclonal antiserum. although most of the antigenic changes solely detected by mono clonal antibodies may be innocuous, there is also the possibility that these changes deter mine viral tropism for different target cells in vivo and hence their subsequent virulence. however, while there is considerable antigen variation between different strains of coxsackievirus b, repeated passage in intermediate hosts seems to stabilize their properties since there is preferential survival of strains that are tropic for some target cells such as pan creatic islet cells and other endocrine tar get cells. thus, coxsackievirus b repeatedly passaged in mouse pancreata or beta cell cul tures acquired a specific tropism for those cells [ ] . the nature of the host immune response also influences the outcome of experimental coxsackievirus infection. this important point is borne out by all the earlier work on this model (reviewed by woodruff [ ] ). both humoral and cell-mediated immune responses contribute to host defense. in animal models, humoral mechanisms are decisive in limiting primary infection. cell-mediated immunity also contributes to host defense but some times at the price of initiating immunopathologic reactions against virus-infected myocar dial cells. thus cell-mediated responses are a mixed blessing for the infected host. there is good experimental evidence that cellmediated immunity may reduce the virulence of coxsackievirus infections. khatib et al. [ ] found that the virulence of one strain of coxsackievirus b was increased in a strain of swiss mice given antithymocyte serum at the time of infection. in addition, immunosuppressive treatment with cyclophosphamide converted infection by an avirulent strain of b coxsackievirus into one produc ing myocarditis, probably because the virus was thereby allowed to replicate to abnor mally high titers [ ] . however, the dual contribution of viral cytopathic effects and cell-mediated immunity is well illustrated by experiments in which athymic balbc-nu/nu mice were infected with coxsackievirus b [ ] . in the immunodeprived mice, the virus produced mild degenerative or necrotic changes only. however, intense inflammatory changes were induced after the adoptive transfer of spleen cells of immunocompetent nu/+ mice immunized with the same virus. in another athymic strain nu/nu mice could not eliminate infectious virus from their hearts, and this persistent infection was associated with myocardial abnormalities characteristic of cytopathology and also with increased mortality rates [ ] . euthymic nu/+ mice were infected for much shorter periods, yet developed persistent myocardial abnormali ties attributable to the cellular response. these observations emphasize that the myocardial changes are determined partly by viral factors and partly by the character of the t-cell response. the immunopathologic consequences of t-cell immunity often offset any advantage to the host. this outcome is suggested by the frequent presence of mononuclear cell infil trates at sites of coxsackievirus replication in myocytes indicative of myocarditis. in vitro studies of sensitized lymphocytes in coxsackie virus infections emphasize the complexities of cell-mediated immunity to these agents and point to some pathways that may lead to immunopathologic catastrophes. in general, the briskness of the cytotoxic t-cell reaction to infected myocardial cells correlates with the severity of the myocarditis, which sug gests that hypersensitivity to viral antigens contributes to the immunopathologic conse quences of infection. however, there are also important observations that sensitized lym phocytes in coxsackievirus infections are able to kill uninfected myocardial cells and fibroblasts (woodruff [ ] ), and there is now good evidence that this autoreactivity contributes to the myocarditis. in addition, paque et al. [ ] have described experiments in which peritoneal exudate cells from mice infected with coxsackievirus b were tested for their reactivity with cardiac antigens using the mi gration inhibition assay. they found that the migration of these cells was specifically inhib ited by cardiac antigens isolated from mice infected with coxsackievirus b but not by antigens prepared from uninfected hearts or from hearts infected with an unrelated virus. since the cardiac extracts failed to bind virusneutralizing antibodies, they concluded that virus-induced tissue antigens but not virusrelated antigens were responsible for this inhibition. paque et al. have extended these observations to baboons infected with this virus [ ] . in these experiments, sensitivity directed solely at antigens isolated from hearts infected with coxsackievirus was shown by a proliferative response of lympho cyte monitored by the incorporation of tritiated thymidine as well as by inhibition of migration. further evidence for autoimmunity to myocyte antigens is provided by experiments in which spleen cells of mice infected with coxsackievirus b were allowed to adsorb to uninfected or infected myocyte monolayers [ ] . one population of t lymphocytes adsorbed to and lysed uninfected myocytes and were assumed to be autoreactive. in contrast, lymphocytes lysing infected cells were considered to be virus specific. the autoreactive lymphocytes were injected into recipient mice infected with the same virus but deprived of t lymphocytes by prior ex perimental manipulation. the transferred cells induced myocarditis in mice not other wise able to mount a t-cell response to infected myocytes. these observations raise important questions about the nature of the autoantigens, the manner in which auto immune reactions are induced, and the fac tors that dictate their immunopathologic importance. so far, there is little information about the precise nature of the antigens in apparently uninfected heart cells which attract the t-cell response. these might be related to sites of viral transport across cell membranes during the initial infection or during the export of newly synthesized viral particles. nor is it clear whether the response is directed at viral antigens or, as seems more likely, normal host cells whose production or expression is al tered by viral infection. furthermore, while autoreactivity persists beyond the time in which viral persistence can be detected by conventional techniques, there is still the strong possibility that in reality it is sustained by viral persistence in latent or defective form. appropriate experiments with viral probes or with techniques for reactivating vi rus have not yet been reported. the sequence of events by which transient or persistent coxsackievirus infection induces an autoreactive t-cell response also remains largely unexplored. autoreactivity arises early in infection and is more vigorous in female than in male mice. the ability of t cells to gain access to infected myocardial fibers may also influence the outcome of the infection. huber and job [ ] in described two strains of coxsackievirus b type which are indistin guishable in terms of their antigenic proper ties and ability to grow to high titers in myo cardial cells. judged by in vitro techniques these strains are equally efficient in inducing cytotoxic t lymphocytes. however, it is likely that in mice infected by the virulent strain, these cells are able to reach and damage myo cardial cells, whereas lymphocytes in mice infected by the nonvirulent strain lack this ability. however, other crucial pieces of in formation are lacking, such as the expression of viral antigens in infected cells, local inter feron production, the expression of la anti gens by myocardial cells, and the kinetics of the infiltrating cell populations. it is also possible that the extent to which coxsackievirus infection interferes with the re gulation of the immune response also deter mines the severity of the immunopathologic damage. as with other models of experimental virus infection, coxsackieviruses induce sup-it has long been recognized that some strains of echo virus induce pm in mice [ ] . how ever, the pm results from acute necrosis of muscle fibers, and there is little evidence that the pm is mediated by immune mecha nisms. indeed, paralysis only occurs in mice in which viral replication has reached very high titers. thus, the disease is easily induced in newborn mice in which more than pressor t cells which affect immune responses in general and may also influence the character and efficiency of specific antiviral immune re sponses [ ] . deficient suppressor mechan isms could allow persistent autoreactive tcell responses that damage heart fibers even after infectious virus has been eliminated and viral antigens are no longer expressed. there is little evidence to date that coxsackieviruses interfere directly with lym phocyte subpopulations. however, it has been shown that these viruses readily establish per sistent infections in human lymphoid cell lines and particularly in b-cell lines [ ] . only a minority of the cells are infected, hinting that in vivo infection might be difficult to identify if only a minority of circulating lym phocytes are infected. these observations also suggest that the infection may be maintained by mutants of the original infecting virus. the relevance of this experimental model of lym phocyte infection to putative defects in immunoregulation has not yet been established. in addition to specific immune responses, other nonspecific factors influence the out come of experimental coxsackievirus infec tion. thus, reyes et al. [ ] have shown that myocardial damage is accentuated in mice infected with coxsackievirus b that were given forced exercise in the form of daily swimming. observations of this kind rein force the suspicion that excessive physical ac tivity may predispose to myocarditis and pm in clinical practice. % of skeletal muscle tissue is destroyed, older mice rapidly acquiring resistance to this process [ ] . similarly, acute myositis has long been recognized as a consequence of coxsackievirus b in neonatal mice, the dis order resulting from the cytopathic effects of viral growth [ ] . interestingly, even in this model, the myositis is selective for spe cific muscle groups, the hip extensors and hindquarter knee flexors being particularly susceptible. a more persuasive animal model of virusinduced pm is that described by strongwater et al. [ ] . in this model, the intraperitoneal inoculation of the tucson strain of coxsackievirus bl induced proximal hindquarter weak ness that persisted for more than weeks. moreover, the criteria for pm were satisfied by the characteristic emg and histologic changes. furthermore, these changes per sisted long after infectious virus could no the extraordinary diversity of clinical syn dromes associated with the acquired immu nodeficiency syndrome (aids) has alerted clinical investigators to the possibility that demonstrable or covert infection by retroviruses could be etiologically related to pm, or indeed any chronic disorder whose cause has continued to baffle them. however, pm has not emerged as a frequent complication of aids despite the close monitoring of such patients for neurologic disorders. thus, of patients with aids or generalized lymphadenopathy studied at one center between and , only five patients had muscle problems: two had persistent myalgias, two had myopathy, and only one had pm [ ] . more recently, however, two additional pa tients with pm associated with aids were reported [ a] , as discussed in chapter . yet in a primate model of acquired immunodefi ciency, % of monkeys infected with the d retrovirus called saids d showed muscle weakness and wasting, elevated serum con centrations of sarcoplasmic enzymes, and bi opsy features of pm [ ] . the etiologic agent could be transmitted experimentally to healthy rhesus monkeys by tissue homogenates, blood, saliva, and urine. reverse transcriptase assays revealed the presence of virus in muscle biopsies, but immunofluorescent studies with antibody to the agent longer be detected, suggesting that the disor der was mediated by immune mechanisms. infectious virus could only be recovered from the affected muscles for weeks after infec tion, during which period viral antigen could also be detected by immunofluorescence. no tably, electron microscopy did not reveal characteristic viral particle during this period of demonstrable infection. this model has many features in common with human pm and could also explain why it might be diffi cult to demonstrate virus infection in man. showed that the agent was localized to the infiltrating inflammatory cells and interstitial fibroblasts rather than to muscle fibers. inter estingly, the retrovirus replicated in myotubes and fibroblasts in culture but did not have any cytopathic effect on these cells. the predomi nantly immunopathologic features of this model make it an attractive analogue of the human disease. however, generalized immu nodeficiency is not a feature in the vast major ity of patients with pm/dm. nevertheless, these observations emphasize the protean na ture of retroviral infection and indicate that this etiologic possibility should not be lightly discarded. in the past, retroviral infection has been invoked rather simplistically in the pathogenesis of inflammatory connective tis sue diseases, such as pm/dm. it is worth noting that harbers et al. [ ] microinjected cloned retroviral dna into mouse zygotes and found that virus-specific rna was subse quently expressed at different concentrations in the organs of infected mice. in one mouse, retrovirus specific information was expressed at far higher concentrations in muscle than in other tissues. while such viruses can be ac quired by horizontal infection, it is also possi ble that any involvement of retroviruses in the pathogenesis of pm might be more complicated. the pathogenesis of autoimmune diseases in general continues to be keenly debated and frequently reviewed [ ] . viruses are com monly invoked in most schemata albeit in concert with genetic and immunologic factors [ ] . it is instructive to apply these general hypotheses to pm/dm in order to see how viruses could invoke this autoimmune disease and the extent to which there is supporting data ( table . ). it has long been a popular notion that virus infection might induce autoantigens and thereby make infected cells targets for auto immune attack. there is in fact little evidence that viruses induce novel antigens or alter the structure or expression of normal antigens. nevertheless, there is good evidence that vi rus infection affects the recognition of surface antigens by the immune system, thereby in creasing the possibility that these will induce an immune response. classically, lindenmann and klein [ ] in found that influenza virus infection of target cells increased the immunogenicity of host cell antigens expressed by the infected cells. the mechanisms of this phenomenon have been extensively studied. for example, it has clearly been shown that vaccinia virus infection of tumor cells increases cytotoxic t-cell and antibody responses against the in fected cells [ ] . there are also several models of experimental viral infection that provoke autoimmune responses against the infected organs, with pathologic consequences. mice infected with reovirus type i develop both transient diabetes and a runting syndrome [ ] . the infected mice produce serum autoantibodies reactive with cytoplasmic an tigens in beta cells of the pancreatic islets of langerhans, the anterior pituitary gland, and gastric mucosa. additional autoantibodies are generated against both insulin and growth hormone, and serum concentrations of these hormones are abnormally low. another perti nent example is provided by experimental in fection with the jhm strain of murine coronaviruses, which is neurotropic and which induces either acute or subacute encephalomyelitis, depending on viral and host genetic factors [ ] . subacute encephalomyelitis is determined not only by the antiviral immune response but also by an autoimmune reaction to brain antigens, including myelin. an important issue in such models is the role of viral replication in the target cells. in the reovirus model of experimental autoim mune endocrine disease it seems likely that virus in the target organs is mandatory since reovirus type , which does not infect the anterior pituitary gland, does not induce autoantibodies to growth hormone. how ever, the period of virus infection may be transitory since the autoimmune process per sists long after local infection can readily be detected. moreover, an immunopathologic basis for the syndrome seems certain, since it can be prevented by immunosuppressive treatment with antilymphocyte serum [ ] , the extent to which persistent infection of brain cells by jhm virus is essential for the subacute demyelinating disease in susceptible hosts is still debatable. one possible mechanism by which tran sient virus infection of target cells may induce a persistent t-cell response is the inappropri ate expression of la (class , or dr) antigens by the host cell. the expression of such anti gens has mainly been attributed to -interferon released by infiltrating t lymphocytes. in par ticular, this mechanism has been invoked to explain autoimmune disorders of the thyroid gland [ ] . however, more recently in the jhm-induced model of demyelinating encephalomyelitis, it has been shown that viral particles directly induce la antigen expres sion by astrocytes [ ] . moreover, the extent of this interferon production is likely to be genetically controlled. since both viral and host cell antigens are likely to be presented to the immune system in association with la an tigens, it is also evident that genetic factors control the generation of cytotoxic t cells directed at both sets of antigenic determinants. there is so far little evidence that similar mechanisms operate in pm. in particular, there is no information about the steps that initiate the infiltration of muscles by lympho cytes. analysis of the infiltrating cells by immunocytochemical techniques has shown that the majority of the infiltrating cells are t lymphocytes bearing antigen determinants characteristic of activated lymphocytes [ ] . furthermore, these cells predominate in un treated acute rather than chronic disease [ ] . interferons have also been detected in muscle biopsies [ ] . however, the antigens against which these cells react have not been charac-viral etiology of polymyositis terized. it is possible that after isolation and expansion by cloning techniques the infiltrat ing t cells may prove specifically reactive to muscle cells, using the kind of assays that cambridge and stern [ ] have shown are true of circulating blood lymphocytes in pm. cellular infiltrates resembling those en countered in pm/dm are also provoked by exercise, emphasizing that these do not pro vide specific clues to the nature of the local initiating event, infectious or otherwise [ a] . antimyosin antibodies have been detected by radioimmunoassay in % of sera from pa tients with pm [ ] , but the nature of the stimuli provoking these autoantibodies is un known. there have been claims that the stain ing pattern of autoantibodies in patients with acute myocarditis can distinguish between patients with preceding coxsackie b infection and those with other etiologies, but these an tibodies were present in low titer, and their reactivity showed considerable overlap with antigens in other tissues [ ] . it is purely conjectural that transient infec tion of muscle cells initiates the immunopathologic events of pm/dm. however, many vi ruses are able to replicate in vascular endothelial cells [ ] . these cells could serve as sites for the initial growth of viruses implicated in the pathogenesis of pm/dm. indeed, it has been shown that -interferon induces la ex pression on cultured human vascular endothelial cells and dermal fibroblasts [ ] , fur thermore, ashida, johnson, and lipsky [ ] have shown that these cells function as antigen-presenting cells in model systems. it is feasible, therefore, that muscle fibers become secondarily inflamed as the result of changes in the microenvironment of local blood ves sels and that persistent local immune responses are thereby established. indeed, fully developed lymphoid follicles have been noted as a histologic feature of pm/dm [ ] , it is customary to invoke genetic factors to account for the rarity of such events. how ever, in contrast to the strong hla associa tions with most organ-specific autoimmune diseases, there is only weak evidence for such associations in pm/dm [ ] . claimed associ ations with hla-b and hla-dr in both adult and juvenile dermatomyositis are not compelling and have been observed in rela tively small numbers of patients. the specificities of the autoantibodies characteristic of pm/dm have been invoked as evidence for a possible viral etiology for this group of diseases. several authors, nota bly plotz [ ] and bernstein et al. [ ] , have stressed that the autoantibodies associated with inflammatory connective tissue diseases, including pm/dm, are directed at a relatively small number of cellular components. these autoantigens have been fairly well character ized as proteins, often present in complexes with additional proteins or nucleic acid mole cules. the antinucleoprotein autoantibody peculiar to many connective tissue diseases reacts with small ribonucleoproteins (rnps) in which the antigens are commonly the small nuclear rna species, ul, u , u , u , or u [ ] . in addition, the antibodies characteristic of pm/dm commonly react with cytoplasmic antigens, notably aminoacyl-trna synthetases. these antigens are associated with mechanisms of protein synthesis [ ] . in the first stage of protein synthesis, rnapolymerases catalyze the formation of messenger rna complementary to the dna sequence. this messenger specifies the sequence in which amino acids are assembled in the na scent polypeptide chain. the amino acids are initially attached to trna molecules before their polymerization into polypeptides. a specific set of enzymes termed aminoacyl-trna synthetases couple each amino acid to its matching trna. anti-la autoantibody reacts with precursor forms of ribosomal rna and trnas for five different amino acids [ ] . the transcription of these rna sequences is controlled by rna polymerase iii. similarly, anti-ro autoantibodies precipi tate rnps containing rnas whose transcrip tion is also dependent on polymerase iii [ ] . the jo- antigen is a protein-rna complex incorporating the synthetase needed to com plex histidine with the corresponding trna [ ] , moreover, anti-jo autoantibody blocks the incorporation of histidine into nascent proteins, a reaction that depends upon this enzyme. since these protein synthetic steps are also utilized in the synthesis and assembly of some virus particles, it is conceivable that these autoantibodies might represent an im mune response by the host aimed at disrupt ing viral synthesis. anti-la antibody precipi tates not only cellular rnps but also rnp complexes containing rnas involved in the synthesis of adenovirus and epstein-barr vi rus (ebv) [ ] . many plant viral rnas and a few animal viral rnas contain a trna-like structure at their free end which may utilize trna synthetases to regulate viral assembly [ ] , equally, these autoantibodies might be directed at enzymes and proteins involved in protein synthesis which have been rendered autoantigenic by the transcription of viral se quences [ ] . this mechanism is analogous to that likely to explain lupus-like syndromes provoked by drugs such as hydralazine. however, it is only an assumption that the antigen specificities of the autoantibodies hint at a viral etiology. these autoantibodies may simply reflect an autoimmune response to a spectrum of antigens common to many tissues regardless of the provoking cause. similarly, there is an obvious analogy with experimental reovirus infection in that the autoantibodies might determine patterns of tissue damage but be of little help in determin ing the nature of the initial insult. a similar dilemma arises from the convinc ing observations that the sera of patients with autoimmune connective tissue diseases con tain antibodies to antigens coded by retroviruses. rucheton et al. [ ] have shown that sera from patients with anti-rnp and anti-la autoantibodies react with several viral polypep tides coded by the viral gag gene. how ever, as the authors emphasize, these antibod ies could be of secondary importance, simply reflecting the nonspecific activation of endo genous viral antigens rather than a response to exogenous retroviral infection. the advent of monoclonal antibodies has provided a powerful means of testing the long-held belief that autoimmune diseases may arise from cross-reactions between mi croorganisms and tissue antigens. molecular mimicry of this kind can now be sought by direct experiment and by computer tech niques comparing known microbial and tis sue antigenic sequences. these techniques have been used to test the idea that molecular mimicry between viruses and muscle antigens might account for the continued immune re sponses characteristic of pm/dm. srinivasappa et al. [ ] screened monoclonal antibodies to different viruses for their reactivity with different organs from normal mice. they found that . % of the antiviral antibodies reacted with specific cells in these organs and that several of the antibodies reacted with antigens in more than one organ. however, no cross reactions with muscle-specific anti gens were uncovered in this survey or referred to in the accompanying review of similar searches. immunofluorescence studies have revealed a monoclonal antibody to coxsackievirus b that reacts exclusively with a bands in the myofibrils of myocardial muscle, al though only one of antibodies tested showed this pattern of reactivity [ ] . walker and jeffrey [ ] adopted a different strategy by using a computer alignment procedure to seek microbial sequences comparable with amino acid sequences of histidyl-trna synthetase and alanyl-trna synthetase since these are known to be the target molecules for autoantibody responses in pm/dm. close matches were discovered between histidyl-trna synthetase and protein sequences of ebv. similarly, of the closest matches with alanyl-trna synthetase, more than half were with viral proteins, including those coded by ebv, some influenza virus hemagagglutinins, and a protein coded by adenovirus . these studies also uncovered sequence similarities with the myosin light chain tropomyosin and the skin component keratin. however, as em phasized by the authors, the trna synthetases are universally distributed, and it is diffi cult therefore to understand why skeletal muscle fibers should be a specific target organ in this disease. furthermore, sequence homologies may be entirely fortuitous or result when part of a cellular protein is incorporated into viral protein during the course of viral replication. furthermore, similarities in se quence do not take into account conformational differences which may in reality deter mine the ability of an antibody to fit a given epitope [ ] . moreover, regions of high mo bility in a protein also determine antibody binding so that comparison of sequences can never replace the need for experimentally studying the ability of an antibody which reacts with one structure to react with an other that appears to mimic it [ ] . it is also important to test experimentally the idea that any observed cross-reactivity accounts for persistent autoimmune reactions. thus fujinami and oldstone [ ] in reported sequence homology between amino acid se quences in myelin basic protein and hepatitis b virus polymerase; lymphocytes from rab bits immunized with the viral sequence pro duced a proliferative response in vitro to chal lenge with basic myelin protein and also developed histologic changes reminiscent of experimental allergic encephalomyelitis. it is now accepted that antibodies to novel sequences in the variable portion of the anti body molecule (idiotypes) contribute to the regulation of antibody production. further more, since there are sequence similarities be tween the provoking antigen, receptors for those antigens on cell surfaces, and the idiotype, anti-idiotype antibodies might be expected to recognize antigen receptors. this pathway for inducing autoantibodies reactive with cell membrane antigens has been recog nized as a possible source for autoimmune diseases and, indeed, anti-idiotype autoanti bodies have been shown to have immunopathologic consequences in some experimen tal models [ , ] . the complexities of possible derange ments of the idiotype network have been in creased by the recent demonstration that a human monoclonal antibody reactive with a membrane protein of multiple organs con tains an idiotype which in turn stimulates an anti-idiotype antibody, and that an antibody raised against this anti-idiotypic antibody it self recognizes the protein determinant that initiated the whole chain of events [ ] . ago nized readers are referred to ancient chain songs dealing with kids brought to market. hybridoma techniques have revealed monoclonal antibodies that react with epitopes shared by multiple organs and tis sues. one such epitope in sle is the phosphodiester bond [ ] , while monoclonal auto antibodies reactive with pituitary, thyroid, pancreas, and stomach have also been de scribed [ ] . antibodies to these idiotypes have been produced experimentally and used to screen the sera of patients with autoim mune diseases for antibodies bearing these idiotypes. if autoimmune diseases are pro voked by a persistent autoantibody response to idiotypes, one might predict that the sera of patients with autoimmune diseases would contain a higher concentration of antibodies with this idiotype than do sera of normal individuals. however, essani et al. [ ] found that idiotypic markers thought to be related to autoimmune diseases are expressed in normal individuals and that antibodies bearing these markers do not necessarily bind to autoantigens. similarly, the number of circulating b lymphocytes reactive with anti-idiotype antibody was identical in patients with autoimmune diseases and in controls. similar observations showing the public nature of autoantibody idiotypes have been made by other authors [ ] . thus far theories about the pathogenesis of autoim mune diseases based on generation of antiidiotype antibodies have not provided any etiologic clues in autoimmune diseases in gen eral. with respect to pm/dm, those autoreactive antibodies whose idiotypes have been analyzed do not react with muscle. however, the idiotypes of muscle-reactive antibodies in pm/dm have not been examined. there are also caveats concerning the idea that analyz ing autoantibody idiotypes might further im plicate viruses that are putatively involved in the etiology of pm. mcclintock et al. [ ] in reported that anti-idiotypic antibodies raised to mouse monoclonal antibodies reac tive with independent epitopes on coxsackievirus b did not block the binding of this virus to cells bearing appropriate receptors. the original studies of mice with an autoim mune polyendocrine disorder provoked by infection with reovirus type revealed that spleen cells fused with a myeloma cell line produced monoclonal antibodies reactive with cells in the anterior pituitary gland, pan creas, small intestine, and stomach [ ] . ad sorption studies on antigen affinity columns confirmed that the antibody was indeed re acting with antigens common to the different target organs. it subsequently became evident that cells producing monoclonal antibodies with the same repertoire of multiple organ reactivity can be isolated from the spleens of normal mice [ ] . similarly, dighiero et al. [ ] have established hybridomas with spleen cells of normal mice synthesizing monoclonal autoantibody reactive with myosin, doublestranded dna, actin, tubulin, and spectrin. in clinical studies, ebv transformed blood lymphocytes from both normal individuals and patients with autoimmune diseases pro duced monoclonal autoantibodies reactive with antigens in multiple organs [ ] . the target antigens were detected in thyroid, pan creas, stomach, stratified squamous epithe lium, and nerve axons. the reactions could only be detected by the highly sensitive avidin-biotin-immunoperoxidase assay. fur thermore, this group [ ] used the same tech nique of screening ebv-transformed b cells to reveal that b cells from patients with autoim mune diseases produce monoclonal antibod ies reactive with both pancreas and thyroid. evidently b cells in patients with autoimmune disease synthesizing serologically recogniz able autoantibodies may also synthesize autoantibodies reactive with different organs which are undetectable by standard tech niques. thus the association of pm/dm with other autoimmune diseases could result from the simultaneous production of muscle-re active autoantibodies that have not yet been characterized. it also follows that pm/dm, like other autoimmune diseases, could result from the inappropriate activation of b cells already programmed to react with muscle an tigens. the multiple ways in which this acti vation could be achieved include the bypass ing of suppressor t-cell mechanisms and the inappropriate presentation of muscle-specific antigens to b cells by t cells reacting with surface antigens expressed by muscle fibers [ ] . viral infections could provide the ini tiating signal; experimentally bromberg et al. [ ] have shown that herpes simplex, new castle disease, and vaccinia viruses can induce immune responses against membrane anti gens that are normally weakly immunogenic. there is only meager evidence to support the alternative possibility that viruses putatively inducing pm/dm latently infect suppressor cells, thereby leading to their functional abla tion. hollingworth et al. [ ] showed that herpes simplex virus failed to replicate in pha-stimulated blood lymphocytes from pa tients with pm/dm, suggesting that some cells might harbor virus particles that inter fered with the growth of the challenge virus. since viral infection of a minority of circulat ing lymphocytes has marked immunodepressive effects [ ] , this possibility can not be lightly discarded. it has recently been shown that measles virus has a strong tropism for lymphocytes and that continued infection of these cells may contribute to the pathogenesis of the persistent viral disease subacute sclerosing panencephalitis [ ] . pm/dm commonly occurs in association with other inflammatory connective tissue diseases in which autoimmune reactions are promi nent. thus, theories attributing autoimmune diseases to the uncontrolled proliferation of autoreactive lymphocyte clones are also ger mane to the pathogenesis of pm/dm. classic ally, the emergence of such clones has been considered to result from the failure of regula tory mechanisms that normally lead to their elimination. alternatively, mutations in the progenitor cells for autoimmune reactive lymphocytes might lead to their autonomous proliferation in a manner independent of con ventional antigenic stimulation. indeed, the proliferation of autoreactive cells has often been considered intermediate between a con-ventional immune response and overt malig nant proliferation. theoretically it is now possible to test the strength of such hypothe ses by comparing the characteristics of autoantibody secreting cells with frankly malig nant lymphoma cells (table . ). there is so far no evidence that autoreactive cells origi nate from a single progenitor cell in a manner analogous with the b cells of patients with chronic myeloid leukemia; the latter point has been clearly established in black female patients with the disease who are heterozy gous for the enzyme glucose- -phosphate hydrogenase [ ] . nor have gene rearrange ments of the kind observed in neoplastic lymphoid cells [ ] been detected in the genes coding for the antigen receptors of autoreac tive t cells or the immunoglobulin coding genes of autoreactive b cells. gene probes for t-cell receptors have been used to document the maturation of mutant cells lacking hypoxanthine-guanine phosphoribosyltransferase (hprt) activity [ ] . furthermore, assays for rearrangements of genes coding for t-cell antigen receptors have provided insight into a clinically benign but biologically lymphoproliferative disorder, lymphomatoid papulosis [ ] . immunoglobulin synthesis involves a se ries of differentiation steps in which the prod- these mutations usually involve a sin gle base substitution, but it is not known whether these are entirely spontaneous or de pendent on antigenic stimulation. similarly, there is a high rate of mutation in the genes coding for the variable portions of the beta chain of the antigen receptor of t cell al though at a lower rate than that occurring in genes coding for the variable portion of the immunoglobulin molecule. thus, classic the ories postulating that autoimmune diseases arise from mutation in antibody synthesizing clones must be reconciled with observations showing that mutation is a physiologic step in the generation of antibody diversity. further more, such theories must explain the circum stances that confer continuous and appar ently autonomous proliferation on such clones. certainly a single somatic mutation can result in the production of an antibody with autoantibody specificity [ ] , but anal ogous mutations in human autoantibodyproducing cells have not yet been described. in malignant lymphomas, substantial evi dence has been adduced implicating the inap- propriate activation, mutation, or translocation of oncogenes in abnormal b-cell proliferation; moreover, many of these mod els postulate that these transforming events are initiated or enhanced by exogenous viral infection. there is as yet no evidence that similar events govern the proliferation of autoreactive b cells in connective tissue dis eases. the activation of the c-myc gene in sle, for example, seems simply to reflect the extent of b-cell proliferation rather than an unusual event initiating this proliferation [ ] . nevertheless, the wealth of interaction observed between exogenous infectious agents and the deregulation of myc-genes in b cell tumours [ ] means that such ideas should not be lightly discarded in other lymphoproliferative disorders. b-cell differentiation and maturation involves multiple steps, each of which is governed by interactions with ex ternal ligands, such as b cells and their prod ucts, activated components of the comple ment sequence, and other as yet unknown factors. it is noteworthy that the b-cell hyperplasia characteristic of mouse strains prone to spontaneous autoimmune disease is deregu- there is so far little evidence that pm/dm is caused directly or indirectly by viral infec tions. conventional methods have failed to isolate viruses in the vast majority of cases and serologic surveys have proved uncon vincing. the case for continuing to seek a viral etiology rests mainly on animal models. myocarditis and pm induced by coxsackievirus infection is mediated by immunopathologic mechanisms that may continue to dam age muscle fibers long after infectious virus and even viral antigens have been eliminated. clearly, if analogous mechanisms operate in human disease, it will prove extremely diffi cult to identify the provoking agent by the time the disease is clinically apparent. fur thermore, the combination of viral and host genetic factors necessary to induce muscle in flammation may be extremely rare and im-lated at one or more points in their matura tion [ ] . there is little information about the detailed regulation of human b cells syn thesizing conventional or autoantibodies. however, the introduction of improved tech niques for propagating human b cells in cul ture should remedy this situation. there is evidence, for example, that lupus b cells spon taneously synthesize b-cell growth factors and are refractory to exogenous growth fac tors [ ] . the failure of normal repair processes may increase the possibility for recombination events between immunoglobulin-coding genes and genes related to controlled proliferation. this mechanism has also been invoked as a factor in mouse strains susceptible to plasmacytoma induction [ ] , and it is note worthy that lymphocytes from patients with pm/dm are abnormally susceptible in vitro to irradiation, ultraviolet irradiation, and the mutagen methylnitrosourea [ ] . in general, the relevance of such concepts to the pathogenesis of pm/dm has not been experimen tally tested. possible to define by orthodox biologic and immunologic techniques. indeed, the succes sive steps leading to autoimmune inflamma tion of muscle in pm/dm or inflammation of organs in other autoimmune diseases are tortuous to unravel even when the disease is deliberately incited. the hope of progress lies in improved techniques for isolating, expand ing, and characterizing the effector cells from the blood and local lesions of patients with this group of disorders. the association of pm/dm with other auto immune diseases encourages the belief that genetically determined mutations in t or b lymphocytes may be responsible for the dis order. if pm/dm results from the quasi-malig nant proliferation of autoreactive lymphocyte clones, such hypotheses can only be tested by detailed analysis of the multiple steps leading to the emergence of such clones. such analo gies imply that viruses might not act by simple cause and effect. instead, it is possible that they might act at several stages in the emer gence of such clones. viruses could promote recombinational events between transform ing genes and genes coding for immunoglobu-lin or t-cell receptor sequences. alternative ly, viruses could act by overcoming restric tions to lymphocyte proliferation normally imposed by a variety of regulatory ligands. however, overall, the stimulus for further in vestigating a viral etiology for p m / d m still rests on faith and metaphors. immunological features of polymyositis/dermatomyositis viral myocarditis picornavirus-like crystals in subacute polymyositis chronic myopathy associ ated with coxsackievirus type a : a com bined electron microscopical and viral iso lation study transient acute myositis in childhood hepatitis b antigen and polymyositis inclusion-body myositis: clinicopathological studies and isolation of an adenovirus type from muscle biopsy speci men polymyositis ac companying coxsackie virus b infection piconavirus-like inclusions in polymyosi tis: aggregation of glycogen particles of the same size myo sitis and acute respiratory infection acute myositis associated with influ enza b infection vasculitis and myositis sec ondary to rubella vaccination infectious myositis and related syndromes benign postinfection polymyositis echovirus disease in hypogammaglobulinemic patients persistent and fatal central nervous system echovirus infections in patients with agammaglobulinemia isolation of influenza virus from muscle in myoglobinuric polymyositis lack of asso ciation of a/nj/ (swine flu) vaccine and polymyositis detection of coxsackie-bvirus-specific rna sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy antibodies to coxsackie b viruses in congestive car diomyopathy the heart and cardiac conduction system in polymyositis-dermatomyositis: a clinicopathologic study of autopsied patients coxsackie b neutralisation titres in polymyositis/dermatomyositis antibody to coxsackie-b virus increased incidence in sera from children with recently diagnosed juve nile dermatomyositis coxsackie b virus specific igm re sponses in patients with cardiac and other diseases coxsackie-b-virus-specific igm responses in children with insulindependent (juvenile-onset: type i) diabetes mellitus myositis, myoglobinemia and myoglobinuria associated with enterovirus echo infection infection of cultured human mus cle cells by influenza virus permeability changes elicited by influenza and sendai viruses: separation of fusion and leakage by ph-jump experiments properties of coxsackievirus b variants which are amyocarditic or myocarditic for mice high frequency of antigenic variants among naturally occurring cox sackie b virus isolates identified by monoclonal antibodies detection of conserved and nonconserved epitopes on coxsackievirus b: frequency of antigenic change virus-induced diabetes mellitus: glucose abnormalities produced in mice by the six members of the coxsackie b virus group alterations in coxsackievirus b heart muscle disease in icr swiss mice by antithymocyte serum the role of t lymphocytes in hepathogenesis of coxsackievirus b heart disease cocksackievirus b persistence and myocarditis in n:nih(s) ii nu/nu and +/nu mice assessment of cell-medi ated hypersensitivity against coxsackie virus b viral-induced myocarditis utiliz ing hypertonic salt extracts of cardiac tis sue assess ment of cell-mediated immunity against coxsackievirus b -induced myocarditis in a primate model {papio papioic) coxsackievirus b- myocarditis in balb/c mice: evidence for autoimmunity to myocyte antigens differences in cytolytic t cell response of balb/c mice infected with myocarditic and non-myocarditic strains of coxsackievirus group b, type impairment of immunocompetent mouse spleen cell func tions by infection with coxsackie virus b group b coxsackieviruses readily establish persistent infections in human lymphoid cell lines a mouse model of dilated-type cardiomyopathy due to coxsackievirus b factors determining pathogenicity of variants of echo virus for newborn mice age dependence of paralysis in duced by echovirus type in infant mice selec tive polymyositis induced by coxsackie virus bl in mice a murine model of polymyo sitis induced by coxsackievirus bl (tucson strain) neurological manifestations of the acquired immunodeficiency syndrome (aids): ex perience of ucsf and review of the litera ture polymyositis in patients with aids polymyositis in an immunodefi ciency disease in monkeys induced by a type d retrovirus microinjection of cloned retroviral genomes into mouse zygotes: integration and expression in the animal the autoimmune diseases virus-induced autoimmunity viral oncolysis: increased immunogenicity of host cell anti gen associated with influenza virus the augmentation of tumour-specific immu nity by virus help. i. demonstration of vac cinia virus-reactive helper t cell activity involved in enhanced induction of cytotoxic t lymphocyte and antibody responses virusinduced diabetes mellitus xx polyendocrinopathy and autoimmunity adoptive transfer of ere-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis virus-induced dia betes mellitus: autoimmunity and polyendocrine disease prevented by immunosuppression epithelial cells expressing aber rant mhc class ii determinants can pres ent antigen to cloned human t cells viral particles induce la antigen expression on astrocytes characterization of polymyosi tis infiltrates using monoclonal antibodies to human leukocyte antigens monoclonal antibodies to human leuko cyte antigens in polymyositis and muscular dystrophy localization of interfer ons and interleukin w in polymyositis and muscular dystrophy the uptake of tritium-labelled carnitine by monolayer cultures of human fetal muscle and its po tential as a label in cytotoxicity studies cellu lar infiltrates in human skeletal muscle: exercise-induced damage as a model for inflammatory muscle disease radioimmunoassay for antibodies to human skeletal muscle myosin in serum from patients with polymyositis diagnostic rele vance of humoral and cell-mediated im mune reactions in patients with acute viral myocarditis virus infection of endothelial cells lymphocytes recognize human vascular endothelial and dermal fibroblast la anti gens induced by recombinant immune in terferon hu man endothelial cell-lymphocyte interac tion polymyositis with infiltration by lymphoid follicles autoimmune diseases of muscle hypothesis: viruses and autoantibodies. autoantibodies and anti-idiotype antibodies to anti-viral antibodies cellular protein and rna antigens in auto immune disease precursor molecules of both human s ribosomal rna and trans fer rnas bound by a cellular protein reac tive with anti-la lupus antibodies basic genetic mechanism genes for two small cytoplasmic ro rnas are adjacent and ap pear to be single-copy in the human ge nome myositis autoantibody inhibits histidyl-trna synthetase: a model for autoimmunity possible functional role of viral trna-like struc tures presence of circulating antibodies against gag-gene mulv proteins in patients with autoim mune connective tissue disorders molecular mimicry: frequency of reac tivity of monoclonal antiviral antibodies with normal tissues monoclonal antibody to coxsackievirus b reacts with myocardium polymyositis and molecular mimicry: a mechanism of auto immunity on the use of se quence homologies to predict protein structure: identical pentapeptides can have completely different conformations which structural features determine protein antigenicity? amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity idiotypes and autoimmunity multiple organ-reactive igg antibody induced by an antiidiotypic antibody to a human mono clonal igm autoantibody polyspecificity of monoclonal lupus auto antibodies produced by human-human hybridomas human multiple-organ reactive mono clonal autoantibody recognizes growth hormone and a , molecular weight protein anti-idiotypic antibodies against a human multiple organreactive autoantibody: detection of idiotopes in normal individuals and patients with autoimmune diseases anti-idiotypic antibodies to mono clonal antibodies that neutralize coxsackievirus b, do not recognize viral receptors multiple organ-reactive monoclonal autoantibodies lymphocytes capable of mak ing monoclonal autoantibodies that react with multiple organs are a common feature of the normal b cell repertoire murine hybridomas secreting natural monoclonal antibodies reacting with selfantigens epstein-barr virus-transformed lympho cytes produce monoclonal autoantibodies that react with antigens in multiple organs hu man monoclonal autoantibodies that react with both pancreatic islets and thyroid mecha nisms of autoimmunity: a role for crossreactive idiotypes viral antigen act as helper determinants for anti body responses to cell surface antigens effects of immunosuppression on herpes simplex virus growth in lymphocytes of patients with connective tissue diseases interactions of viruses with the immune system detection of measles virus rna in lym phocytes from peripheral-blood and brain perivascular infiltrates of patients with subacute sclerosing panencephalitis chronic myelocytic leuke mia origin of some lymphocytes from leukemic stem cells rearrangement of genes for the antigen receptor on t cells as mark ers of lineage and clonality in human lymphoid neoplasms use of t-cell receptor gene probes to quantify the in vivo hprt mutations of human t-lymphocytes clonal t-cell populations in lymphomatoid papulosis: evidence of a lympho-proliferative origin for a clinically benign disease somatic muta tion of the t- heavy chain gives rise to an antibody with autoantibody specificity induction of c-myc expression early in the course of b-cell activation: studies in normal humans and patients with systemic lupus erythematosus mechanisms in b-cell neoplasia spontaneous production of b cell growth factors by sle lymphocytes defective repair of o -methylguanine in autoimmune diseases key: cord- - tionxun authors: fenner, frank; mcauslan, b.r.; mims, c.a.; sambrook, j.; white, david o. title: the nature and classification of animal viruses date: - - journal: the biology of animal viruses doi: . /b - - - - . - sha: doc_id: cord_uid: tionxun nan virology began as a branch of c h apt er pathology, the study of disease. at the end of the nineteenth century, when the microbial the nature and classification etiology of many infectious disof animal viruses eases had been established, pathologists recognized that there since been discovered, but two still apply: (a) unlike even the smallest microorganisms (chlamydiae), viruses contain no functional ribosomes or other cellular organelles, and (b) in rna viruses the whole of the genetic information is encoded in rna, a situation unique in biology. other distinctions apply to some but not all viruses, e.g., the isolated nucleic acid of viruses of several genera is infectious (i.e., the virus can be generated intracellularly from a single molecule of nucleic acid), and viruses of most genera contain either no virus-coded enzymes, or one or more enzymes that belong to particular classes (neuraminidases and nucleic acid polymerases). it is impossible to define viruses satisfactorily in a sentence or even a paragraph, bearing in mind both their intracellular states and the extracellular particles or virions. virions consist of a genome of either dna or rna enclosed within a protective coat of protein molecules, some of which may be associated with carbohydrates or lipids of cellular origin. in the vegetative state and as "provirus" (see chapter ), viruses may be reduced to their constituent genomes, and the simplest "viruses" may be transmitted from one host to another as naked molecules of nucleic acid, possibly associated with certain cellular components. at the other extreme, the largest animal viruses, e.g., the poxviruses and the leukoviruses, are relatively complex. lwoff's concept that "viruses are viruses" has had important theoretical and practical consequences; on the one hand, it emphasized their similarities irrespective of the nature of the host (animal, plant or bacterium), and, on the other hand, it led to the possibility of freeing viruses from the rules of bacteriological nomenclature. however, the operational division of viruses made according to type of host continues to be used by the majority of virologists most of the time, and it is significant that the international committee on nomenclature of viruses (icnv), although dedicated to a universal classification, operates through subcommittees on bacterial, invertebrate, plant, and vertebrate viruses (wildy, ). the simpler viruses consist of nucleic acid and a few polypeptides specified by it. more complex viruses usually also contain lipids and carbohydrates; in the great majority of viral genera these chemical components are not specified by the viral genome but are derived from the cells in which the viruses multiply. in exceptional situations, cellular nucleic acids or polypeptides may be built into viral particles. viruses contain only a single species of nucleic acid, which may be dna or rna. viral nucleic acid may be single-or double-stranded, the viral genome may consist of one or several molecules of nucleic acid, and if the genome consists of a single molecule this may be linear or have a circular configuration. as yet, no animal viral nucleic acid has been found to be methylated, or to contain novel bases of the type encountered in bacterial viruses or mammalian transfer rna's, but some virions contain oligonucleotides rich in adenylate, of unknown function. the base composition of dna from animal viruses covers a far wider range than that of the vertebrates, for the guanine plus cytosine (g+c) content of different viruses varies from to %, compared with to % for all chordates. indeed, the g+c content of the dna of viruses of one genus (herpesvirus) ranges from to %. the molecular weights of the dna's of different animal viruses varies from just over to about million daltons; the range of molecular weights of viral rna's is much less, from just over to about million daltons. the nucleic acid can be extracted from viral particles with detergents or phenol. the released molecules are often fragile but the isolated nucleic acid of viruses belonging to certain genera is infectious. in other cases, the isolated nucleic acid is not infectious even though it contains all the necessary genetic information, for its transcription depends upon a virion-associated transcriptase without which multiplication cannot proceed. all dna viruses have genomes that consist of a single molecule of nucleic acid, but the genomes of many rna viruses consist of several different molecules, which are probably loosely linked together in the virion. in viruses whose genome consists of single-stranded nucleic acid, the viral nucleic acid is either the "positive" strand (in rna viruses, equivalent to messenger rna) or the "negative" (complementary) strand. preparations of some viruses with genomes of single-stranded dna consist of particles that contain either the positive or the complementary strand. viral preparations often contain some particles with an atypical content of nucleic acid. host-cell dna is found in some papovaviruses, and what appear to be cellular ribosomes in some arenaviruses. several copies of the complete viral genome may be enclosed within a single particle (as in paramyxoviruses) or viral particles may be formed that contain no nucleic acid ("empty" particles) or that have an incomplete genome, lacking part of the nucleic acid that is needed for infectivity. terminal redundancy occurs in the dna of some vertebrate viruses, but most sequences are unique. the largest viral genomes contain several hundred genes, while the smallest carry only sufficient information to code for about half a dozen proteins, most of which are structural proteins of the virion. the major constituent of the virion is protein, whose primary role is to provide the viral nucleic acid with a protective coat. as predicted by crick and watson ( ) , from a consideration of the limited amount of genetic information carried by viruses, the protein shells of the simpler viruses consist of repeating protein subunits. sometimes the viral protein comprises only one sort of polypeptide chain, although, more commonly, there are two or three different polypeptides. the proteins on the surface of the virion have a special affinity for complementary receptors present on the surface of susceptible cells. they also contain the antigenic determinants that are responsible for the production of protective antibodies by the infected animal. viral polypeptides are quite large, with molecular weights in the range , - , daltons. the smaller polypeptides are often but not always internal, the larger ones often but not always external. there are no distinctive features about the amino acid composition of the structural polypeptides of the virion, except that those intimately associated with viral nucleic acid in the "core" of some icosahedral viruses are often relatively rich in arginine. viral envelopes usually originate from the cellular plasma membrane from which the original cellular proteins have been totally displaced by viral peplomers and a viral "membrane protein" (see fig. - ) . the peplomers consist of repeating units of one or two glycoproteins, the polypeptide moiety of which is virus-specified while the carbohydrate is added by cellular transferases. in many enveloped viruses, the inside of the viral envelope is lined by a viral protein called the membrane or matrix protein. not all structural viral proteins are primary gene products, since with many viruses the viral mrna is translated into a large polypeptide that is enzymatically cleaved to yield two or more smaller virion proteins. cleavage is often one of the terminal events in the assembly of the virion and it can occur in situ after most of the proteins are already in place. although most virion polypeptides have a structural role some have enzymatic activity. many viruses contain a few molecules of an internal protein that functions as a transcriptase, one of the two kinds of peplomers in the envelope of myxoviruses has neuraminidase activity, and a variety of other enzymes are found in the virions of the larger, more complex viruses. in addition to polypeptides that occur as part of the virion, a large part of the viral genome (most of it, with the large dna viruses) codes for polypeptides that have a functional role during viral multiplication but are not incorporated into viral particles. few of these "nonstructural viral proteins" have been characterized. except for the large and complex poxviruses, which constitute a special case, lipid and carbohydrate are found only in viral envelopes and are always of cellular origin. the lipids of viral envelopes are characteristic of the cell of origin, though minor differences between the viral envelope and the normal plasma membrane may be demonstrable. about to % of the lipid is phospholipid and most of the remainder ( - %) is cholesterol. some of the viral carbohydrate occurs in the envelope as glycolipid characteristic of the cell of origin, but most of it is part of the glycoprotein peplomers that project from the viral envelope. during the years that followed the introduction of negative staining for the electron microscopic study of viruses (brenner and home, ), a general the structure of animal viruses picture was obtained of the structure of representatives of most of the groups of animal viruses that were known at the time (review: home and wildy, ). three structural classes were distinguished: isometric particles, which were usually "naked" but in some groups were enclosed within a lipoprotein envelope; long tubular nucleoprotein structures, always (with viruses of vertebrates) surrounded by a lipoprotein envelope; and in a few groups, a more complex structure. accepting a number of new terms defined by lwoff et al. ( a) , caspar and his colleagues analyzed the principles underlying the structure of simple viruses (review: caspar, ). their basic concepts remain valid, but subsequent work has rendered some of the original definitions ambiguous; where necessary these have been modified. virion (plural virions) is used as a synonym for "virus particle." the protein coat of an isometric particle or the elongated protein tube of viruses with helical symmetry is called the capsid. it may be "naked," or it may be enclosed within a lipoprotein envelope (peplos) which is derived from cellular membranes as the virus matures by budding. where the capsids directly enclose the viral nucleic acid, as is usual with tubular capsids but less common with isometric capsids, the complex is called the nucleocapsid. with most isometric particles and in all complex virions, the capsid encloses another protein structure containing the viral genome, called the core. capsids consist of repeating units of one or a small number of protein molecules. three levels of complexity can be distinguished. chemical units, the ultimate gene products, are single polypeptides that may themselves constitute the structural units, or several polypeptides may form homo-or heteropolymers which constitute the structural units. the structural units, or groups of them, may be visualized in the electron micrographs as morphological units. morphological units that form part of a capsid are called capsomers; those projecting from the envelope are the peplomers (sometimes called "spikes," an unsatisfactory term since they are never pointed and may, indeed, have knob-shaped ends). the chemical units are sometimes held together by disulfide bonds to form the structural units, hence the practice of using reducing agents in polyacrylamide gel electrophoresis when analyzing viral proteins to determine their constituent polypeptides. the structural units are held together to form the capsid by noncovalent bonds, which may be polar (salt and hydrogen bonds) or nonpolar (van der waals and hydrophobic bonds). the capsids of some viruses are readily disrupted in molar calcium or sodium chloride, suggesting electrovalent bonds between the structural units; others are unaffected by salt and can only be disrupted by detergents, suggesting that they are hydrophobically bonded. it has been found that the isometric virus particles that have been adequately studied by x-ray diffraction and electron microscopy have capsids in which the capsomers are arranged with icosahedral symmetry. according to caspar and (b) . the capsids consist of morphological suhunits called capsomers, which are in turn composed of structural suhunits that consist of one or more chemical suhunits (polypeptide chains). many icosahedral viruses have a "core" (not illustrated), which consists of protein(s) directly associated with the nucleic acid, inside the icosahedral capsid. in viruses of type b the envelope is a complex structure consisting of an inner virus-specified protein shell (membrane protein, made up of structural suhunits), a lipid layer derived from cellular lipids, and one or more types of morphological subunits (peplomers), each of which consists of one or more virus-specified glycoproteins (modified from caspar et ah, ) . klug ( ) , this occurs because the icosahedron is that polyhedron with cubic symmetry which, if constructed of identical subunits, would least distort the subunits or the bonds between them. a n icosahedron ( fig. - ) has equilateral triangular faces, vertices, where the corners of triangles meet, and edges, where the sides of adjacent pairs of triangles meet. it shows twofold symmetry about an axis through the center of each edge ( fig. - a) , threefold symmetry w h e n rotated around an axis through the center of each triangular face ( fig. - b) , and fivefold symmetry about an axis through each vertex ( fig. - c ). each triangular face may be thought of as containing, and being defined by, three asymmetric units (i.e., units that have no regular symmetry axes themselves) so that a minimum of sixty asymmetric units are required to construct an icosahedron. the triangular faces of an icosahedron can be subdivided into smaller identical equilateral triangles, to form a solid called an icosadeltahedron. only certain subdivisions are possible; the number of new triangles per facet is called the triangulation number (t), and t = h + hk + k , where h and k are any pair of integers. when h = k (t - , , , etc.), or when either h or k = (t = , , , , etc.), the triangles are arranged symmetrically on the underlying icosahedral face, but with other values for h and k (e.g., h = , k = and t = ) they are in a skew arrangement. a complete description then requires determination of the hand of the structure (right-dextro or left-zevo). the hand of the icosahedral shells of some papilloma viruses has been investigated by klug and finch ( ) and finch and klug ( ) , who concluded that the human papilloma virus (human wart virus) had a t = d icosahedral surface lattice whereas rabbit papilloma virus had a t = l lattice. in an icosadeltahedron with a triangulation number of , each icosahedral face has and the whole solid x = asymmetric units. these structural units may differ in shape and clustering so that the morphological units (capsomers) visible by electron microscopy may differ greatly in viruses with the same triangulation number. there are three basic types of clustering pattern: . the three units defining each triangular face may cluster at the center of the triangle, forming trimer capsomers ( fig. - d ). . the structural units may cluster at the vertices of the triangles, so that where five triangles meet at the vertices of the icosahedron there are pentamer capsomers, and where six triangles meet on faces of the icosadeltahedron there are hexamer capsomers ( fig. - e) . . pairs of structural units from adjacent triangles may cluster on the edges between the triangle to give dimer capsomers ( fig. - f ). the pattern seen on the surface of the virion need not reflect the way in which the structural units are bonded together, and gives no clue as to whether the structural units are constituted by single chemical units or are homo-or heteropolymers of the chemical units. however, the number of structural units in each capsomer can be guessed at from the arrangement and size of the capsomers ( fig. - ) . all known animal viruses whose genome is dna have isometric (or complex) capsids, as do all those whose genome is double-stranded rna and the viruses of two major families (picornaviridae and togaviridae) whose genome consists of a single molecule of single-stranded rna. tobacco mosaic virus occupies a unique position in virology. not only was it the agent whose "viral" nature was first appreciated (beijerinck, ), and the first virus to be crystallized (stanley, ), but more is known of its physical and chemical structure than any other virus (reviews: caspar, ; kaper, ). the virus particles are nonenveloped straight rods, which consist of repeating polypeptide (chemical) units, which are the structural units and also, without clustering, constitute the capsomers. these protein molecules are arranged in a helical manner so that except at the ends of the particles every capsomer is in a structurally equivalent position in relation to the long axis of the rods. many plant viruses and a few bacteriophages have similar nonenveloped tubular virions, their capsomers being arranged in helices whose pitch is characteristic for the virus group. such viruses are structurally defined by their length and width, the pitch of the helix, and the number of capsomers in each turn of the helix. tubular nucleocapsids are found in many groups of viruses of vertebrates, but only among those whose genome consists of single-stranded rna. none of these occurs as "naked" virions; the flexuous helical tubes are always inside lipoprotein envelopes. the diameters of the nucleocapsids of several viruses have been measured, but in only a few cases is the length or the pitch of the helix known. the best studied example, the nucleocapsid of sendai virus, a paramyxovirus, is a helix about /xm long and nm wide, with a pitch of . nm (finch and gibbs, ) . there are about hourglass-shaped structural units in the nucleocapsid, with either eleven or thirteen units per turn of the helix. the structural units are single polypeptides with a molecular weight of about , daltons, arranged with their long axes at an angle of about ° to the long axis of the nucleocapsid, which therefore has a herringbone appearance in electron the structure of animal viruses micrographs (see plate - ). the contact surface between adjacent turns of the basic helix is conical, so that contact is maintained even when the nucleocapsid is sharply flexed, and the viral rna is thus protected. unlike cells, which contain several different species of nucleic acid that subserve different functions, the only nucleic acid in viruses, apart from small amounts of host frna in leukoviruses, is their genome. it may consist of either dna or rna, it may be single-or double-stranded, it may be linear or cyclic, and the genome may consist of one or several molecules of nucleic acid. although the only detailed studies have been made on a few plant and bacterial viruses (review: tikchonenko, ), it is clear that the interaction between the viral nucleic acid and the capsomers is different in nucleocapsids with helical and icosahedral symmetry. in tobacco mosaic virus, there is a maximum regular interaction between the single strand of viral rna and the protein subunits which form a protective coat around it. a similar relationship probably exists in most animal viruses with tubular nucleocapsids, but in some viruses (e.g., influenza virus) the integrity of the tubular structure is destroyed by treatment with rnase but not by proteases, suggesting a different relationship of rna and protein. in icosahedral viruses, on the other hand, there can be no such regular relationship of the nucleic acid and each polypeptide subunit. in the simplest isometric viruses, the folding of the flexible single-stranded rna may have some regularity in relation to the capsomers and their constituent chemical subunits. x-ray diffraction studies of turnip yellow mosaic virus (klug et al., ), for example, show that a significant portion of the single rna chain is deeply embedded within the protein shell, large segments being intimately associated with the structural units, which as hexamers and pentamers make up the capsomers. the presence of the rna in and about these positions enhances the definition of capsomers seen in electron micrographs (finch and klug, ) . except for some togaviruses, even the simple isometric viruses of vertebrates have a more complex structure than this, since they contain several virus-coded poly pep tides. one or more of these poly pep tides are known to be "internal" to the capsid and it is thought that these rather than the capsomers interact with the viral rna. reovirus particles have two concentric protein shells, each consisting of well-defined morphological units. the proteins of the larger dna viruses are arranged in several layers, not all of which display symmetry. the internal proteins of many dna viruses are highly basic and are thought to be bonded to the viral nucleic acid, constituting a core within the isometric capsid. although occasionally used in a more general way to refer to the outer viral coats of some complex viruses like the poxviruses (mitchiner, ), we think that it is desirable to restrict the use of the term "envelope" to the outer lipoprotein coat of viruses that mature by budding through cellular membranes. enveloped viruses contain - % of lipid, all of which is found in the envelope. chemical analyses show that the lipid is derived from the cellular membranes through which the virus matures by budding, but all the polypeptides of viral envelopes are virus-specified. herpesvirus is the only virus of vertebrates that matures by budding through the nuclear membrane, and its envelope contains several virus-specified glycoproteins. all other enveloped viruses bud through cytoplasmic membranes, and contain one or more different polypeptides. the togaviridae have an isometric core to which a lipid layer is directly applied, and virus-specified glycoprotein peplomers project from this. all animal viruses with tubular nucleocapsids are enveloped, and in these the lipid layer from which glycoprotein peplomers project is probably applied to a protein shell (the membrane protein; see fig. - ), which may be relatively rigid, as in rhabdovirus, or readily distorted (as in the myxoviruses) so that in negatively stained electron micrographs the virions appear to be pleomorphic. viruses that have large genomes have a correspondingly complex structure. apart from the undetermined nature of the "cores" of many of the isometric viruses (e.g., herpesvirus and adenovirus), the virions of the two largest animal viruses (poxvirus and iridovirus) have highly complex structures, which are described in the appropriate sections of chapter . the rna viruses that have the largest (single-stranded) genomes, those of the leukovirus genus, also have a highly complex structure with an envelope enclosing an icosahedral capsid that, in turn, surrounds a tubular nucleocapsid. the aim of classification in biology is to make an ordered arrangement of a particular class of biological objects that will indicate their similarities and differences. adoption of a system of classification also involves consideration of the nomenclature of the objects to be classified. linnaeus introduced a latinized binomial nomenclature into biology years ago, and phylogenetic classifications of animals and plants based on the theory of evolution have since been introduced. international codes of nomenclature with rigid sets of rules, and judicial commissions to pass judgement on proposed names, have been set up for the naming of plants and of animals. an international code of nomenclature of bacteria and viruses was approved in and has since been revised (buchanan et ah, ). although they are primarily concerned with nomenclature, all these codes involve agreement upon a system of classification. codes are based on "acceptances," i.e., beliefs we would like to justify but are unable to prove, the principal one being that we are able to arrange living things in an orderly system that is indicative of both rank in a hierarchy and phylogenetic relationships (cowan, ) . classifications of animals and plants attempt to be scientific by deriving their taxa from a consideration of phylogenetic relatedness. more recently this approach has been reinforced by tests for genetic relatedness, i.e., the information content of the genetic material of the agents concerned. this has been tested by homology experiments with dna's ex- tracted from the cells of a variety of animals (mccarthy, ) , and it is to be expected that the phylogenetic and the molecular biological approaches will eventually be combined. the classification of bacteria into the same hierarchical pattern as that of plants and animals (phyla, subphyla, classes, orders, suborders, families, genera, and species) has led to a chaotic situation (cowan, ) . some bacterial taxonomists are looking to numerical methods, readily exploited with the aid of electronic computers, for the solution of their problems (sneath, ) . disadvantages of this approach are that the weighting of characters tends to be involuntary, and that pleiotropism may lead to some characters being scored more than once. most virologists believe that certain characters of viruses, such as the type, amount, and conformation of the viral nucleic acid, are taxonomically more important than characters like host range or pathogenic potential. molecular biology provides an alternative to phylogenetic relationships for making a scientific classification of microorganisms, viz., by the determination of genetic relatedness, using both the genetic material and the polypeptides that it specifies (mandel, ) . there are two groups of agents, the mycoplasmas and the viruses, for which detailed "official" classifications are still in the process of formation. because of their small genomes, they are particularly suitable for molecular taxonomy, i.e., classification based on the molecular weights and base ratios of their genomes, and on the results of nucleic acid hybridization experiments. applied to mycoplasmas, this approach has disproved claims that these microorganisms were derived from certain bacterial species (razin, ) . nucleic acid hybridization experiments have now been performed with many different viruses; detailed references to the results obtained will be given in chapter . in general, they have provided some useful data on relationships within genera and species, but not at higher taxonomic levels. with the methods used thus far many viruses now allocated to the same genus have shown little or no homology of their nucleic acids. indeed, nucleic acid hybridization may be too critical a method to be useful except for the comparison of closely related viruses, and less exacting tests for the similarity of viral genomes may be more pertinent when considering different viral species. bellett ( a,b) analyzed the data available in on the molecular weights and base ratios of the nucleic acids of different viruses. his results on the "clustering" of the viruses of vertebrates are consistent with the genera proposed by the icnv (wildy, ). however, newer knowledge about the fragmented nature of the genomes of some rna viruses and of the varied modes of their transcription and translation (baltimore, b) suggests that these data, where available, should be added to the parameters used by bellett. the differences between the molecular weights of the dna's of the dna viruses of vertebrates are such that sophisticated analysis is not needed to define the currently accepted families and genera. until about , little was known about viruses other than their pathogenic behavior. most early proposals for viral classification were confined to either plant or animal viruses and were based mainly upon the symptomatology of diseases caused by them, which tended to classify the host responses rather than the viruses. bawden ( ) made the pioneering suggestion that viral nomenclature and classification should be based upon properties of the virus particle. in the early 's bawden's approach was exploited by animal virologists (andrewes, ), and viruses were allocated to groups which were usually given latinized names constructed from a chosen prefix plus the word "virus." thus, myxovirus (andrewes et ah, ) group, ) , and adenovirus (pereira et ah, ) groups were described. in the meantime, a classification using quite different criteria had been established by epidemiologists. since they were so concerned with the transmission of infection, epidemiologists have used a classification based on the mode of transmission of disease; they have grouped viruses together as "respiratory viruses," "enteric viruses," or "arthropod-borne (arbo-) viruses." the last term, in particular, has been widely used, but it is generally agreed that this epidemiological classification, although useful is in no sense taxonomic. concurrently with these suggestions relating to the viruses of vertebrates, lwoff ( ) insisted upon the similarities between viruses, whatever their natural host, and the differences between viruses and all other biological entities. he was instrumental in arranging for the establishment of an international committee (anon., ; lwoff and tournier, ) to discuss nomenclature. its major proposal was to select "type species" upon which names for groups would be based. it also proposed a classification based on (a) the chemical nature of the nucleic acid, (b) the symmetry of the nucleocapsid (helical, cubical, or binal), (c) the presence or absence of an envelope, and (d) certain measurements: for helical viruses, the diameter of the nucleocapsid, for cubical viruses, the triangulation number and the number of capsomers. the official international committee on nomenclature of viruses (icnv), which was set up at the ninth international congress for microbiology in , adopted the physicochemical criteria of lwoff and tournier, but rejected the detailed hierarchical classification. the more important nomenclatural proposals accepted by icnv were: (a) an "effort should be made" toward a latinized binomial system of nomenclature, (b) the "law of priority" is unacceptable, (c) no taxon should be named from a person, and (d) anagrams, siglas, hybrids of names, and nonsense names should be prohibited. before describing the classification of animal viruses that we shall use throughout this book, it is appropriate to consider some of the problems of classification and nomenclature that have not yet been tackled by icnv. one of the most important is the level of taxa that should be used. so far, only three families of animal viruses have been accepted (see below), but it is clear that large and heterogeneous groups currently classed as genera (e.g., poxvirus, herpesvirus, paramyxovirus, leukovirus; wildy, ) should be regarded as families. indeed, it would not be unreasonable to regard all the currently accepted isolated "genera" as families, some of which (e.g., adenovirus) might at this a classification of animal viruses stage contain only a single genus. the conventional physicochemical criteria [(a) nucleic acid: type, strandedness, fragmentation, and molecular weight; (b) virion: shape, size, and symmetry] are suitable for classification at this level of family/genus, perhaps assisted by the serological cross-reactivity of "group" antigens where these have been recognized. at the other end of the nomenclatural spectrum, there is hopeless confusion in the ways in which the terms "species," "type," "subtype," and "strain" are used. for example, "types" of influenza virus exhibit no serological crossreactivity and their nucleic acids do not hybridize; they should be regarded at least as distinct species. on the other hand, many alphaviruses and flaviviruses with distinct names, which exhibit extensive serological cross-reactivity, should perhaps be regarded as types within the same species. serological cross-reactivity and nucleic acid hybridization tests are probably most useful for making comparisons at this "species" level. the icnv, working under the chairmanship of professor p. wildy, presented its first report at the tenth international congress for microbiology in mexico city in , and has published valuable basic data on forty-three viral groups encompassing viruses of bacteria, invertebrates, plants, and vertebrates (wildy, ) . in spite of the problems referred to above, we shall follow the classification set out in the report, amplifying it with proposals that have come forward since then, but maintaining accepted usages of the terms "type," "strain," etc. only two families were established by icnv in (wildy, ): papovaviridae and picornaviridae, and subsequently the family togaviridae was defined. most accepted "groups" of vertebrate viruses were given generic names. no species names were adopted by icnv, although "t> ~>e species" were designated for several of the genera. the family papovaviridae (sigla: pa = papilloma; po = poly oma; va = vacuolating agent, sv ) encompasses two genera, polyomavirus (poly = many; oma = tumor) and papillomavirus (papilla ^nipple; oma = tumor), which differ substantially in size and nucleic acid content of the virion (table - ) but share many other properties. an important property of many papovaviruses is their capacity of produce tumors. in nature, some produce single benign tumors (which may undergo malignant change) and are highly host specific ; others may cause primary malignant tumors within a short period of their inoculation into newborn rodents. the adenoviruses (adeno = gland) are nonenveloped icosahedral dna viruses which multiply in the nuclei of infected cells, where they may produce a crystalline array of particles. many serological types have been isolated from human sources. these have an antigen that is shared by all mammalian strains, but differs from the corresponding antigen of avian strains. allocation to the genus is made primarily on the basis of the characteristic size and symmetry of the virion as seen in electron micrographs (icosahedron with capsomers). most adenoviruses are associated with respiratory infection and many such infections are characterized by prolonged latency. some multiply in the intestinal tract and are recovered in feces. many adenoviruses, from both mammalian and avian sources, produce malignant tumors when inoculated into newborn hamsters. in the laboratory, stable hybrids have been produced between certain adenoviruses and the polyomavirus, sv (see chapter ). the herpesviruses (herpes = creeping) are readily recognized by their morphology. their icosahedral capsid is assembled in the nucleus and acquires an envelope as the virus matures by budding through the nuclear membrane. electron microscopic examination by negative staining of many previously unclassified viruses showed that several of them had large icosahedral capsids with capsomers enclosed within lipoprotein envelopes, similar to the type species, herpes simplex virus. when examined further, such viruses were found to be dna viruses that multiplied in the nucleus, and have now been included in the genus herpesvirus. table - shows some of the viruses now regarded as members of this genus; a more complete list is given by andrewes and pereira ( ). there is a group-specific antigen(s) associated with the nucleocapsids and demonstrable by immunodiffusion, and several type-specific antigens associated with the nucleocapsid and envelope. some type-specific antigens crossreact (e.g., herpes simplex viruses type and type and b virus). different herpesviruses cause a wide variety of types of infectious diseases, some localized and some generalized, often with a vesicular rash. a feature of many herpesvirus infections is prolonged latency associated with one or more episodes of recurrent clinical disease. this genus (irido = iridescent) was defined on the basis of several viruses of insects whose structure and nucleic acid content have been carefully studied . nature and classification of animal viruses (bellett, ; wrigley, ) . several dna viruses of vertebrates that are similar in morphology and certain other characteristics have been tentatively grouped with the genus iridovirus (table - ) . like poxviruses, but unlike other dna viruses, iridoviruses multiply in the cytoplasm. their dna consists of a single linear molecule, with a molecular weight of about - million daltons, and the virion is a large and complex nonenveloped icosahedron, with an outer shell composed of about capsomers. the vertebrate ''iridoviruses" may be enveloped. several enzymes are found within mature virions. the best studied of the vertebrate "iridoviruses" are some viruses of frogs, notably fv (review, granoff, ); the most important economically is african swine fever virus (review, hess, ). the poxviruses (pock = pustule) are the largest animal viruses, and contain a larger amount of dna ( - million daltons of double-stranded dna) than any other virus. the structure of the brick-shaped virion is complex, consisting of a biconcave dna-containing core surrounded by several membranes of viral origin. there is a poxvirus group antigen which is probably an internal component of the virion, and can be demonstrated by complement fixation or gel diffusion tests. several enzymes, including a transcriptase, are found within mature virions. multiplication occurs in the cytoplasm and the virions mature in cytoplasmic foci. occasionally, the virion may be released within a loose membrane derived from the cytoplasmic membrane. this is not essential for infectivity, and must be distinguished from the envelope of viruses that mature by budding through cellular membranes. the genus is divided into several subgenera (table - ) , and there are several poxviruses that have still to be classified. the properties outlined for the genus apply to all the subgenera, except that the virions of members of the subgenera b and c (see table - ), and swinepox virus, are narrower than those of other poxviruses, and virions of subgenus b (orf) have a distinctive surface structure. species within each subgenus show a high degree of serological cross-reactivity by neutralization as well as complement fixation tests. genetic recombination occurs within, but not between, subgenera; nongenetic reactivation (complementation) occurs between most poxviruses of vertebrates (chapter ). certain viruses that multiply in insects have many of the attributes of poxviruses and have been tentatively called entomopoxviruses (entomo = insect) (review: bergoin and dales, ). poxviruses cause diseases in man, domestic and wild mammals, and birds. these are sometimes associated with single or multiple benign tumors of the skin, but are more usually generalized infections, often with a widespread vesiculo-pustular rash. several poxviruses are transmitted in nature by arthropods acting as mechanical vectors. the viruses of rodents cause acute fulminating disease when inoculated into newborn hamsters (kilham, ) . the adenovirus-associated viruses are not known to cause any symptoms. the picornavirus group (sigla: pico = small; rna = ribonucleic acid), which includes a very large number of viruses, was accepted by icnv as a family, picornaviridae, with three genera: enterovirus (entero = intestine), rhinovirus (rhino = nose), and calicivirus (calici = cup). newman et al. ( ) believe that this subdivision of the family is unduly restrictive; on the basis of particle density, base composition of the viral rna's, and stability at various phs they differentiate cardioviruses (cardio = heart) from enterovirus, and foot-and-mouth disease virus and "equine rhinovirus" from the genus rhinovirus (table - primarily inhabitants of the intestines, and a large number of serotypes have been found in the feces of man and of various animals. the enteroviruses of man have been subdivided into three major subgroups : poliovirus, three serotypes; echovirus (acronym: echo = enteric cytopathogenic /luman orphan), thirty-four serotypes; and coxsackievirus (coxsackie = town in new york state), twenty-four serotypes of type a and six of type b. the polioviruses, which show some serological cross-reactivity, are distinguished by their capacity to paralyze humans. coxsackieviruses were originally defined in terms of their capacity to multiply in infant mice, but subsequently some echoviruses were found to do the same. it has been recommended that all future en-short descriptions of the major groups of rna viruses teroviruses that are discovered should be numbered sequentially from , irrespective of subgroups (rosen et al., ) . most infections with enteroviruses are inapparent, a few are associated with gastrointestinal disorders, and some may cause generalized infections with rash, central nervous system involvement, including poliomyelitis and aseptic meningitis, or specific damage to the heart. genus: rhinovirus [r/ : . - . / : s/s: v/ ]. the rhinoviruses resemble the enteroviruses in several characteristics but they are acid labile (ph ) and have a buoyant density (in cscl) of . - . g/cm . most have a low ceiling temperature of growth and are characteristically found in the upper respiratory tract of man and various animals. there are a large number of different serotypes of human rhinoviruses, and there are several serotypes of foot-and-mouth disease virus, which resemble rhinoviruses in some respects, but not in others (table - ) . most rhinoviruses cause mild localized infections of the upper respiratory tract, but foot-and-mouth disease virus causes a severe generalized disease with rash in cattle. during the last quarter century intensive world-wide efforts have been made to recover viruses which would multiply in both arthropods and vertebrates, and some different agents with these biological properties are now known. they have been called "arthropod-borne viruses," a name which was shortened to "arborviruses" and then (in order to avoid the connotation of "tree") to "arboviruses." the arboviruses have been defined, on epidemiological grounds (mode of transmission), as a group comparable to the "respiratory viruses." arboviruses are viruses which, in nature, can infect arthropods that ingest infected vertebrate blood, can multiply in the arthropod tissues, and can then be transmitted by bite to susceptible vertebrates (world health organ., ). for many years arboviruses have been recovered from vertebrate tissues and suspensions of arthropods by the intracerebral inoculation of mice, and advantage has been taken of certain chemical and physical properties found to be commonly associated with them to avoid confusion with murine picornaviruses. the property generally tested was sensitivity to lipid solvents. many arboviruses have lipoprotein envelopes and their infectivity is destroyed by these reagents (theiler, ; casals, ) . there was thus a tendency to equate sensitivity to lipid solvents with "arbovirus." during the last decade it has been recognized that the arbovirus group is quite heterogeneous in its physicochemical properties (see table - ). some members are not enveloped (orbivirus, nodamura virus), and those sensitive to lipid solvents belong to at least three major groups (togaviridae, rhabdovirus, and bunyamwera supergroup). this preamble has been necessary because in the past the term "arboviruses" has been regarded as applying particularly to viruses with the physicochemical properties of the group a and group b arboviruses. these viruses now form two genera (alphavirus and flavivirus) of the family togaviridae (toga = cloak). (table - ) and show serological cross-reactivity by the hemagglutinin-inhibition test. the arthropod vectors are mosquitoes, but some alphaviruses may be transmitted congenitally by vertebrates. in nature, they usually cause inapparent infections of birds, reptiles, or mammals, but some can cause generalized infections associated with encephalitis in man and in other mammals. genus: flavivirus [r/ : / - : s/s: v,l/ , di, ac]. this genus (flavi = yellow) comprises the group b arboviruses. all members show serological crossreactivity. the arthropod vectors may be ticks or mosquitoes, and some of them may be transmitted by the ingestion of contaminated milk. they differ from the alphaviruses in that budding usually occurs into cytoplasmic vacuoles rather than from the plasma membrane. most cause inapparent infections in mammals and less commonly in birds, but generalized infections of man may occur with visceral symptomatology (e.g., yellow fever), rashes (e.g., dengue), or encephalitis (e.g., japanese encephalitis). other possible members of the family togaviridae. on the basis of the physicochemical definition proposed, several other viruses that are not transmitted by arthropods should probably be included in this family. generic names have not yet been proposed for these viruses, which include rubella and equine arteritis viruses, and the two serologically related viruses of hog cholera and bovine mucosal disease. in early classifications, some members of two very different genera, now distinguished from each other as orthomyxovirus (ortho = correct; myxo = mucus) and paramyxovirus, were grouped together as myxovirus (andrewes et ah, ). the common properties were an rna genome, a tubular nucleocapsid, and a pleomorphic lipoprotein envelope that carried the properties of hemagglutination and enzymatic elution. the term "myxovirus" is now only used as a vernacular expression to encompass the viruses that have these properties {viz., influenza, mumps, newcastle disease, and parainfluenza viruses); it has no taxonomic status. type a influenza viruses have been recovered from a number of different species of animal (birds, horses, and swine) as well as man; types b and c are specifically human pathogens. they are an important cause of respiratory disease in man and other animals, and some of the avian influenza viruses may cause severe generalized infections. in contrast to the orthomyxoviruses, the paramyxoviruses (para = alongside; myxo = mucus) are enveloped viruses whose rna occurs as a single linear molecule with a molecular weight of about million daltons (table - ) . the tubular nucleocapsid has a diameter of nm and is about . μπι long. it is enclosed within a pleomorphic lipoprotein envelope nm or more in diameter; long filamentous forms with the same diameter also occur. three serologically related viruses, those of measles, distemper, and rinderpest, have been tentatively allocated to the paramyxovirus genus on the basis of the morphology of the virion and nucleocapsid; they do not have a neuraminidase; respiratory syncytial virus is different again. some paramyxoviruses cause localized infections of the respiratory tract and several produce severe generalized diseases; among the latter some are characteristically associated with skin rashes. the genus arenavirus (arena = sand) was defined in terms of the electron microscopic appearance of the virions in thin sections, and serological crossreactivity (rowe et al., a) . the pleomorphic enveloped virions are - nm in diameter (sometimes larger), and have closely spaced peplomers. the structure of the nucleocapsid is unknown, but in thin sections the interior of the particle is seen to contain a variable number of electron-dense granules - nm in diameter, hence the name. all members of the genus are associated with chronic inapparent infections of rodents; some cause acute generalized diseases in other hosts (e.g., lassa fever virus in man). b characteristics : single-stranded rna probably in several pieces, total molecular weight . million daltons; lipoprotein envelope - nm in diameter; multiply in cytoplasm; mature by budding from plasma membrane. all members share a group-specific antigen. envelope encloses "granules" - nm in diameter; some of these are cellular ribosomes. the bunyamwera "supergroup" of arboviruses (bunyamwera, a locality in africa) was established by casals (world health organ., ) to bring together a number of minor arbovirus groups linked by distant serological reactions between occasional "bridging" viruses. the subgroups of viruses included are shown in table - . all these viruses, numbering well over , are known or suspected to be arthropod-borne. morphologically, those that have been studied have enveloped roughly spherical virions - nm in diameter with a tubular nucleocapsid. several other arboviruses serologically unrelated to those of the "supergroup" have a similar morphology (table - ). their genome consists of single-stranded rna probably occurring in several pieces; its molecular weight has not been determined. the outstanding characteristic of the genus leukovirus (leuko = white) is that all members contain an rna-dependent dna polymerase ("reverse transcriptase"). the viruses contain three or four pieces of single-stranded rna, with a total molecular weight of to million dal tons, associated with a helical nucleocapsid, which is enclosed within a capsid with cubic symmetry. this is, in turn, enclosed within a lipoprotein envelope about nm in diameter, containing peplomers which confer the type specificity. leukoviruses mature by budding from the plasma membrane. genome is a linear molecule of single-stranded rna, - million daltons molecular weight, consisting of three to four linked pieces and probably associated with tubular nucleocapsid. structure of virion is complex, the nucleocapsid being enclosed within a capsid of cubic symmetry, which is enclosed in an envelope that carries type-specific antigens. virion also contains species-specific (e.g., feline or murine) and interspecies-specific (e.g., avian or rodent) antigens. as table - illustrates, the genus leukovirus accepted by icnv is clearly an inadequate taxon for the variety of viruses that now fulfill the physicochemical criteria set out above. the term "oncornaviruses" (nowinski et al., ) is also not suitable for the taxon as a whole or any subgroup of it, for not all the viruses conforming to the physicochemical specifications of the genus leukovirus are tumor viruses, many "rna tumor viruses" are not transforming (temin, ) , and in any case the leukemia-sarcoma viruses and the mammary tumor virus (both of which produce tumors) belong to different subgenera. in order not to prejudge a classification of the group, we shall adhere to the accepted generic name, but indicate four subgenera. subgenus a includes the "c-type particle" viruses, some of which cause leukemia-sarcoma and are currently being subjected to intensive study. the mammary tumor virus, which is the best known representative of the subgenus b, differs from viruses of subgenus a in morphology and maturation ("b-type particles") and shows no serological cross-reactivity with the murine viruses of subgenus a. subgenus c includes a group of serologically related viruses that cause slowly progressive diseases in sheep. they have all the physicochemical properties of leukoviruses. although they do not cause neoplastic disease, they will transform cells that are nonpermissive for viral growth (takemoto and stone, ). the viruses of subgenus d (foamy agents) include a number of viruses of monkeys, cats, and cattle, that have no known pathogenic potential but have been frequently isolated from tumors (as "passenger viruses") or healthy animals. they have a different morphology from other leukoviruses (clarke et al., ) and produce an intranuclear antigen as well as cytoplasmic antigens in infected cells (parks and todaro, ), but they contain a reverse transcriptase and are much more resistant to uv irradiation than other rna viruses. the rhabdoviruses (rhabdo = rod) are enveloped rna viruses with singlestranded rna of molecular weight million daltons. the rna is associated with a very regular double-helical nucleocapsid nm in diameter, enclosed within a bullet-shaped shell that measures about x nm (table - ) . several arboviruses belong to this genus, which also includes rabies virus and the virus of hemorrhagic septicemia of trout. it has been claimed that rabies virus can be adapted to multiply in drosophila melanogaster (plus and atanasiu, ). several viruses with a somewhat similar morphology cause diseases of insects and plants (table - , and see table ii of howatson, ), but it may well turn out that these resemblances are superficial. examination of the nature of their genomes and polypeptides is necessary before it can be confidently stated whether these viruses rightly belong to the genus rhabdovirus or even to an enlarged family that might be called rhabdoviridae. nature and classification of animal viruses the mammalian serotypes share a common antigen, which differs from the group antigen of the avian serotypes.clover wound tumor virus, which multiplies in plants and leafhoppers, resembles the reoviruses of vertebrates morphologically and chemically but does not cross-react with them serologically. bluetongue virus, an arbovirus, was found to resemble the reoviruses in some properties but not in others (review: howell and verwoerd, ). subsequently, a large number of similar viruses have been recognized (table - ) and the name "orbivirus" (orbis = ring) was suggested for them (borden et a\., ). reoviruses and orbiviruses may eventually be grouped together in the same family for which the name "diplornavirus" has been suggested (verwoerd, ) . apart from its "illegality" (according to icnv rules), the occurrence of other quite different viruses with genomes of double-stranded rna (like some of the viruses of fungi and insects) cautions against ready acceptance of this term.all members of the genus multiply in arthropods as well as vertebrates. some of them (bluetongue and colorado tick fever viruses) cause severe generalized diseases with viremia in some vertebrates. it is pleasing to note that several of the viruses listed as "unclassified" in the first edition of this book have now been allocated to genera (lymphocytic choriomeningitis of mice, arenavirus; mouse hepatitis virus, coronavirus; rubella virus, togaviridae; visna virus, leukovirus; and african swine fever virus, iridovirus). a few unclassified viruses remain that warrant special mention here, such as the human hepatitis viruses, the agents of the subacute spongiform encephalopathies (scrapie, etc.), lactic dehydrogenase elevating virus (ldv), and the marburg agent. experiments with human volunteers many years ago (neefe et al, ) and again more recently (krugman et al., ) have shown that the diseases commonly known as infective hepatitis and serum hepatitis are caused by two viruses that differ serologically, in their clinical expression, and in their usual routes of transmission. because both can be transmitted orally it is better to use noncommital names for them, and "serum hepatitis" is now termed hepatitis b; infective hepatitis, hepatitis a. study of these viruses has been greatly inhibited by the lack of susceptible laboratory animals (chimpanzees may get clinical hepatitis; marmosets and rhesus monkeys subclinical infection, while other laboratory animals are insusceptible), and the difficulty of obtaining reproducible cytopathic changes in cultured cells. the recognition in the sera of cases of serum hepatitis of lipoprotein particles of characteristic serological specificity, called "australia antigen/ hepatitis-associated antigen (haa), and now hepatitis b antigen (hb-ag), has led to a great expansion in studies on the incidence and pathogenesis of hepatitis b, but the actual virions have not yet been unequivocally demonstrated (see chapter ). serologically unrelated particles of similar morphology have been reported to occur in feces from patients with hepatitis a (cross etal, ). four diseases of similar nature, scrapie of sheep, transmissible encephalopathy of mink, and kuru and creutzfeld-jakob disease in man appear to be caused by similar agents, which differ from all known viruses by being nonimmunogenic. the causative agents are filtrable, highly heat-resistant, and highly resistant to ionizing radiation. it has been suggested that they may be small molecules of naked rna, protected by being closely associated with cellular membranes (diener, a), but a definitive description of these agents is still awaited. this virus, which occurs as an inapparent infection in many laboratory mice and as a contaminant of cells and viruses derived from or passaged through mice (review: notkins, ), shows some resemblances to the togaviruses. it appears to have an isometric core and a lipoprotein envelope, and its rna is infectious. however, the viral rna is large, perhaps million dal tons (darnell and plagemann, ). in germany, in , a small outbreak of a serious new disease occurred in laboratory workers who had handled the tissues of recently imported vervet monkeys (review: siegert, ). the causative agent grows in cultured cells and kills guinea pigs. studies with inhibitors suggest that it contains rna; of known viruses it most closely resembles rhabdoviruses in structure but is much larger and more pleomorphic (murphy et ah, b) . the foregoing account has shown how varied are the agents that we classify as viruses, for reasons based on their composition and their mode of intracellular replication. we can only speculate about their origins and relationships to each other, except in cases where the relationship is very close. it seems likely that different viruses belonging to any one genus, and in at least some cases, different genera allocated to a particular family, may be phylogenetically related. no use- . nature and classification of animal viruses fui suggestions can be made concerning the relationships between families or genera (except in some of the cases where genera have been allocated to the same family), a fact which underlines the undesirability at this stage of our knowledge of erecting any taxa at levels higher than the family.two suggestions have been made concerning the origin of viruses: (a) that they are the result of progressive parasitic degeneration of microorganisms (green, ) and (b) that they have developed from components of the cells of their hosts (andrewes, ; luria and darnell, ) , or are indeed still a permanent part of the host's genome (todaro and huebner, ) . with our present knowledge of the morphological and chemical complexity of the poxviruses, it is not difficult to envisage these agents as being the next degenerate step in the series: bacterium, rickettsia, chlamydia. although they resemble bacteria in most important respects, rickettsia and chlamydiae are, like viruses, obligate intracellular parasites lacking the metabolic equipment for independent multiplication.on the other hand, some dna viruses could well have arisen from episomes, by the acquisition of genetic information specifying a protein coat. even this may not be essential, if diener's ( b) observations on potato spindle tuber virus are confirmed and generalized. the two alternatives are not mutually exclusive; some viruses may have evolved from cellular organelles like chloroplasts or mitochondria, themselves probably derived from bacteria (swift and wolstenholme, ). it is difficult to see where most rna viruses could have originated except from cellular rna's.comparing the nearest neighbor nucleotide doublet frequencies of the nucleic acids of several large and small viruses, subak-sharpe ( ) noted that the patterns shown by small viruses with genomes of less than million daltons (two enteroviruses, three parvoviruses, two polyoma viruses, and two papilloma viruses), closely resembled the pattern of mammalian dna. on the other hand, the doublet frequency patterns of several viruses with large genomes (two herpesviruses and a poxvirus) differed strikingly from that of mammalian dna. the doublet patterns of three adenoviruses resembled each other and showed a slight resemblance to the pattern of mammalian dna, which could derive from some earlier natural fusion of genomes, like that recognized as a laboratory artifact with adenoviruses and sv (see chapter ). this evidence supports the notion that the small viruses may have originated from vertebrate cells whereas the herpesviruses, poxviruses, and probably the adenoviruses did not. these large viruses may have originated from the nucleic acid of cells of a different phylum, or as suggested earlier, by parasitic degeneration of microorganisms. key: cord- -z w wkm authors: beeler, judy a. title: human and animal viruses date: - - journal: maintaining cultures for biotechnology and industry doi: . /b - - / - sha: doc_id: cord_uid: z w wkm this chapter provides an overview of the human and animal viruses. viruses held to a low number of passages in animals or cell cultures represent a viral population that is similar to that found in nature, and freezing these pools guards against genetic mutations that occur during subsequent passage. aliquots of viral stocks frozen at a designated passage level can then be used for multiple and repeatable experiments with the same viral population. furthermore, it is important that consistency should be maintained during the production of viral vaccines; new lots of final product are prepared with frozen viral seed stocks that consistently reproduce the desired immunogenic and attenuation characteristics. to better appreciate the requirements for freezing and freeze drying of human and animal viruses, some consideration is given to understanding the structural and functional organization of this diverse group of microorganisms. the classification of viruses is based on morphological and physiochemical properties. thus, viruses are divided into those with dna or rna genomes and subdivided into families based on size and structural properties. several methods for preservation of viruses are included in the chapter. shackell's successful treatment of rabies virus in represents the first published account of preservation of an animal virus using a process that was an early version of freeze-drying (shackell, ) . prior to this, viruses were maintained by passage in animals. however, freeze-drying provided a way to maintain virally infected material over long periods of time with relative ease when compared to serial passage in a susceptible animal host. the first experiment to demonstrate that this method could provide long-term stability was reported for a bovine virus that had been freeze-dried in and was shown to be viable after being maintained at room temperature for years (fasquelle and barbier, ) . later, as mechanical freezers became more available, freezing viruses at - ~ or less was adopted as a practical and reliable method of virus preservation. influenza virus-infected mouse lung tissue and yellow fever virus were successfully frozen at - ~ by turner ( ) , and horsfall ( ) , using turner's methods, demonstrated successful storage of influenza, mouse pneumonitis, and canine distemper viruses. following these early successes, viral repositories, diagnostic and research laboratories, and vaccine manufacturing facilities have relied on freezing and freeze-drying for preservation of viruses and viral vaccines. the reasons for preserving viruses are similar to those for the preservation of other microorganisms and cells: successful preservation allows for long-term maintenance of consistent stocks and for reproducibility in the testing of viral samples and manufacture of vaccines. for example, field and clinical isolates need to be preserved for transport to testing laboratories; harvested virus from animals or cell cultures may not be processed immediately and require preservation until used. viruses held to a low number of passages in animals or cell cultures represent a viral population that is similar to that found in nature, and freezing these pools guards against genetic mutations that may occur during subsequent passage. aliquots of viral stocks frozen at a designated passage level can then be used for multiple and repeatable experiments with the same viral population. furthermore, it is extremely important that consistency be maintained during the production of viral vaccines; new lots of final product can be prepared with frozen viral seed stocks that consistently reproduce the desired immunogenic and attenuation characteristics. for example, the world health organization (who) maintains aliquots of the original attenuated poliovirus strains developed by dr. albert sabin in the s. these aliquots are frozen and, when amplified properly and kept to the prescribed low passage level, should produce a vaccine today with the same properties that characterized the original sabin vaccine. similarly, viruses that are currently under development for use as vectors for gene therapy must be highly characterized and homogeneous for safe and effective use and need to be maintained as frozen stocks. in order to better appreciate the requirements for freezing and freezedrying of human and animal viruses, some consideration must first be given to understanding the structural and functional organization of this diverse group of microorganisms. the classification of viruses is based on morphological and physiochemical properties. thus viruses are divided into those with dna or rna genomes and further subdivided into families based on size and structural properties. there are families of dna viruses and families of rna viruses (see table ). the genomes of viruses within a given family are similar in terms of their polarity, genome organization, and method of replication and have morphologic features in common, including size, shape, nucleocapsid symmetry, and presence or absence of an envelope. other characteristics that help to classify viruses include sensitivity to low ph, heat, and lipid solvents. sensitivity to ph is determined by subjecting a virus to a ph of . . loss of titer greater than one log indicates that the virus is ph sensitive and acid labile. heat treatment of a virus for min at ~ is a test of thermal stability. if the virus survives this treatment, with no more than a one log loss of infectivity, it is considered to be thermostable. treatment with lipid solvents, commonly ether and despite the similarities among the physical properties of these families of viruses, relatively few general rules for freezing or freeze-drying viruses can be made. for example, it is common to use membrane-stabilizing agents to preserve viruses with a lipid envelope. picornaviruses, which lack a lipid membrane, maintain infectivity in the presence of mgc . however, the optimal conditions for freezing specific viruses need to be determined empirically. significant reduction of morbidity and mortality for many viral infections has been accomplished by the successful application of viral vaccines. in the case of smallpox, eradication of this important human pathogen has been realized. polio has now been targeted as the next viral disease that can be eradicated by immunization. vaccine production requires the use of the "seed lot system" for consistent reproduction and quality control of vaccine lots. vaccine virus seeds are usually frozen at two passage levels prior to vaccine lot production and these stocks of virus are designated the master seed and the production or working seed, respectively. multiple aliquots of these seeds are prepared for reference as well as for characterization and standardized testing for possible contaminants. finally, it is also important to distribute the storage of the master virus seed and working seed among multiple freezers in order to be assured of preserving part of the stock in the event of a mechanical failure. the seed lot system establishes a viral stock that can be standardized and provides a source of viruses so that vaccine lots manufactured over decades will be closely related to the original seed lot. this is especially important for live-attenuated vaccines that in most cases have been derived by multiple serial passage of a clinical isolate of a virus. deviation from the attenuated passage number may result in undesirable phenotypic changes of the attenuated vaccine virus. this was the case with yellow fever vaccine when uncontrolled passage of this virus resulted in a vaccine with a higher than normal incidence of side reactions (fox et al., ) . presently, researchers are using the tools of molecular biology to produce a new class of recombinant viral agents that will be used for immunization or genetic therapy. for example, mammalian retroviruses that integrate into the host cell genome are being developed as vectors for delivery of human dna sequences. the newly acquired dna is expressed in the form of protein(s) that may be deficient in the host cell or may require de novo synthesis. this form of genetic therapy has been demonstrated routinely in vitro and in vivo in animal experiments and is being proposed as therapy for some human genetic diseases (for review, see roemer and friedmann, ) . in addition, recombinant viral vectors are also paving the way for a novel type of therapy in which cancer cells can be specifically targeted for infection by a recombinant virus; following infection the inserted gene is expressed and the gene product selectively makes the infected cancer cells more susceptible to subsequent chemotherapy or to natural immune surveillance in the host (collins et al., ; culver et al., ) . alternatively, recombinant vectors may express antisense rna that can bind to and block the activity of the host gene sequences. successful experiments of this kind require viral vectors that are thoroughly characterized and safe. seed stocks of the recombinant viral vectors constructed to express the desired nonviral gene sequences must be frozen or freeze-dried in order to be preserved for future experiments and clinical trials. a somewhat similar approach is being developed for vaccination using viral vectors as delivery vehicles for genes important for immunization. for example, one approach uses a highly attenuated vaccinia virus that carries, as part of its genome, various immunogenic proteins that would evoke protection against infection (moss and flexner, ) . other viral vectors used similarly include adenovirus and canarypox. antigens used for viral diagnostic purposes must also be quality controlled and, in general, would also be produced from a virus seed lot system. variation of quality or quantity of viral antigens in diagnostic kits and reagents may directly influence results in the diagnostic laboratory. regulation of the manufacture of diagnostics is similar to those for vaccine manufacture. regulations concerning the manufacture of biological products are published by the united states public health service ( cfr ) and outline the requirements for control of reagents and raw materials in the manufacture of diagnostics to screen blood and blood products. for these purposes, it is essential to use viral seed stocks that are appropriately characterized and preserved by freezing. viability of viruses is usually determined by the use of infectivity assays. these include assays whereby viral cytopathology is measured in cell cultures or, when this is not possible, potency may be determined by inoculation of animals or eggs. by far the most accepted and accurate quantitative measure of viral infectivity is the plaque assay. most viruses form plaques in susceptible host cells. generally, after virus inoculation on suitably sensitive cell monolayers, an agar overlay is applied so that viral cytopathology is localized. after an incubation period, viable cells are stained with a vital dye, revealing areas of cell death, the plaque-forming unit. most viral freezing and recovery experiments use the plaque assay to titer virus before and after treatment as a measure of viral activity. in this way, one can determine the efficiency of the freezing or freeze-drying process. electron microscopy (em) has also been used on occasion to visualize the effects of freezing and freeze-drying. these em experiments are limited by the purity and titer of the viral preparation as well as the subjective interpretation of observations. the success of freezing or freeze-drying of viruses is dependent on the proper preparation of the harvested virus. the source of virus may be animal tissues or organs, animal sera, virus-infected cell cultures, and, on occasion, egg allantoic or amniotic fluids. the harvested tissue will depend on the particular host-range of the virus being grown. harvesting methods are also dependent on the properties of viral replication. some viruses, e.g., herpes viruses, stay cell associated whereas other are readily released from the cell into the culture fluids. in general, viruses grown after passage in vivo require disruption of the infected animal tissue or organ by homogenizers or aerosol-controlled blenders. diluent consisting of buffer or cell culture media is added for release of the virus into the aqueous phase during cell disruption. depending on the virus, serum may be required as an additive to the diluent for optimal freezing ( table ). viruses that are found in blood may be harvested and frozen directly in the serum after separation from blood cells or by cocultivating infected peripheral blood lymphocytes (pbls) with susceptible cell lines. these preparations may be frozen without further treatment, although it is generally recommended that serum or pbls be frozen and thawed only once to achieve optimal virus recovery. similarly, cell culture harvests may require disruption of the cell monolayer as a first step. cells may be scraped from the culture vessel surface into the culture fluids or these fluids may be removed and the cells treated separately. in either case, cells are disrupted and virus is released. in certain cases, virus-infected cells are harvested for a seed preparation. gentle scraping and harvesting of these cells into the culture fluids or into flesh buffer or cell culture medium is required. the specific details for optimal harvesting procedures for individual viruses may be found in the literature. usually animal serum is used as a component of cell culture medium. fetal bovine serum (fbs) is the most frequently used additive by cell ( ) culturists and virologists because it is nontoxic to most cells and the protective effect during freezing is consistent. therefore, fbs doubles for cell nutrition as well as a cryoprotectant for freezing and freeze-drying. concentrations used for cell culturing and growing viruses range from . to % in cell culture medium. in many cases the cell culture medium containing serum constitutes the cyroprotective diluent that will be used for freezing. however, further addition of serum is required for many viruses (see table ). infectious materials should be handled according to the biosafety guidelines that apply for each virus. the rate of freezing and the final temperature must be considered for virus preparations being frozen for long-term storage. to maintain viral infectivity throughout this process, the integrity of the viral capsid, the viral envelope (if there is one), and the viral nucleic acid must be preserved. dimmock ( ) demonstrated that viral infectivity may be reduced by heat damage to both viral proteins and nucleic acid. he showed that the stability of these components varies with elevated temperature so that inactivation at a particular temperature takes place through whichever component is least stable at that temperature. in general, most viruses may be "snap frozen" in a dry ice/ethanol bath prior to storage at temperatures _<- ~ it may be preferable to freeze some viruses, such as cytomegalovirus or varicella-zoster virus, as viable infected cells. in this case, a slow controlled rate of freezing is recommended: cooling should be done at l~ to- ~ and then at ~ to - ~ (simione and brown, ) . it has also been reported that influenza and measles virus may benefit from this two-step freezing process (rowe and snowman, ) . storage temperature is also critical for some viruses with envelopes, such as influenza virus, respiratory syncytial virus, and measles virus. these all require storage at - ~ or lower for optimal recovery after long-term storage (rightsel and greiff, ; greiff et al., ; law and hull, ) . it is common practice for today's virologist to store frozen viruses in ultralow-temperature freezers at temperatures of - to - ~ freezing in liquid nitrogen or its vapor phase, a common practice for preservation of mammalian cells, is not necessary for most viruses. liquid nitrogen storage may be used as a "backup" system for viral seed repositories. no known conventional virus requires - ~ (liquid) or - ~ (vapor) nitrogen for short-or long-term storage. cryoprotectants in one of the first comprehensive studies of freezing and freeze-drying with representative members of most of the rna and dna virus families, rightsel and greiff ( ) demonstrated the importance of the suspending medium used for freezing and freeze-drying. medium containing skim milk, calcium lactobionate, normal serum albumin, dimethyl sulfoxide (dmso), glycerol, or magnesium chloride promoted better freezing and freeze-drying recovery with a range of viruses, especially enveloped rna viruses. wallis and melnick ( ) further demonstrated that dmso or serum acted as a cryoprotectant for four different enveloped viruses, herpes virus, measles virus, sindbis virus, and vesicular stomatitis virus. under the conditions described dmso performed somewhat better than serum as a cryoprotectant. the nonenveloped viruses, vaccinia, adenovirus, and poliovirus, did not require dmso or serum for retention of viability during freezing. the authors concluded that enveloped viruses were similar to mammalian cells and that dmso stabilizes the viral envelope much as it stabilizes the mammalian plasma membrane. however, another member of the herpes virus family, varicella-zoster (vz), is especially sensitive to freezing and freeze-drying. grose et al., ( ) used sucrose, potassium phosphate, sodium glutamate, and albumin in a cryoprotective "cocktail" for vz virus. for both cell-associated and cell-free virus, the cocktail was fully protective, with % recovery of infectivity after freezing to - ~ and freeze-drying. additionally, these authors found that glutamate and albumin could be removed from the cocktail without loss of protection. in contrast, scott and woodside ( ) showed that sodium glutamate was essential for the stability of freeze-dried herpes virus. certain viruses are stabilized by the addition of divalent cations. for example, respiratory syncytial virus was found to be stable for up to month at ~ following the addition of magnesium sulfate, and the titer of virus was maintained through several cycles of freezing and thawing when mgso was added (fernie and gerin, ) . likewise, poliovirus was stabilized in the presence of mgc (melnick et al., ) . alternatively, mm sodium phosphate at ph . stabilized poliovirus at - ~ for up to months (mauler and gruschkau, ) . cryoprotectants and the rate of freezing minimize the formation of ice crystals that can damage the viruses; similarly, it is common practice to rapidly thaw frozen viral stocks at a previously determined optimal temperature to minimize ice crystal formation. freeze-drying, or lyophilization, is a method for preserving viruses and other biological materials by removing water from frozen samples via sublimation. the result is a dried preparation that is usually more stable than wet-frozen preparations at various temperatures and for longer periods. the process is usually divided into three stages: prefreezing, sublimation drying under vacuum (primary drying), and desorption or secondary drying. prefreezing can be accomplished in special freezers or in the freeze-dryer itself. a freeze-dryer has a vaccuum pump that removes air from a chamber that holds vials of the material to be freeze-dried. water vapor generated through this process is condensed into a refrigerated trap and is not allowed to enter the vacuum pump. the rate of sublimation can be controlled by regulating the heat transfer to the frozen material. after final drying, inert gas is used to fill the vials and protect the freeze-dried material from the deleterious effects of oxygen. the freeze-drying cycle requires optimization for each virus. a typical cycle that has been used successfully for dengue liveattenuated virus vaccines (all four serotypes) consisted of freezing to - ~ for hours and allowing the condenser to reach a temperature of - ~ (k. eckels, unpublished data). vacuum was drawn to /zm of hg, at which time heat was applied at a controlled rate overnight (approximately hours). final drying occurred at - ~ for hours followed by backfilling the vials with dry, sterile nitrogen and capping. vaccine vials were finally stored at - ~ another cycle that preserves viral infectivity has been used for dengue candidate vaccines; this consisted of freezing to - ~ overnight followed by raising the temperature to - , , and finally to ~ over a -day period while vaccuum was applied (k. eckels, unpublished data). in both cases, care was taken to avoid subjecting this thermolabile virus to prolonged periods of high temperatures that could result in inactivation of viral infectivity. greiff and rightsel ( ) have successfully freeze-dried influenza and measles virus with cycles extending over a -hour period with shelf and product temperatures not exceeding ~ greiff ( ) has outlined an approach for the development of a successful freeze-drying cycle. similarly, by varying drying time, shelf temperature, and vacuum pressure an optimal lyophilization cycle for varicella-zoster vaccine was determined (bennett et al., ) . freeze-drying methods and cycles used by the american type culture collection (atcc) for their virus stocks can be found in simione and brown ( ) . stabilizers added to the viral suspension medium for freeze-drying are many of the same used for freezing. these additives promote preservation of viral infectivity by acting as antioxidants and by providing "bulk" to the virus preparation to allow easier reconstitution. also, they should be nonhygroscopic to avoid moisture contamination of the freeze-dried mate-rial. for vaccines, the stabilizers must also be nonimmunogenic and nonreactive in the vaccine recipient. one of the first stabilizers used for viral freeze-drying was . % gum acacia to preserve the infectivity of vaccinia virus (rivers and ward, ) . peptone and normal horse serum were also successfully used for freeze-drying of vaccinia virus (collier, ; sparkes and fenje, ) . calcium lactobionate and human serum albumin were cryoprotective for measles virus during freeze-drying (greiff and rightsel, ) , whereas cell culture medium alone was less protective. a combination of sucrose, phosphate, sodium glutamate, and albumin was used for freeze-drying pseudorabies and herpes viruses (scott and woodside, ; calnek et al., ) . varicella-zoster virus was also freeze-dried successfully in this combination of stabilizers, but grose ( ) found that he could eliminate glutamate and albumin from the mixture and still retain % recovery of this very labile virus. dmso cannot be used for stabilization in freeze-dried preparations because it becomes concentrated to toxic levels (greiff and rightsel, ) . a group of viruses that requires special attention to stabilizing media is the enteroviruses, for example, poliovirus and hepatitis a virus. early work with poliovirus demonstrated that it had a high degree of lability when freeze-dried, even with stabilizers. the lability is due to the presence of inorganic salts that may be present in the culture medium. when these are removed by dialysis prior to freeze-drying, losses do not occur (berge et al., ) . the atcc uses ultrafiltration, a more rapid type of dialysis to remove salts prior to freeze-drying of enteroviruses (simione and brown, ) . vaccines that require freeze-drying have special stabilizer requirements that allow them to be used safely in humans. animal serum cannot be used as a stabilizer and must be completely removed from the vaccine prior to bottling. when a protein substitute is required, human serum albumin has been used successfully for many vaccines. more recently, hydrolyzed gelatin has been used as a replacement for albumin (table ). both of these stabilizers are acceptable for human vaccines because they usually do not stimulate deleterious immune responses in the vaccinee. sugars such as sucrose, lactose, and sorbitol are also used either alone or in combination with a protein stabilizer. cell culture medium is often used as a buffer because many live vaccines are harvested in their culture medium without further processing. optimal moisture content of the freeze-dried product needs to be empirically determined for each viral preparation. a good freeze-drying cycle can attain a moisture content of % or less in the final product. however, depending on the virus, a higher moisture content may be desirable. a varicella virus vaccine with - % moisture following freeze-drying was found to be more stable than vaccines containing less moisture (bennett et al, ) . various viruses have been freeze-dried to approximately % moisture and shown to be stable over periods of time at ~ but better long-term stability can be achieved by storage at - ~ stability assays for retained infectivity following freeze-drying are done on rehydrated vials of freeze-dried virus. a volume of sterile, distilled water is used to reconstitute the vial, with titration of the virus done immediately in the appropriate plaque or infectivity assay. the best control for baseline infectivity would be the virus harvest that was assayed prior to freezedrying. often, frozen and thawed specimens are used for baseline titers. freeze-dried viruses are normally stored at ~ or - ~ accelerated stability studies can be done to test thermostability for shorter periods at elevated temperatures. often an ambient temperature of ~ or temperatures of - ~ are used for accelerated studies. these temperatures are chosen to study thermostability for viruses, mainly vaccines, that may not have continuous refrigeration available up to the time of use. this is a problem for many live viral vaccines that are being used in developing countries (widdus et al., ) . the largest application of freeze-drying of viruses occur for liveattenuated viral vaccines that are thermolabile. the first viral vaccine to be freeze-dried was yellow fever vaccine (penna, ) . recent advances have resulted in more stable, freeze-dried yellow fever vaccines (robin et al., ; burfoot et al., ) . the who standard for stability of this vaccine is no more than one log loss of potency held at ~ for weeks. a recent collaborative study of yellow fever vaccines from manufacturers demonstrated that of vaccines met this requirement. similar who stability standards are in place for live, freeze-dried,measles vaccine. a method to freeze-dry and stabilize trivalent, live, attenuated, oral polio vaccine is being sought so that this vaccine can be delivered and used in developing countries without significant loss in potency. it has been proposed that thermoresistant mutants of polioviruses might serve as the prototype for the development of heat-stable vaccine strains (kew, ) . alternatively, organic compounds such as win and r , which bind to polivirus vp , have been found to increase the half-life of poliovirus antigen -to -fold at temperatures up to ~ (rombaut et al., ) . these drugs stabilize the conformation of the viral capsid and exhibit a potent antiviral effect that would not make them suitable as stabilizers for a live virus vaccine. in the future, similar compounds could be designed to stabilize but not inhibit the replication of live poliovirus strains. other licensed, freeze-dried live-viral vaccines are those for mumps and rubella, whereas vaccines for varicella (chicken pox) and cytomegaloviruses are being developed that will require stabilization by freeze-drying. table lists licensed vaccines as well as those still being tested and the available published data on freeze-drying of these vaccines. american type culture collection parklawn drive rockville, maryland telephone: - - - (united states and canada, only) or - - - fax: - - - request for international reference materials should be made with a statement of intent from the investigator. the catalogue of animal viruses and antisera, chlamydiae, and rickittsiae atcc preservation methods: freezing and freeze-drying diagnostic procedures for viral, rickettsial, and chlamydial infections freezing and drying diagnostic procedures for viral, rickettsial, and chlamydial infections diagnostic procedures for viral, rickettsial, and chlamydial infections biological product freeze drying and formulation diagnostic procedures for viral, rickettsial, and chlamydial infections diagnostic procedures for viral, rickettsial, and chlamydial infections temperature-stable vaccines for developing countries world health organization edwards freeze-drying handbook diagnostic procedures for viral, rickettsial, and chlamydial infections atcc preservation methods: freezing and freeze-drying temperature-stable vaccines for developing countries: significance and development strategies the author would like to thank dr. kenneth eckels for guidance and sharing data on freeze-drying dengue viruses and drs. karen goldenthal, ron lundquist, and hana golding for reviewing the manuscript. key: cord- -jqgervt authors: fenner, frank; bachmann, peter a.; gibbs, e. paul j.; murphy, frederick a.; studdert, michael j.; white, david o. title: laboratory diagnosis of viral diseases date: - - journal: veterinary virology doi: . /b - - - - . - sha: doc_id: cord_uid: jqgervt tests for the specific diagnosis of a viral infection in an animal are of two general types: ( ) those that demonstrate the presence of the virus and ( ) those that demonstrate the presence of specific viral antibody. the provision, by a single laboratory, of a comprehensive service for the diagnosis of viral infections of domestic animals is a formidable undertaking. there are about individual viral species in some different viral families that infect the eight major domestic animal species. if antigenic types within an individual viral species are considered and the number of animal species is broadened to include turkey, duck, and zoo and laboratory animals, then the number of individual viruses exceeds . it is, therefore, not surprising that few single laboratories could have available the necessary specific reagents, skills, and experience for the diagnosis of such a large number of infections. tests for the specific diagnosis of a viral infection in an animal are of two general types: ( ) those that demonstrate the presence of the virus, and ( ) those that demonstrate the presence of specific viral antibody. the provision, by a single laboratory, of a comprehensive service for the diagnosis of viral infections of domestic animals is a formidable undertaking. there are about individual viral species, in some different viral families, that infect the eight major domestic animal species (cattle, sheep, goat, swine, horse, dog, cat, and chicken) . if antigenic types within an individual viral species are considered, and the number of animal species is broadened to include turkey, duck, and zoo and laboratory animals, then the number of individual viruses exceeds . it is therefore not surprising that few single laboratories could have available the necessary specific reagents, skills, and experience for the diagnosis of such a large number of infections. one consequence of this great variety of viruses is that veterinary diagnostic laboratories tend to specialize, e.g., in diseases of food ani- mais, or companion animals, or poultry, or in "exotic" viruses. within these specialized laboratories there is considerable scope for the development of rapid diagnostic methods that short-circuit the need for the isolation of viruses, which is expensive, time-consuming, and rarely necessary. many viral diseases can be diagnosed clinically, others with the assistance of the pathologist; but there are several circumstances under which laboratory confirmation of the specific virus involved is desirable or, indeed, essential. the industrialized countries of europe, north america, australasia, and japan are free of many devastating diseases of livestock that are still enzootic in other parts of the world, such as foot-and-mouth disease, african swine fever, rinderpest, and fowl plague. all industrialized countries maintain or share the use of specialized biocontainment laboratories (such as those at plum island in the united states and pirbright in the united kingdom) devoted to diagnosis and research on such "exotic" viruses. clearly it is of the utmost importance that the clinical diagnosis of a suspected exotic virus should be confirmed quickly and accurately (see chapter ). several animal viral diseases such as rabies, rift valley fever, and eastern, western, and venezuelan encephalomyelitis are zoonotic and are of sufficient human public health significance to require the maintenance of specialized diagnostic laboratories. for example, confirmation of the diagnosis of rabies in a skunk that has bitten a child provides the basis for postexposure treatment of the human patient (see chapter ). confirmation and early warning of an equine encephalomyelitis virus epizootic allows implementation of mosquito control and other measures such as restriction of the movement of horses. for diseases in which there is lifelong infection, such as bovine and feline leukemia, equine infectious anemia, and herpesvirus infections, a negative test certificate is often required as a condition of sale, particu-larly export sale, for exhibition at a state fair, or show, or for competition, as at race meetings. males used for semen collection and females used in embryo transfer programs, especially in cattle, and blood donors of all species, are usually screened for a range of viral infections to minimize the risk of transmission to recipient animals. for retrovirus infections, marek's disease, pseudorabies, and certain other diseases, it is possible to reduce substantially the incidence of disease or eradicate the causative virus from the herd or flock by test and removal programs. laboratory diagnosis is essential for the effective implementation of such operations. provision of a sound veterinary service in any state or country depends on a knowledge of prevailing viral diseases; hence, epidemiologica! studies to determine the prevalence and distribution of particular viral infections are frequently undertaken, usually based on the detection of specific antibody. many relatively nonspecific disease syndromes, such as respiratory disease (e.g., kennel cough and shipping fever), diarrhea, and some skin diseases, may be caused by a variety of agents, viral and bacterial. proper management of individual cases or infected herds or flocks may require specific viral diagnosis. specific diagnosis of the kind outlined above can be achieved by one of three methods: ( ) isolation and characterization of the causative virus, ( ) direct demonstration of virions, viral antigens, or viral nucleic acids in tissues, secretions, or excretions, and ( ) detection and measurement of antibodies (table - ). each group of methods has its place. viral isolation remains the benchmark against which other methods are measured, and is essential when decisions of major economic importance depend on the diagnosis, e.g., with suspected exotic diseases such as foot-and-mouth disease or fowl plague. on the other hand, the direct demonstration of virions or viral components may provide a much more rapid and cheaper method of specific diagnosis than viral isolation, particularly when large numbers of samples must be tested. epidemiologica! surveys, eradication programs, and the provision of certificates of freedom from specific infections are often based on serological methods or rapid tests for viral antigen. it requires at least as much effort, and often more, to process a negative specimen as it does one from which virus is isolated. the chance of isolating a virus depends critically on the knowledge, care, and attention of the veterinarian who collects the specimen (see plate - ). obviously such a specimen must be taken from the right place and at the right time. the right time is always as soon as possible after the onset of clinical signs; virus is usually present in maximum amount at about this time and diminishes, sometimes quite rapidly, in the ensuing few days. specimens taken as a last resort when days or weeks of empirically chosen antibiotic therapy have failed are almost invariably a waste of effort. the site from which the specimen is collected will be influenced by the clinical signs and a knowledge of the pathogenesis of the suspected disease (table - ). having collected the appropriate specimen(s), it should be properly labeled and sent to the laboratory, with a history, including the provisional diagnosis. where ambient temperatures are moderate and transit time to the laboratory is less than day, ice or cold packs (< °c) in a styrofoam box are frequently used. if the environmen tal temperature is high and transit times longer than a day, dry ice (- °c) may be used, although wet ice with provision to replenish it in transit is better. if exotic or zoonotic viruses are suspected, the styrofoam boxes must be replaced by or enclosed within sturdier, dou ble-walled containers with absorbent padding. appropriate permits must be obtained for interstate and international transportation, and in "blood: refers to clotted sample for serology and sample with anticoagulant added for other tests. large animals, - ml; small animals, - ml; others as appropriate. if possible remove clot before dispatch. such circumstances the collection and transport arrangements need to be discussed with the laboratory and/or the appropriate government regulatory agency. for particularly labile viruses such as respiratory syncytial virus, herpesviruses, or coronaviruses, it may be an advantage to take the cell culture to the animal. isolation and identification requires at least a week, usually longer, and it is expensive. however, it is probably the most sensitive available method, if properly collected material is used, and it provides material for further study. the sooner the specimen is processed and inoculated after arrival at the laboratory, the better. if delays of more than day are anticipated, the specimen may be frozen to - °c. swabs are processed by twirling them in the transport medium and expressing the fluid by pushing the swab firmly against the side of the container. feces are dispersed on a vortex mixer. tissue specimens are finely minced with scissors and ho mogenized in a glass or mechanical homogenizer. prior to inoculation, contaminating microorganisms are removed by filtering through membrane filters of average pore diameter . μιη, although such filters allow the passage of mycoplasmas. once virus is successfully isolated and grown to a high titer, the suspension can be refiltered through . μιη filters to exclude mycoplasmas. feces and tissue homogenates should be diluted at least : and centrifuged at g for minutes to obtain a supernate that can be filtered. if the concentration of virus is suspected to be very low, high concentrations of antibiotics may be preferable to filtration. whatever the origin of the specimen, some of the original sample and some of the filtrate should be retained at °c or frozen until the isolation attempt is finalized. virus can be grown from the suitably prepared specimen by inocula tion into cell cultures, laboratory animals, or the species of host animal from which the specimen was obtained. by far the most widely used substrate is cultured cells. choice of cultured cells. the choice of the optimal cell culture for the primary isolation of a virus of unknown nature from clinical specimens is largely empirical. primary or low-passage, homologous, monolayer cell cultures derived from fetal tissues probably provide the most sen sitive substrate for isolation of the greatest variety of different viruses. continuous cell lines derived from the homologous species are almost as good. often the nature of the disease from which the material was obtained will suggest what species of virus may be found, and in such cases the optimum cell culture for that virus can be chosen, in parallel, perhaps, with a second type of culture with a wide spectrum. cell lines offer some advantages and are available for most domestic animals ex cept avian species (table - ) . monolayer cell cultures for virus isolation should be grown in sealed containers, such as plastic flasks or glass tubes with screw caps. openculture systems such as petri dishes or microtiter trays should not be used because of the risks of cross-contamination. for some viruses, rolling the cultures on a drum improves isolation rates. special types of cultures are utilized for particular viruses. for exam ple, betaherpesviruses and gammaherpesviruses may be recovered from monolayer cultures of tissue taken directly from the diseased animal, whereas inoculation of established monolayer cell cultures with cell-free material may be negative. for some corona viruses and rhinoviruses that do not grow well in monlayer cultures, growth may occur in expiant cultures (i.e., small cubes of tissue from the trachea or gut), probably because these do not dedifferentiate in culture (see chapter ). recognition of viral growth. cultures are usually incubated at °c, despite the fact that the normal body temperatures of all domestic animal species are somewhat higher. cultures are observed daily for cytopathic effects. the speed and nature of the cytopathic effect caused by different viruses varies considerably. cytopathic effect must always be based on comparison with uninoculated cell cultures; this is particularly important for viruses requiring incubation periods of longer than a week. where none or a doubtful cytopathic effect is observed, it is usual to make a second or even a third ("blind") passage. when cytopathic effect is observed, there is a range of options: . the speed and appearance of the cytopathic effect, coupled with the case history, may immediately suggest the diagnosis. . after suitable manipulation, material from the cell culture may be examined by electron microscopy. . infected monolayers on glass coverslips or special slide/culture chambers may be fixed and appropriately stained, and the cells examined for inclusion bodies, syncytia or other characteristic cell changes. better, they may be stained with fluorescent antibody, which may provide an immediate definitive diagnosis. where prior experience and knowledge suggest it, such slide cultures may be included at the time of primary inoculation, with a consequent saving in time. table - ). their growth in monolayer culture may sometimes be recognized by means of hemadsorption. most viruses that hemagglutinate will also hemadsorb; the growth of paramyxoviruses, orthomyxoviruses, and, to a lesser extent, the flaviviruses and toga viruses, is routinely recognized in this way (see plate - ). nowadays laboratory animals play a minor role in the virus diagnostic laboratory. however, some virologists still regard intracerebral inoculation of baby mice as the method of choice for the isolation of rabies virus, flaviviruses, and toga viruses. the developing chick embryo occupies a special place. intraamniotic inoculation provides the most sensitive method for influenza viruses and for several avian viruses, and species diagnosis of orthopoxviruses can be made directly from the type of pock produced on the chorioallantoic membrane. in addition, chick embryos are extensively used as a source of primary monolayer cultures (fibroblasts, kidney cells) for the isolation of avian viruses. in veterinary medicine it is feasible to consider using the natural host species, especially susceptible young animals (e.g., calves, pigs, chicks), for the recovery of a virus from suspect material. such animals, if free of antibodies, must be considered a highly sensitive substrate. however, their use would now be contemplated only for viruses not yet cultivable, or where cell culture procedures were negative in circumstances that strongly indicated a viral etiology, and/or where there might be serious repercussions if the diagnosis were missed. a newly isolated virus can usually be provisionally allocated to a particular family, and sometimes to a genus or species, on the basis of the clinical findings, the host cell used for virus isolation, and the visible result of viral growth (cytopathic effect, hemadsorption, hemagglutination, electron microscopy of the cytopathogenic agent, etc.). definitive identification, however, usually depends on serological procedures. by using the new isolate as antigen against known antisera, e.g., in a complement fixation test, the virus can often be placed into its correct family or genus. having allocated it to a particular family (e.g., adenoviridae), one can then go on to determine the species or serotype (e.g., canine immunodiffusion antibody neutralizes infectivity of virion; inhibits cytopathology, reduces plaques, or protects animals antibody inhibits viral hemagglutination antigen-antibody complex binds complement, which is thereafter unavailable for the lysis of hemolysissensitized sheep red blood cells antibody-aggregated virions are visible by electron microscopy antibody labeled with fluorochrome binds to intracellular antigen; fluoresces by uv microscopy peroxidase-labeled antibody binds to intracellular antigen; colored precipitate forms on adding substrate enzyme-labeled antibody (or antigen) binds to antigen (or antibody); substrate changes color radiolabeled antibody (or antigen) binds to antigen (or antibody), e.g., attached to solid phase antibodies and soluble antigens produce visible lines of precipitate in a gel adenovirus ) by more discriminating serological procedures. this sequential approach is applicable only to families with a common family antigen. the range of available serological techniques is now extremely wide (table - ). some are best suited to particular families of viruses. each laboratory makes its own choice of favored procedures, based on considerations such as sensitivity, specificity, reproducibility, speed, convenience, and cost. currently most serological procedures are carried out with "hyperimmune" sera comprising a polyclonal mixture of antibodies, sometimes after they have been absorbed to eliminate antibodies of certain specificities. monoclonal antibodies with defined specificity are now becoming available. these make it possible to proceed quickly to very specific diagnosis even to the level of subtypes, strains,or variants, e.g., rabies viruses from different geographical areas. family-, genus-, and typespecific monoclonal antibodies are also being developed. as their properties are defined and they become commercially available, we can expect monoclonal antibodies to be widely used for all methods of serological identification. . immunofluorescence, used here for determining the site of assembly of components of influenza virus. antibody against the nudeoprotein antigen shows nuclear accumulation at hours after infection of chick cells. procedure: guinea pig antiserum to nudeoprotein antigen is added to a monolayer of infected chick cells, then fluoresceinconjugated rabbit anti-guinea pig igg. (courtesy dr. n. j. dimmock.) immunofluorescence. the simplest way of identifying a newly iso lated virus is by fluorescent-antibody staining of the infected cell monolayer itself (plate - ). this can provide definitive diagnosis within an hour or so of recognizing the earliest suggestion of cytopathic effect. immunofluorescence is best suited to the identification of monotypic genera, or genera of which only a single species affects that particular species of animal, or to epidemic situations when a particular virus is suspected; otherwise, replicate cultures must be screened with a range of antisera. the advantages and disadvantages of monoclonal anti bodies, in comparison with polyclonal or "absorbed" sera, discussed below in the context of radioimmunoassays, apply equally to other serological procedures, including immunofluorescence and neutrali zation. electron microscopy and immunoelectron microscopy. these pro cedures (see plate - ) are most useful in the rapid identification of cell culture virus isolates, as well as directly on specimens (see below). elec tron microscopy allows identification only to the level of family, whereas immunoelectron microscopy using suitable specific antibody may per mit finer distinctions to be made. complement fixation. for the complement fixation test, the acute and convalescent sera are heated ( °c for minutes) to inactivate complement, then serially diluted in a plastic tray. two to four units of antigen (e.g., a crude preparation of live or inactivated virus) are then added to each serum dilution together with units of complement, derived from a guinea pig. the reagents are allowed to interact at °c overnight, to allow the complement to become "fixed/' sheep erythrocytes, "sensitized" by the addition of rabbit antiserum to them ("hemolysin") are then added and the trays are incubated at °c for minutes. in those cups where the complement has been fixed by the virusantibody complex, the hemolysin fails to lyse the sheep erythrocytes; where complement is still present, they are lysed. crude cell culture supernatants often used for complement fixation tests contain not only mature virions but a range of soluble antigens, both structural and nonstructural. since many of these are shared by many or all viruses within a particular genus or family, e.g., mastadenovirus, they will cross-react with antibodies raised against any other member of the genus or family. this property makes complement fixation a useful method for preliminary screening of an isolate-to place it within the correct family or genus. immune-adherence hemagglutination is basically a somewhat simplified version of the complement fixation test; currently it is applied more often to the detection of antibody than that of antigen. hemagglutination and hemagglutination inhibition. virions of several viral families bind to red blood cells and cause hemagglutination. if antibody and virus are mixed prior to the addition of red blood cells, hemagglutination is inhibited (table - ; plate - ). the hemagglutination titer of certain viruses, e.g., canine distemper virus, may be increased by dissociation of the virions with detergents. antisera may have to be pretreated to remove nonspecific inhibitors of hemagglutination (see chapter ). the hemagglutination inhibition test is sensitive and, except in the case of the togaviruses, highly specific, since it measures antibodies binding to the surface protein most subject to antigenic change. moreover, it is simple, inexpensive, and rapid, and is therefore the serological procedure of choice for identifying isolates of hemagglutinating viruses. virus neutralization. the infectivity of a virus may be neutralized by specific antibody by a variety of mechanisms (see chapter ). serum must first be "inactivated" by heating at °c for minutes to remove nonspecific virus inhibitors. serum-virus mixtures are inoculated into appropriate cell cultures, which are then incubated until the "virus titer only" controls develop cytopathic effects (plate - ). antibody, by neutralizing the infectivity of the virus, protects the cells against viral destruction. in keeping with the general trend toward miniaturization, most neutralization tests are now conducted in disposable nontoxic sterile plastic trays with, say, flat-bottomed wells in each of which a cell monolayer can be established. virus-antiserum mixtures can be added to established monolayers, or, more usually, serum dilutions are made in the wells, a standard amount of virus is added, and the mixture incubated, after which cells are added. in the standard neutralization test the end point is read by cytopathic effect, the titer of the serum being defined as the highest dilution that inhibits the cytopathic effect. in a version of the neutralization test known as the plaque reduction assay, cell monolayers inoculated with virus-serum mixtures are overlaid with agar or methylcellulose and incubated until plaques develop (see plate - ); the end point is usually taken to be the highest dilution of serum reducing the number of plaques by at least %. if a newly isolated virus proves to be "untypeable," i.e., not neutralizable by antisera against any of the known serotypes, it may be a novel serotype, or it may indicate a mixed infection with two distinct viruses, or aggregation of virions in the specimen. aggregates can be removed by vigorous agitation, filtration, or, in the case of some nonenveloped viruses, dispersed with sodium deoxycholate, prior to repeating the neutralization test. for most routine diagnostic purposes it is usually not necessary to "type" the isolate antigenically, even to the degree just described. sometimes, however, important epidemiological information can be obtained by going even further, to identify differences between "variants" or subtypes within a given serotype (see table - ). short of determining the complete nucleotide sequence of viral nucleic acid, the most useful methods of doing this are by oligonucleotide fingerprinting of viral rna or the determination of restriction endonuclease fragment patterns of viral dna. with rna viruses, viral rna is labeled with p during replication of the virus in culture; the labeled rna is phenol-extracted from purified virions, digested with ribonuclease tl, and the resulting oligonucleotide fragments separated by two-dimensional polyacrylamide gel electrophoresis, or by cellulose acetate electrophoresis followed by deae-cellulose chromatography. autoradiography reveals a // fingerprint ,, unique to that particular viral strain. an example of the epidemiological use of this technique to trace the origin and spread of foot-and-mouth disease virus in europe in is described in chapter . similarly, viral dna prepared from virions or infected cells can be cut with appropriately chosen restriction endonucleases and the fragments separated by agarose gel electrophoresis. when stained with ethidium bromide or silver, restriction endonuclease fragment patterns (also called fingerprints) are obtained. the method has found application with all dsdna virus families, particularly in epidemiological studies, but also in understanding pathogenesis. depending on the viral family, the resolution of these methods is such that different isolates of the same viral species may be distinguishable, unless they come from the same epizootic. minor degrees of genetic drift, often not reflected in serological differences, can sometimes be detected in this way. the isolation and identification of a particular virus from an animal with a given disease is not necessarily meaningful in itself. fortuitous subclinical infection with a virus unrelated to the illness in question is not uncommon. koch-henle postulates (see chapter ) are as apposite here as in any other microbiological context, but are not always easy to fulfill. in attempting to interpret the significance of any virus isolation, one must be guided by the following considerations: . the site from which the virus was isolated is important; e.g., one would be quite confident about the etiological significance of equine herpesvirus isolated from the tissues of a -month-old aborted equine fetus with typical gross and microscopic lesions, or of distemper virus isolated from the cerebrospinal fluid of a dog with encephalitis, because these sites are usually sterile, i.e., they have no normal bacterial or viral flora. on the other hand, recovery of an enterovirus from the feces, or a herpesvirus from a nasal or throat swab may not necessarily be significant, because such viruses are often associated with inapparent infections at these sites. . interpretation of the significance of the isolation in such instances will be facilitated by recovery of the same virus from several cases of the same illness during an epizootic. . knowledge that the virus and the disease in question are often causally associated provides confidence that the isolate is significant. it is appropriate to conclude this section with some remarks about safety precautions in virus diagnostic laboratories in general and regulations about exotic viruses in particular. diagnostic virology is one of the less hazardous human occupations, but over the years a number of deaths have been caused by laboratory-associated infections. some of the commoner hazards are listed in table - . it is important to note that many of the procedures that may be dangerous for laboratory workers, particularly aerosolization, may also be sources of laboratory contamination-something that may give rise to mistaken diagnoses and sometimes a great deal of misdirected administrative action. precautions to avoid these hazards consist essentially of good laboratory technique, but special measures may be needed. mouth-pipetting is banned. gowns must be worn at all times, and gloves for anything besides personal hazard, exotic animal viruses pose special community risks such that major developed countries with large livestock indus- tries support special laboratories for their investigation. these are the so-called maximum containment laboratories, popularly designated by their location, e.g., plum island in the united states, pirbright in the united kingdom, and geelong in australia. "restricted" animal viruses in the united states and australia, the importation, possession, or use of which is prohibited or restricted by law or regulation, are listed in table - . we use the term direct identification, in contrast to virus isolation, to encompass a variety of methods that can be used to detect and often identify the etiological agent by the direct demonstration of virions or viral constituents in the tissues, secretions, or excretions of infected animals. although they do not provide the laboratory worker with a culture of the causative virus for further study, these direct methods have great advantages in terms of speed, cost, and the number of samples that can be examined. they can be subdivided into methods used to detect virions, viral antigens, or viral nucleic acids. the introduction of negative staining procedures, together with a realization that in many clinical situations the concentration of virions frequently exceeds the critical lower limit of per milliliter required for visualization in the electron microscope, has led to the use of this instrument for rapid viral diagnosis (plate - ). the procedure is particularly suited to enteric infections, in which a crude fecal suspension can be clarified by low-speed centrifugation, followed by high-speed centrifuga tion to yield a pellet for negative staining. in addition to its value in the recognition of known viruses, this technique has led to the discovery of new viruses of etiological importance in diarrheal diseases which were, and in some cases remain, uncultivable (e.g., some adenoviruses, astroviruses, caliciviruses, coronaviruses, parvoviruses, and rotaviruses). the procedure is also suited to viral infections of the skin and mucous membranes, the appropriate specimen being scabs, vesicular fluid, or scrapings made with a scalpel. also, as described earlier, electron microscopy can be used for the rapid identification of viruses isolated in cell culture, allowing immediate and definitive diagnosis to the family or sometimes the genus or species level. the sensitivity of electron microscopic methods can be enhanced by the use of immune serum, by a procedure known as immunoelectron microscopy. the sample, usually clarified by low-speed centrifugation, is mixed with antibody, and after overnight interaction, the immune complexes are pelleted by low-speed centrifugation and the pellet negatively stained. the antibody used may be serum from an old animal hyperimmune to a large number of viruses, or it may be type-specific polyclonal or monoclonal antibody, or such antibodies may be used sequentially. solid-phase immunoelectron microscopy procedures have also been developed, in which virus-specific antibody is first bound to the plastic supporting film on the copper grid. sensitivity is enhanced by a double-layering procedure, in which staphylococcal protein a (which binds the fc moiety of igg) is bound to the film, then virus-specific antibody, to which the sample is then added. . negative staining for electron microscopy. (a) direct staining: virions of bovine papular stomatitis virus. an electron-opaque stain ( % phosphotungstic acid, ph . ) was mixed with scrapings from the lesion and applied to plastic film supported by the copper electron microscope grid (xl , ). (b) immunoelectron microscopy: an isolate of foot-and-mouth disease virus type o was incubated with homotypic antiserum and stained with phosphotungstic acid (χΙΟΟ,ΟΟΟ). note aggregation of virions by antibody. [from e. p. ] . gibbs et al., vet. ree. , ( ) .] these methods are based on direct interaction between virions or viral antigens, in situ in tissues or in excretions or secretions, and specific antibodies which are prelabeled in some way so as to permit the ready recognition of the interaction. the methods are specified by the method of labeling used: immunofluorescence, immunoperoxidase staining, radioimmunoassay, or enzyme-linked immunosorbent assay (elisa). vir- al antigens can also be detected by such time-honored serological procedures as precipitation and complement fixation. immunofluorescence. its specificity, sensitivity, rapidity, and relative simplicity make immunofluorescence a procedure of singular importance in the rapid diagnosis of viral infections. the prototypic example of immunofluorescence is the diagnosis of rabies, for which it has been the standard test for more than years (see chapter ). it is now being used for a wide range of viruses. immunofluorescence can be applied to smears and frozen sections of tissues or organs. two alternative staining procedures are used: ( ) direct immunofluorescence, in which the antiviral antibody is conjugated to the fluorescent dye, fluorescein, and ( ) indirect ("sandwich") immunofluorescence, in which an antiimmunoglobin specific for the animal species providing the antiviral antibody is conjugated to fluorescein ( fig. - ) . for instance, an acetone-fixed smear or frozen tissue section is treated with virus-specific antibody (prepared, say, in rabbits), then rinsed before the second antibody, a fluorescein-conjugated anti-rabbit immunoglobulin made in goats, is added. indirect procedures have two significant advantages over direct-staining procedures: . if antibodies to different viruses are raised in a single animal species, e.g., rabbits, then only a single conjugated antibody is required. . the amount of bound labeled antibody is greatly augmented, hence the method is much more sensitive. although simple in principle, the effective use of immunofluorescence demands careful attention to many technical details if false positive results are to be avoided. in addition to immunofluorescent staining of specimens taken directly from clinical cases, the method is an important adjunct in the identification of viruses isolated in cell cultures. it may also be used in reverse, for the detection of antibody in serum. slides containing viral antigen, either smears, sections, or, more usually, cell cultures, are prepared in large numbers and stored at - °c. for use, they are flooded with the serum under test and a second fluorescein-conjugated antispecies antibody is used to detect the bound antibody. special care needs to be exercised in the application of immunofluorescence to herpesviruses, in that some herpesvirus-infected cells are known to express fc receptors on their plasma membrane; such receptors bind all igg molecules, not only those with herpesvirus specificity, hence additional controls are required. staining. an alternative method for locating and identifying viral antigen in infected cells employs an enzyme-labeled antibody. the procedure requires less expensive equipment than immunofluorescence-an ordinary light microscope is used-and produces a morphologically clearer, nonfading, permanent preparation. the procedures and principles are similar to those of immunofluorescence. the conjugated antibody, bound to antigen by a direct or indirect procedure, is detected by adding a substrate appropriate to the particular enzyme; in the case of peroxidase this is h mixed with a benzidine derivative which forms a colored, insoluble precipitate in the presence of enzyme. a disadvantage of the technique is that endogenous peroxidase present in the cells of many tissues, particularly leukocytes, produces false positive results. this problem can be circumvented by meticulous technique and adequate controls. in radioimmunoassay the label is a radioactive element, commonly i. the method is exquisitely sensitive, enabling viral antigens to be detected at concentrations as low as ~ m. many protocols for radioimmunoassay s have been devised. both direct and indirect methods can be used, the principles being the same as for immunofluorescent staining (fig. - which the "capture" antibody (or antigen) is bound to a solid substrate, typically a polystyrene tube or bead, or to the wells of a plastic microtiter plate. in the simplest format ( fig. - , left) the sample suspected to contain virus or viral antigen is allowed to bind to the bound antibody, then after washing, i-labeled antiviral antibody ("detector" antibody) is added. after a further washing, the bound labeled antibody is mea sured in a gamma counter. a more commonly used protocol is the indirect radioimmunoassay, in which the detector antibody is unlabeled but a further layer, i-labeled anti-igg, is added as "indicator" anti body. (the antiviral antibodies constituting the capture and detector antibodies must be raised in different animal species; see fig. - , right.) enzyme-linked immunosorbent assays (elisa). elisa (also known as enzyme immunoassay, eia) offers the same sensitivity as radioim munoassay without the inherent disadvantages of expensive isotopes of short half-life and the need for safe handling and disposal and a costly gamma counter. the basic principles are similar to those of radioim munoassay ( fig. - ) . antibody is bound to a solid phase, usually the wells of a microtiter tray. samples suspected to contain antigen are added to the wells. after an appropriate reaction time, the wells are rinsed and a second virus-specific antibody that has been conjugated to an enzyme is added. after allowing this to bind, the contents of the well are rinsed and a substrate for the enzyme is added. the assay is read by a color change in the substrate and can be made quantitative by serially diluting the antigen to obtain an end point or by photometrically reading the amount of color change, a reflection of the amount of enzymeconjugated antibody bound. as in radioimmunoassay, there are many variations in protocol, e.g., exploiting the very high affinity of avidin for biotin ( fig. - , right) . moreover, if antigen is bound to the plate first, the procedure is equally suitable for the detection and quantitation of viral antibody. elisa procedures have been developed for a wide variety of applications in veterinary medicine. at one level, kits have been marketed for the rapid diagnosis of a number of important viral diseases by veterinary practitioners themselves. at another level elisa procedures have been automated by the introduction of automatic dispensing, washing, and spectrophotometric reading and recording instruments, that permit hundreds of samples to be processed in a day, e.g., in the testing of swine for pseudorabies antibodies. immunodiffusion (precipitation-in-gel). if wells are cut in agar and antibody and antigen are placed in separate wells, the two diffuse toward each other (immunodiffusion) and form visible bands of precipitate (plate - ). several ingenious applications of the procedure have been developed and the test is widely used for the diagnosis of some diseases of domestic animals (e.g., bovine leukemia, equine infectious anemia; see chapter ). although now considered too cumbersome a procedure for general use in the rapid detection of viral antigen, the complement fixation procedure is still employed for the rapid and specific detection of foot-and-mouth disease viral antigen in vesicular fluid, providing both rapid diagnosis and specific typing of the virus involved. if dsdna is separated ("melted") into single strands by heat or alkali treatment, the single strands will, under appropriate conditions, reanneal to each other, or competitively to an identical or related complementary strand. if the original dna is labeled with either p or s, it may be used as a probe for the detection of related dna in infected cells. this procedure, known as in situ hybridization, can be made even more specific by using probes that are shorter than the full-length genome. it can also be made more sensitive by maximizing the amount of label incorporated into the probe. hybridization is detected by autoradiography. recently, nonradioactive hybridization procedures, based on the incorporation of biotin-conjugated nucleotides into the dna probe, have been developed; avidin, which binds strongly to biotin, is subsequently added. the avidin is detected by elisa, immunofluorescence, or immunoperoxidase staining. in situ hybridization procedures are particularly useful when viral dna is present in cells but is not expressed, as with integrated retroviral dna, or episomal dna in some papo va virus-infected cells. probes can be made highly specific by selection from a collection of cloned fragments of the whole viral genome. the probes are labeled by in vitro nick translation procedures, and are then applied to nitrocellulose blot transfers of the animal tissue (active hybridization) or to nitrocellulose blots taken from gels on which viral nucleic acid has been separated (southern blotting), or from nitrocellulose onto which viral nucleic acid-containing samples have been spotted (dot-blot hybridization). these procedures have proved of great value in virus research, but it remains to be seen to what extent nucleic acid probes and hybridization procedures displace other methods for rapid diagnosis of viral infections. it may have advantages over virus isolation in the case of viruses that are noncultivable, slow growing, dangerous, or nonviable as a result of suboptimal conditions of transport or storage. detection of viral antibody can be used for the diagnosis of viral infections, either in individual animals or in populations. the method is particularly useful in the latter context, since serum samples are readily obtained with simple equipment, in contrast to special requirements, time, and effort needed for collecting samples for virus isolation. furthermore, tests for antibody such as elisa lend themselves to automation, so that large numbers of samples can be tested. they form the basis of epidemiological surveys and of control and eradication programs, but have major limitations in diagnosis. for diagnosis in the individual animal, paired sera are tested for specific viral antibody, the first sample being taken when the animal is first examined (acute-phase serum), and the second sample - weeks later (convalescent-phase serum). a rise in antibody titer between the first and second samples is a basis, albeit in retrospect, for a specific viral diagnosis. sometimes the demonstration of antibody in a single serum sample is diagnostic of current infection, e.g., with retroviruses and herpesviruses, since these viruses establish lifelong infections. however, in such circumstances there is no assurance that the persistent virus was responsible for the disease under consideration. detection of antiviral antibody in presuckle newborn cord or venous blood provides a basis for specific diagnosis of in utero infections. it was used, for example, in showing that akabane virus was the cause of arthrogryposis-hydranencephaly in calves (see chapter ). since transplacental transfer of immunoglobulins is rare in domestic animals (see table - ), the presence of either igg or igm is indicative of exposure of the fetus to antigen. serological methods based on the detection of virus-specific igm may also be used for the specific diagnosis of recent viral infection, since antibodies of the igm class appear first after primary infection and de-clines to relatively low levels, compared to igg, by about months after infection. however, the method has not yet been much exploited in veterinary medicine. technical advances such as miniaturization (microtiter plates), automation for large numbers of samples, monoclonal antibodies, and the development of diagnostic kits such as latex agglutination assays for detecting specific igm, have resulted in a revolution in the approach to diagnostic serology in human medicine. the costs, coupled with the technical problems associated with the large number of animal hosts and their many viruses, have delayed the development of these procedures in veterinary medicine, but their use can be expected to expand considerably in the future. however, screening programs to establish regional or national prevalence rates for particular viruses, based on detection of specific antibody in single serum samples, are an essential feature in defining the epidemiology of viruses of domestic animals. biosafety in microbiological and biomedicai laboratories oligonucleotide fingerprinting of viral genomes diagnostic virology using electron microscopic techniques rapid virus diagnosis radioimmunoassay in diagnostic virology new developments in practical virology laboratory diagnosis of viral infections diagnostic procedures for viral and rickettsial infections manual of clinical microbiology recent advances in virus diagnosis guide to the collection and transport of virological specimens laboratory diagnosis of viral diseases laboratory safety: principles and practices manual of clinical laboratory immunology automated systems in viral diagnosis immunochemistry of viruses. the basis for serodiagnosis and vaccines enzyme immunoassays for the detection of infectious antigens in body fluids: current limitations and future prospects key: cord- -fcno z authors: nan title: molecular aspects of viral immunity date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: fcno z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv ). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between and days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd + and cd + t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd + and cd + cells were detected by immunohistochemistry at days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il- , il- , il- , il- , il- , tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il- , tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd + cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp was both necessary and s f i c i e n t to cause retention of class i and icp expression in fibroblasts caused the cells to resist lysis by cd + t lymphocytes. icp is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp are being assessed in proteosome and tap transporter assays. gst-icp fusion proteins tightly bind a . kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp binds to a single cellular protein of = kda on ligand blots. these proteins are good candidates as cellular targets of icp and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp is very effective i n blocking cd + t lymphocyte responses in vivo, perhaps explaining the predominance of cd + vs. cd + anti-hsv ctl i n vivo. we expect that icp may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma , john p. carroll , sara r. abromson-leeman , joseph c. laning , martin e. dorf , and thomas l. benjamin . idepartment of pathology, emory university school of medicine, atlanta, ga , and department of pathology, harvard medical school, boston, ma . susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h- haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c wbida mouse and the highly resistant but mhc-identical (h- k) c bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c wbida x c bwcd.i) x c bwcdj backcross mice cosegregated with mtv- . inheritance of mtv- showed perfect concordance with absence of peripheral vp + t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv- showed no evidence of recombination between pyvs and mtv- . strongly biased usage of vp by (a) polyoma-specific cd + ctl from virus-infected c bwcdj mice and by @) cd + t cells infiltrating a polyoma tumor in a virus-immune c bwcd.i host provide further evidence that t cells bearing this mtv- sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv- sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h- , non h- , level of cd + t cells, of cd + t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd + t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd + t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd + t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd + t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type (ad ) and human papillomavirus type (hpv )) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad e a protein to which ctl clones were directed that could eradicate ad e induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad e b protein. these ad eib specific ctl clones, however, were as effective as the ad e a specific ctl clones in the eradication of ad e induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad e induced tumor cells with the activated ras oncogen the possibility of ad e b specific ctl to recognize the ad e induced tumors was eliminated whereas the ad e a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv induced mouse tumor model an immunosubdominant ctl epitope was identified in the e protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv , that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a * , a * , a" , a* and a * ). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of . class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment . , biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual ' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are - 'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the -month old infant, an rsv vaccine will need to be effective when given to -month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: ) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: ) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd + cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd ' and cd + t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd ' t cells were more pathogenic than cd + t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m ( k) protein were predominantly cd ' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd + t cells, and ctl. mice primed to g protein gave rise to predominantly cd ' t cells producing th cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type cytokines, notably il- , appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd t cells and protective effects by cd ' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d ' t cells and b cells (immune complexes) in maintaining cd + t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn . the analysis of the development and memory phases of the cd + "helper" n h ) and cd + cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type ) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose ( mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd + th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd -high, l-selectin-low, cd d (vla- ) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il- production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b . and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il- vs il-i /ifny are present at the initiation of the response of either cd or cd cells they dictate that the effectors generated will be polarized either towards il- and il- secretion or il- and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd effector cells not re-exposed to ag, produce no cytokines and they die within - days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il- produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il- and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to . , the s ikes re ain their neutral configuration ( ). probably as a consequence, the viral infectivity is totally preserved after an exposure of hours at p . an cf t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein ( ). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position , which is part of antigenic site , is important, although not essential, for the viral virulence. similarly, the arginine , which belongs to antigenic site , is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in ). viral strains mutated at arginine have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody ( ). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus ( ). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg a antibody; (c) activated (le., ll- rc) t cells infiltrating the cns are exclusively of the cd + phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids - of the vp capsid protein. a thl line specific for vp - exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein . prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by amino acids between residues and . au of the tg(mhu m) mice developed neurologic drsease - days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu m) mice with cjd prims produced mhu mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu m) mice wen differenl about % of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane > days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene - -fold was not deleterious to themiw but it did shorten scrapie incubation times from - d to - d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon (corresponding to codon in hurp) pmdnced spontaneous, fatal neurcdegeneration between and d of age in two lines of tg(mohp-pio l) mice designated and . genetic crosses of tg(moprp-p l) mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between and days of age. the t g~o p r p -p l o l l ) ~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast the tg and mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg overexpress the mutant transgene - -fold and develop fatal neurodegeneration behveen and d of age. tg mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md - . cd ' lymphocytes (tcd +) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of to residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors ( residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh -terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the residue precursor than from the nh -terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv- envelope protein that contain overlapping determinants each presented by different class i mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv- envelope to provide help for neutralizing antibody (ab) and cd + cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p peptide from the v loop of hiv- iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p segment of both iiib and mn strains of hiv- in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all responses, neutralizing ab, ctl, and th helper cells. the ctl specific for the mn strain p peptide crossreacted with strains sc, sf , , and cdc . the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it to -fold more potent in binding to the class i mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv- sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a , with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/ . cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m ) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/ and a/hawaii/ . this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/ virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il- , months after immunization were also observed . immunization of rabbits with dna encoding l , the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type (hsv- ), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv- . dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, - , dna cell biol, the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e ) codes for more than polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e gpl k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by distinct viral polypeptides, of which (the ad e . k or the complex of the . k and . k proteins) are coded in the e region. the e polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e . k or ad e gpl k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e genes in viral constructs, several transgenic mouse lines containing the ad e genes have been constructed for these experiments. the e genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e transgenic animals (h- b'd) have been transplanted allogeneically to h- d recipients and remained viable, secreting insulin until the end of the experiment at days; in contrast, control nontransgenic islets of the same genotype were rejected by - days. the e genes behind the native e promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e promoter of the transgene is responsive to stimulation by the ad e a following infection with an e minus ad and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr .) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than , cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in , a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus . k promoter and the s segment under control of the k promoter. the m segment, which encodes the g and g envelope proteins, was included because of our findings that: ( ) immunization with vaccinia or baculovirus-expressed g and g induced a neutralizing and protective immune response in hamsters; and, ( ) neutralizing antibodies to g or g could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd t cells and in the induction of virus-specific ctl activity. thereafter, the cd t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl- in the t and b cells and in c bl/ ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd , thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma thfectious disease unit, massachusetts general hospital, boston, ma §tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo for gag and / for envelope. cd + lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd + cells. suppression mediated by unstimulated cd autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd + cells activated by an anti-cd -specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv- molecular clone (nl ) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p and gp were observed in chimpanzees infected with n u and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv- , and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to years with cd cells > /mm . methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn- infected autologous cd cells, using target cells infected with recombinant vaccinia viruses expressing hn- proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for years, who by qc-pcr had <i viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv- variants, and both the p and p epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr region of the tcr in an individual with a vigorous response to gp suggested that approximately % of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv- infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf- ) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il- and il- receptor genes. to investigate the role of irf- related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o pfu of coxsackie virus (cvb ). homozygous irf- (+) and heterozygous irf- (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf- -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf- regulated gene products such as inos and ifn are essential for the early defense against cvb -induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd and b-cell populations and a higher proportion of macrophages and cd cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il , il and il , but higher levels of il and ifng in the elderly as compared to the young mice. the addition of mf to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il , il and il levels for both young and old, while il and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus (cvb ), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb is able to infect normal c bl/ mice and immunocompromised (cd ko and m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers - days pi.. large pathological changes were detectable only in heart tissue of cd ko mice, and were maximal at - weeks after infection. these results suggest the possibility that cd ' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd + and cd ' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p lck are resistant to coxsackievirus b induced heart disease. tamara martino, karen aitken, joseph penninger, jan nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus (cvb ) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p kk are resistant to cvb induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb induced heart disease, cytokine expression in the hearts of cvb infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd + t cells from m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd + t cells seen in normal mice days after lcmv infection, previously presumed to be due to the activity of cd + ctl, still occurs in m-/-mice. however, mice show an earlier rise in percentage and absolute numbers of cd + t cells by days after infection. cd + t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd + t cells in normal mice after lcmv infection. cd + ciz activity is lower in m-/mice and have a later peak than cd + ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in -mice. mice can assume some of the cytotoxic functions of the cd + cell population from normal mice, including immune-mediated pathology. further, cd + t cells from m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd + t cells from normal mice. mice genetically engineered to be deficient in -microglobulin in conclusion, these data suggest that cd + t cells from am-/- australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd and its ligand, in the presence of b cell acting cytokines, such as interleukin (il- ), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd l and il- , did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w lpg, u. k. and *nationallnstirute formedica research, london, nw aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il- has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il- on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il- gene was disrupted in /sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il- . mice were infected intranasally with a strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il- deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd + cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd +t helper (th) cells of the - subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within days pi. c t l s were detected by h- specific lysis of ldv-specific target cells. these target cells were generated by transfection of t -nm cells with a retrovirus vector carrying the gene (ow ) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected t -nih cells and cultured for days in the presence of il- . the ctls had largely disappeared in mice by days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v ) or an isogenic single-site attenuated vee mutant (v ) (single amino acid change at e glycoprotein position from glu-lys) and at , , , , , and hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il- , ifn-y, il- , il- , il- , and il- gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il- , ifn-y, il- , and il- were detected in the lymph node, with no changes in either il- or il- ; however, whereas cytokine elevations were detected by hours after injection of v , elevations were delayed until hours following infection with v . cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il- , and il- were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il- and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il- andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il- . to confirm the importance of the immune system in clearing rotavirus infection we infected rag knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. , : - ) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement b binding protein (bc -bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature , : - ) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc bbp ( kotwal, am. biotech. lab., ) and has also been shown to bind to two important complement components, c and c , and block the complement cascade at multiple sites in vitro (kotwal et al., science , - ; mckenzie, kotwal et al., j. inf. dis. , - . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas , : - ) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature , : - ) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, mol. immunol. : ) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h- haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, ) . various immune regulatory molecules were investigated in inbred susceptible /j ( ) mice and resistant c bl/ j (b ) mice which share the same mhc haplotype. when they were given eid, of sendai virus intranasally, mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in mice. virus was eliminated from the infected lung days earlier in b mice than in mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il- , ifn-y, il- , and il- producing cells were comparable for each strains of mice, while more il- producing cells were present in the lung of mice. however, the numbers of these cytokine producing cells peaked in mice days later than in b mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il- , and il- were present in mice than in mice. recall cytokine production in the draining mln showed only the presence of il- , ifn-y, il- , and il- , while no il- was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in mice. therefore, the susceptibility of mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il- , ifn-y, tnf-a or il- in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il- by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il- by rvv was shown to inhibit the host il- response required for the development of ctl's. no production was also significantly suppressed by il- . rvvs expressing il- or il- , although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i (il- ) doses of >i ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd + t cell expansion and ctl activation, and up to log increases in viral replication [orange, wolf, and biron, j. immunol. : , . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il- induced tnf-cx expression and that lcmv infection synergized with il- for this induction. administration of antibodies neutralizing tnf reversed the il- -induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd + t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il- -treated uninfected mice and were dramatically elevated in il- -treated virus-infected mice, including: ) decreases in body weights; ) elevation of circulating glucocorticoid levels: and ) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il- for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a % decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c bu mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl- , , ', '-tetramethyl indocarbocyanine perchlorate-or pkh -gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately % of the transferred bone marrow cells accumulated in spleens after hrs and % of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn- mrna by in sifu hybridization. treatment with anti-agmi or anti-nk .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box , canberra, act, australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c bu (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c bu mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn- and il- . in contrast spleen cells from infected c bu mice produced ifn-y and l- to levels that were only slightly less than uninfected controls. a- production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd stimulated lymph node cells from infected mice produced elevated ifn- suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il- or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl- proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl- does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il- produced a fully productive vsv infection at all times analyzed, up to weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells ( - fold by week and , fold by week ). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il- and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or -galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed or days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th type cytokines when stimulated with rsv infected spleen cells . however, the levels of - and fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il- were higher in the bulk culture from vac-f primed animals. the il- and il- production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed days after intranasal inoculation produced much lower levels of il- and - while the levels of il- and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day and subside as cd + t cell responses are activated at day post-infection. in contrast, m-/-mice, lacking cd + t cells, have dramatically elevated nk cell responses on day postinfection. the m-/-response is evidenced by increased nk cell activity, as well as up to -fold increases in blast and total nki.i+cd -cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)- independent, whereas day nk cell responses in m-/-mice are csa-sensitive and il- -dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il- and transforming growth factor (tgf- ) were examined. induction of il- mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days , , , , , and post-infection demonstrated delayed induction of il- protein as compared to ctl activation. tgf- , evaluated in biological and elisa assays, was induced maximally at days to post-infection. the kinetics of tgf- production by cells from infected m-/mice was similar to that of normal mice. however, cells from m-/-mice produced il- at early but not at late times postinfection. together, these results suggest that either il- is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il- p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days - after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with pfu of either a wild type strain of mv(chicago- ,chi- ) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi- on d ( . pfu/ third of implant), and moron d ( . pfu/ third of implant). hematoxylideosin stained sections of chi- -infected thymuses showed marked distortion of the cortex and medulla by d with thymocyte poilolosis and decreased cellularity. by d , these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd ,cd and cd were analyzed by flow cytomehy. there were significant decreases in the cd +cd + cell pop-ulation by d with complete loss of all such cells by d with chi- , and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae ) or cd co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d . only rare cells stained both with mv ha and cd or cd . mv ha was not expressed over background on any cd + cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl- ) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd were found to drop dramatically. other cell surface antigens such as cd , cd , and cd were unaffected during infection with myxoma virus. further more, the downregulation of cd by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p lck associates with the cytoplasmic domain of cd we have also examined the association of p lck with cd as well as steady state levels of p lck during viral infection. the modulation of surface cd has also been described in hiv infected t cells suggesting that the loss of cell-surface cd may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s o r us - gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd +. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p a(paucisymptomatic) group than in the pl(asymptomatic) and the p b-f groups (symptomatic group). the cohort of children has now been followed during years, and children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c weeks of age) showed no deletion of their cognate vp * t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp + t cells characteristic of c h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv- endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i (pol ) template. the promoter contains a tata box bp upstream of the transcription initiation site, binding sites for several pol i transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol . in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt- , but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i promoter ( pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used ( pglml), in the range of a typical polymerase in (pol ) promoter (va-i). purified tax, transactivates this promoter -to -fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the . m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i promoter and a unique pol i promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i template containing the base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within h of virus infection, while an indirect mechanism, regulated by type interferon (ifn), operates within h of virus infection. cd expression increased by - fold within h of wnv infection on hef, and by - -fold within h. wnv-inactivated, conditioned supematants removed from infected hef cultures after h incubation did not alter cd expression on unqimulated hef. whereas conditioned supernatants from h-infected cultures increased cd expression by about . - -fold after incubation for h, but not after h, similar to cd induction by ulml of ifn-p. increased cd expression on hef by wnv was also cell-cycle dependent. cd increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd by types and ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus and , and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd expression on hef after h. another alphavirus, ross river, was able to induce cd but only by the indirect mechanism of type ifn-dependent release. poly i.c, also, increased cd expression to the same extent as ifn-p after h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd expression in a manner similar to wnv. the ability of flavivhses to induce increased cd expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position of the pi gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position , not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p microglobulin in the er results in stable cell surface expression of the trimeric mhc- molecule. in previous studies we showed that in hpv- positive cervical carcinomas there was a loss of mhc- protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc- is mediated by an hpv- encoded protein. human keratinocytes were transfected withvarious hpv- constructs including pat , the full length genome, pat esx the full length genome with a premature stop codon in e , puc.et , the e and e oncogenes only, and pkve , expressing e from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap- and mhc- heavy chains in the cells with pat as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat esx and puc.et lines showed comparable tap- and mhc- protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap- and mhc- mrna between the different cell lines. the data indicate that expression ofhf'v- e leads to post-transcriptional loss of mhc- , presumably by interfering with tap. to map and characterize functional differences between e a of ad and adl , we previously constructed a series of hybrid ad / e a genes and used them with ad e b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad e a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd + as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin- from a variety of organisms. many of the numerous immunological properties ascribed to interleukin- are shared by the product of bcrfl and this has led to it being termed viral interleukin- . in order to investigate the activity of viral interleukin-i (vil- ) and its interactions with the human interleukin- receptor we have expressed the protein in a bacterial and the eukaryotic cos- expression systems. the bacterially expressed vil-i was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt .l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp- . a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil- protein that interact with the hil- receptor and confer its biological activity. a number of these mutants have been expressed in the cos- expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil- that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the . k or . k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k l protein shows sequence similarity to the eukaryotic initiation factor a (eif a) that is phosphorylated and inactivated by pkr. the k l protein competitively binds the kinase and blocks host eif a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin- , tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of i-ifna to u cells and binding of i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for fnc fp. direct binding of -ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd + t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase , wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa . antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p -microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids - (h -kk), - (h -kd) and - (h -db). the frameshift was introduced amino acids upstream of the h -kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h -i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h -kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h -dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np - srywairtr was studied using hla b -restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b' in vitro, and for presentation to ctl clones by hla positive targets. even conservative amino acid substitutions of the peptide residues p , , and profoundly influenced ctl recognition, without affecting binding to hla ' . these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l gene segment (but not those using tcr va ) were also sensitive to p substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class . these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla , p i , p and p are "flag" residues with tcr accessible side chains. the e / k protein of human adenovirus type (ad ) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e / k protein mediates binding to mhc molecules. apart from a stretch of approximately conserved amino acids in front of the transmembrane segment, e / k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad e / k protein. we show that e / k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in cells. based on the differential binding of monoclonal antibody tw . and cyanogen bromide cleavage experiments, a structural model of e / k is proposed, in which cys and cys as well as cys and cys are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e / k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e / k molecules. mutation of the three other cysteines at position , and had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e / k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd ' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent - % of the u.s. population are being tested to determine which of abundant cmv proteins they recognize by a cd ' ctl response: p , p , p , ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd ' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a . developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p and gb together with either of two lipid-based adjuvants, commercially available d tapm (bcehringer-mannheim) or mf gth (chiron, emeryville, ca). our preliminary studies with hsv- gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa as a restriction element in the transgenic model. the ctl response to sendai virus in c by mice is directed almost exclusively to a single h- kb-restricted epitope derived from the virus nucleoprotein, npj - (sev- ). analysis of independent t cell hybridomas generated from c by mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev- bound to kb has shown that the side chaiis of peptide residues phpi, gl , a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi and a d protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev- . to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev- analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev- . these data are consistent with a critical role for the gi and a d in governing tcr-sev- eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla . restricted ctl from hla-a . uansgenic mice was the m peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/ / -specific murine ctl that recognized an hla-a . restricted epitope distinct from m . using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/ nsl protein. because these ctl did not recognize targets infected with the a/alaska/ / saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a . specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common amino acid sequence and two distinct sets of bmding mtif residues. however, the n name.r peptide was able to sensitize ctl for half maximal lysis at - fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions and and was not recognized at any tested concentration, although it bound with higher &ity to hla-a . than the peptide from a/pw . the a/pr/ nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c bv mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd + immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd ' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h- b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd + ctl response directed against three wellcharacterized epitopes presented by h- db molecules: " - (fqpq-ngqfi), gp - (kavynfatcgi) and gp - (sgven-pggycl). the h- db motif is characterized by a sequence of to a.a. with two anchor residues: asn at position and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the lcmv peptides and known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides ( / ) did not bind to db. the other (including the epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt .aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt .aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently ~ . rt .a alleles are naturally linked with the tapa or the tapb allelic group . we have set out to characterise various alleles for the rt .a molecule, and find that, for the majority of tapaassociated rt .a molecules, acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt .a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b . seventeen variant peptides were examined for their binding to hla b ; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b positive hiv- infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd + human t cell lines and clones specific for different hiv antigens (gp , p , p ), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p is processed by the host cell into the nonamer peptide p - and is presented to cytotoxic t lymphocytes by the h- kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p to show that the rate of p - production is proportional to the amount of antigen secreted into the host cell cytosol. p is degraded in the host cell cytosol with a half life of minutes. the appearance of p - is coupled to the degradation of newly synthesized p . we have determined the rate of intracellular p secretion and by accounting for the rate of p degradation we estimate that approximately p molecules are degraded to produce one p - epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd + and class i hlarestricted by the b molecule. all three clones recognized lllb and rf but not mn strains of hiv- . using vaccinia vectors expressing truncated versions of the hiv- envelope, the clones were found to recognize an epitope within amino acids - , but not including - of gp . further mapping of the epitope with synthetic -mer peptides overlapping by , or -mers overlapping by , was unsuccessful. the sequence of the region of gp recognized by these clones was compared to the predicted hla- peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of allelic variants of ebv-lcl. by restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described ' peptide epitope from pp was not able to prime the autologous ebv-lcl for killing by the pp -specific ctl, whereas a recombinant vaccinia virus expressing whole pp could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp -specific cd ' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b' allele are recognized and killed by the pp -specific t cell clone, and cell lines carrying ' alleles are recognized by the hiv gp -t cell clone. we conclude that the reported cmv pp b" restricted epitope is not correct, since the ctl in question will only recognize ' alleles in combination with the correct pp epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus " - , ha - and ha - was found to be similar. an amino acid peptide corresponding to ha - which contains the - epitope was transported at a similar efficiency as the amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue , this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at . fmolelminute (+/- . ). the km for this peptide was found to be . nm(+/- . ). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., . previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids - ) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct ( - /r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus ( < n < amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs ( - /r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the r construct was presented at a level equivalent to that seen with the wild-type - /rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den- was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den- will interact with mim and lymphocytes in different manners, we used two isolates --plo strain (obtained from a df patient during taiwan outbreaks) and strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd icdi mab and complement prior to adherence procedure and the purity of mim culture was checked by cd surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den- strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den strain. in addition, this dhf-den strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv- ) and lymphocytic (h ) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that ( ) different strains of dengue virus could orchestrate quite differently with immune cells, ( ) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and ( ) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. ( ) when viral yields were enhanced early than day post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv- using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny . primary isolates of hiv- have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd -based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd binding site (cd -bs) on gp . cd -lgg is a recombinant molecule in which the variable domains of both heavy and light chains of lgg were replaced with the first and second immunoglobulin-like domains of human cd . both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg and cd -lgg were added at pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p production was measured over days of culture and an end-point titer of hiv- in the presence and absence of added antibody was determined. both igg and cd -lgg were found to reduce the original hiv titer from seven plasma samples with high virus titer (> tcid /ml) by up to -fold. this is in comparison to soluble cd which only reduced viral infectivity by -fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd -bs of hiv- gp are able to effectively neutralize primary isolates of hiv- and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of overlapping synthetic peptides corresponding to the entire hpv capsid proteins was used to generate hyperimmune sera. several antisera against different peptides were reactive with intact hpv capsids at titers up to : . . hiv- serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md . previously, we showed that hiv- proteins conjugated to . abortus (ba) could generate anti-hiv- neutralizing antibodies in mice even after depletion of cd * t cells. in this study a -mer peptide from the v loop of hiv- (mn) was synthesized ) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v -klh induced mainly igg , whereas v -ba induced all igg isotypes but lgg a predominated. fecal extracts from mice immunized with v -ba were shown by elsa to contain iga antibodies. sera from these mice bound gp , expressed on the surface of infected cells. sera from mice immunized with v -ba inhibited syncytia formed between cd ' t cells and chronically infected [hiv-i (mn)] h cells. inhibition of syncytia, formed by other hiv- lab. strains correlated with the degree of their homology with the v region of hiv-i (mn). to mimic the efffect of hiv- , mice were depleted of cd ' cells using anti-l t at the time of primary or secondary immunization. following primary immunization, cd + t cell depletion abrogated v -klh antibody responses, whereas responses to v -ba were retained and sera from these mice were able to inhibit gp- mediated syncytia. in secondary responses, cd ' t cell-depletion prevented boosting to v -klh, but v -ba increased anti and syncytia-inhibiting antibodies. these results suggest that: . . abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and . that infection with hiv- with subsequent impairment of cd ' t cell function would not abrogate anti-hiv- antibody responses if . abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh - p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn i, departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c bl/ j(b ) mice treated with the mab mel- to l-selectin have been analysed following i.n. infection with sendai virus. mel- treatment caused a - % decrease in the lymphocyte recruitment to the mediastinal (h ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd + ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel- antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel- -treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd + th precursor cells into lymph nodes. facs sorting for l-selectin+, +, and l-selectin-, b + cell populations from the mln and the cln of normal b mice days post sendai virus infection, showed that the afcs were from the l-selectin-, b + cell population, a population which comprised - % ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv- infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position in gp , is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position , upstream from the v loop, does not involve the same site and does not involve v . substitution of thr or ile also confers resistance. replacement of the v loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v peptide of mn but is affected by regions outside v . the other appears to be conformational and outside v , but its functional recognition is influenced by the v loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids - of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus to fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids - of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p< . ). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e a n d e genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e -specific and cd + t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e protein of hpv was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in groups of mice ( ) those not challenged with e ( ) mice not grafted but challenged with e protein in the ear ( ) mice primed by grafting with hpv e expressing cells and challenged with e protein ( ) mice primed by grafting with x hpv e cells on day , grafted again with lo hpv e cells on day and challenged with e protein in the ear. mice optimally grafted and challenged (group ) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group ) exhibited igg antibody levels comparable to the control group ( ). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp ) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr antibodies was also investigated in hepatitis c viremic individuals and non-viremic patients. a high frequency of positive reaction ( %) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp v loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., ) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = , ) and with serum rotavirus iga titers stimulated by the different reassortants (p < , ). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v profiles were determined using synthetic proteins derived from the amino acid sequences of the v region of laboratory strains of hiv- in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v regions of mn, sf , ny and han/sc. less than % of individual in each group recognized the v peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< . ). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p ag inhibition assays. although most patient serum was capable of inhibiting p ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at days post-infection, timepoints from days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about -fold by days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd + lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd were evaluated in uninfected controls (group l), hiv- positive patients with % cd + t cells (group ), and hiv-i-infected patients with ~ % cd + t cells (group ). most of these subjects also had -color staining for cd \cd ro\cd ra. the appearance of positive cd and cd ro on hivinfected and uninfected cells correlated well (r=. p<.ool). the percentage of cells staining cd +\cd +.(bright plus dim) was . ( %cl . - . ) in group , . ( . - . ) in group , and . ( . - . ) in group . the respective values for these groups that were cd +\cd gbwm was . ( . - . ), . ( . - . ), and . ( . - . ). values for cd +\cd ro+ were . ( . - . ), . ( . - . ), and . ( . - . ), respectively. in single factor discriminate function tests, the %cd +\cd + cells best predicted subject group ( % correct), proving to be a better discriminator than %cd +\cd b'h' ( % ~orrect),cd +\cd ~"" ( l%), cd +\cd ro+ ( %) and cd +\cd ro+\cd ra-( %). overall, no advantage was seen to splitting the cd +\cd + cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd +\cd ro+ compartment into cd ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv- infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp were performed at month and and two boosts of recombinant gpl mn/lai at month and in alum or incomplete freund adjuvant( fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from out of donors : seven out of eighteen ( %) were found to present envelope specific cytotoxic activity at months , , or post immunization ; this activity was characterized as a cd +,cd +, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd + cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al . mhc class i restricted cd ' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il- and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd ' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd + and cd ' t cells by their expression on the surface of distinct d r isoforms. cd ra is expressed on naive or virgin t cells, while cd ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd + t cells. the cd ' cells were stained for cd ro' (pe) and cd ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd +/cd ro+ t cells. similarly, cd +/cd rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd +/cd rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd + t cell response in lcmv infection we have observed a profound activation and proliferation of cd + t cells with a - fold increase in total number peaking at day - post infection. in c bw mice, most of the viral antigen is cleared by day seven, and after day the total cd + number per spleen drops about -fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd + cell remains remarkably stable between day - of the acute infection and for many months thereafter. thus, the decline in the cd ' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd + cells from c bl female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd + cells either by magnetic bead depletion of non-cd + cells, or by facs after staining with anti-cd monoclonal antibody these enriched (> % cd +) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at cd + cells, compared to a frequency greater than / ooooo in naive controls. the memory cd + cells were further characterized as cd rb-low ( / ) , cd -high ( / ), lselectin-low ( / ), and cd d-high (vla- -high) (v ). this is close agreement with other phenotyping studies on cd + memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn . the immune response of influenza a and sendai-virusspecific, memory cd ' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c bu mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated or days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd ' t cells from the ln and spleen resulting in dna fragmentation of - % ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb a cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb a cells were treated with pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by pm of nmda was reversed by pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a to carol s. reiss. pediatrics, university of iowa, iowa city, ia. mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. . % of suckling c bu (kbdb) mice inoculated intranasally with mhv-jhm at days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at - weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c bu mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c bv mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids - (cslwngphl, db), - (rcqifani, kb), and - (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c bu mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of - week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of - week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd + t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases research units, national animal disease center, usoa, agricultural research service, ames, ia a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a . kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in -week-old or -day-old pigs, lltba replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba -infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba , -day-old pigs were infected intranasally with lltpa or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about -fold for lltpa . abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba -infected pigs. while replication of lltbres in the brain progressed until death at days post-infection, replication of lltpa in the brain ceased by days post-infection and the pigs exhibited only mild clinical signs. since lltba is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type (hsv- ) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv- in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of - week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day post-infection. in contrast, in a large group of cba mice studied up until days post-infwtion, viral rna could be detected by both techniques in % of mice until as late as days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v region of the siv env gene. the majority (> %) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd + t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd + t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd + t cells with monoclonal antibody gk . . both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd + t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md ; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn- epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn- subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv- infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv- of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp . because of its early expression during the hsv replication cycle, lcp represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein (nsi) and the lcp sequence of hsv- . the nsi-icp protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing pg of nsi-icp . responder cells obtained from draining lymphnodes were re-stimulated in vitro with p cells lransfected with icp and then lesled for cytolytic activity on icp -p and control p . the induction of icp specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h- d haplotype: a dd restricted epitope from the gp protein of hiv- and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately ?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd- mice were inoculated subcutaneously with x ' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo ed, of influenza h s . au corn mice suffered severe disease and % died. only one of ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was , while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, . in a parallel experiment, no influenza infectivity was detected in the lungs of ha vector immunized mice at days postchallenge. in contrast, / pbs-inoculated mice and / inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of . and . x lo pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from to . ug of dna was inoculated by intramuscular and intradermal routes. from . ug to . ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required to , times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations ( ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl e hpv associated cervical cancer cells express hpv e protein and antibody to hpv e can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e protein of hpv , and expressing e protein in the skin, has recently been described ( ) and these mice develop spontaneous humoral immunity to e protein similar to patients with cervical cancer( ). to determine whether immunisation could induce immunity to e sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv e protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv e expressing tumour cells in nrro. we then used similar immunisation with e iquil a to induce e specific immunity in fvb (h- ) mice. h- qskin @s expressing e were not rejected by e immunised h-zq mice, though immunisation induced antibody to e , and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl e lack a tc epitope in the context of h-zq, or that expression ofe in the skin from the e transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types or , and continued expression of the e and e gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e and e from hpv and with the aim of inducing e and e specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e /e reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv € specific ctl from c bu mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi e alone, with similar recognition of the defined immunodominant h- db restricted epitope, e residues - . ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa mice infected with the laboratory strains of a/pr/ / (hln ) or the mouse adapted a/hk/ (h n ) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h and h strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/ were susceptible to subsequent infection with the a/hk/ strain although they were resistant to weight loss and death. other strains such as a/beijing/ or a/georgia/ provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/ challenge than mice previously infected with a/bei/ and a/ga/ , and were similar to mice that had been previously infected with a/bei/ . thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h- -ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd + ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day after infection. there was little effect on virus clearance. as found previously with cd -depleted h- b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd + t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd + cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd + ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd +, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd + responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type (hsv- ) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv- to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day and day resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv- vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv- . viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl - and plasmodium berghei cs protein - were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam cyj) and dissolved in a % dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and % soya oil in water. wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of mice intra-peritoneally or subcutaneously at . and days. days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of w:l the plasmodium berghei peptide encapsulated in micmpartides gave % iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p ccs - gave a level of lysis of % at an e:t ralioof w . these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type poliovirus (shah sauketf) together with the regular type and type canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp . as previously shown for hypsin-mated type poliovirus @vm alone. trypsin used to modify the type component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with -fold concentrated type component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type poliovirus. no unexpected sideeffects were recorded phase i trials comprised adult volunteers with at least years since the last dose of poliovirus vaccine and children who were due to receive the third dose of the regular immunization schedule at about years. in both groups. individuals received tryipv and were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with . ug of an h expressing plasmid dna (pcmvri ). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at days post boost protection against the lethal challenge was complete at days, month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv- t-cell epitopes: generation of hla-b* -restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv- patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys) and synthetic htlv- peptides which seem suitable for vaccine design. htlv- amino acid sequences were searched for eight to mer patterns carrying the anchor residues of the hla-b* peptide motif at positions two and eight to fourteen. candidate peptides were synthesized according to the matched sequence patterns. their hla-b* affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b* cells. the fourth group (controls) were inoculated with h n (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h n group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp la sequence. seventeen patients participating in a phase i gp protocol and patients participating in a phase i gp protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into well plates containing peptides overlapping the la sequence of gp , pulsed on day with tritiated thymidine and harvested and counted on day . results: the percentage of patient's pbmcs from each trial with an lsi to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp or gp proliferate to multiple peptides within the gp molecule. reactivity from the end of c through early c (lai #i - ) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v loop peptide (la # ). although the percent reactivity to the entire gp molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c (lai # - ). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il- production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] and a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna proteins ( a, b, c). and ebna c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna , , lp or lmp . the ebv-specific ctl lines expressed cd /cd and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class- . these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna family of proteins and lmp a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology, genetic therapy, inc., gaithersburg, maryland replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. : - ; ) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of , and . in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: . f . % vs . * . %. . * . % v s . * . %. and . * . % vs . * . % (meanrts.e.m., n= ; p<o.ol, all comparisons) a t e:t ratios of , and , respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping -mer peptides derived from p . two individuals ( and ) had ctl against p . (a(mino) a(cids) - , krwiilglnknrmysftsi), two others ( and ) recognized p . (aa - , frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p . ( ) and p . (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of aa overlapping -mers and to test for hiv- isiv crossreactivity. both two p . epitopes (possibly restricted by hla-a ) and the p . epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p . epitopes is in progress. the sn p . epitope was also non-crossreactive, despite only one aa difference (position , s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p in humans (johnson ji , , , buseyne jv , , and to p . in cynomolgus macaques (gotch jmprim . , ) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd- and cd- -cells, b-cells and adherent cells, while an average of % of virus was associated with lps-stimulated -cells and adherent cells. in the second experiment the combined function of antigen presenting cells, -helper cells and -cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day , and decreased at days , and post-cvb , infection relative to unlnlected animals. conclusions: these data indicate that cvb , associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb , modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb ,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih t fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of . kb in hind i j and of . kb in hind i j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the . kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv - progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v - cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv - was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a kb transcript in northern blot analysis. a kb cdna was isolated and codes f o r a novel polypeptide o f amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/ / in in uitro assays of viral infectivity of the mouse b-lymphoma a - . . we have found that although infection sensitizes the a cells for recognition by both class i and class i restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/ / from a different passage history, we find that n o t only are the infected a cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a - the therapeutic and prophylactic antiviral efficacy of interleukin- (il- ) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il- commenced hours after mice were infected w i t h hsv, and was continued daily for a total of days. il- therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. . x we~ells/well of both adherent and nonadherent cell types were infected with two different den- virus strains : ( ) thiland strain isolated from the dengue hemorrhagic fever(dhf patient and ( ) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den and dhf virus strain replicated much more efficiently ( . x pfu/ml on day p.i. than df strain; (b) nonadherent cells were times less efficient to produce viral yields than adherent cells (dhf and df strains peaked at . ~ ' pfu/ml and . ~ pfu/ml on day p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi- /crmn k gene trigger an inflammatory response by - hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at - hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice - months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for days. by this method, pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted :lo. scid mice were used as a second detection assay for mcmv. using scid mice and different doses of virus, the lds, of mcmv in scid mice was - pfu. mcmv infections were detected when - pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of spleens, kidneys, and salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after - days. in addition, lytic mcmv was detected in scid mice days after injection with ( )' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp , but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp -negative cell line showed no difference from control viruses, and the presence of hsp did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp proteins act as molecular chaperones and that hsp can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs /bl and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days and postinfection, mln or spleen cells from both strains of mice produced il- , il- , il- and ifn-y, but little or no il- or il- . however, following in vivo treatment with anti-ifn-y antibody, production of l- and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs /bl mice showed little change in cytokine profile. an increase in il- and l- production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of l- and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c /bl mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a amino acid type i membrane glycoprotein with potential n-glycosylation sites at asn , asn , and asn . due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble amino acid form of rgp lacking a transmembrane domain (rgpt ). rgpt was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt was secreted only when asn l was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt . although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of his residues was added to the cooh-terminus of rgpt . this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+ -agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev- locus is reduced to % compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci ( , , and ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad . one of the major isletcell antigens, shows a high degree of homology with the c protein of cbv- . studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp and gad , are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p expression in of ( %). p detection by ipox is usually associated with mutated p . sscp for exons - revealed p alterations in of specimens, and loh at exon was seen in only of evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p . we recently reported that sv induces mesotheliomas in rodents, and that sv -like sequences are present and expressed in mpm. of note, sv large t antigen (tag) binds and inactivates wt p i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p may result from its physicdl binding with tag. tag was detected by ipox in ( %) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p (p = . , fisher's exact text), and preliminary experiments suggest co-precipitation of p and tag. finally, we demonstrate that waf- is not detectable in mpm expressing wt p suggesting that p is inactive. we speculate that tag expression in mpm binds to and inactivates p contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in % of cervical carcinomas and possibly % of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type ' and hla-a '. culture media consisted of rpmi- % human serum supplemented in three cases with u/ml il- , in another three cases with u/ml il- and u/ml il- , and in the final three cases with u/ml il- and u/ml il- . the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c a (hpv, hla-a ') transfected with either the hpv type e or e genes. one patient's cells maintained with uiml il- and ulml il- showed hpv e protein specific cytotoxicity in a hla-a restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k- . the ctl efficiently lysed a c a-e clone but in contrast a vector only transfectant was not lysed. the lysis of c a-e was blocked with a-cd and a-cd antibodies at % and % respectively. facs analysis determined that this ctl population was . % cd 'cds' and . % cd 'cd '. limited e recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv- infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv- infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b restricted epitopes: p - gag and amino acids - of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv- infection may be influenced by an effective host immune response against hiv- and/or by viral properties. to investigate the cellular immune response to hn- we analyzed ctl activity against different hiv- proteins in long-term asymptomatic hiv- infection as well as during rapid progression to aids. methods: longterm asymptomatic individuals with cd counts > celllpl after more than years of infection were selected from the amsterdam cohort study on aids versus subjects who progressed to aids < years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv- ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus isolates from an outbreak of disease in southem india. sequence analysis within the ' untranslated region reveals that these isolates fall into two groups that differ by - % (equivalent diversity to that seen between between published sequences of poliovirus and coxsackie a virus). these two groups of viruses also differ in their cell tropism. isolates defined as group by their 'utr sequence grow equally well on ht cells (a human colon carcinoma cell line) and vero cells. isolates of group , with one exception, grow only on ht cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the ' untranslated region sequence of a group virus but the protein profile and cellular tropism of a group virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c -i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py is the mouse mammary tumor virus superantigen (mtv sad gene, which deletes t cells required for py tumor immunosurveillance in h- ' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i and pedatteck (peru). both strains are susceptible to py as % of infected animals develop a full profile of tumors. crosses between cs br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech , yield f progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs br) x c br) and ((czech i x cs br) x c br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i and peru mice by pcr using primers specific for mtv sag indicates that both strains are negative for proviral mtv sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i genomic dna. these results indicate that, like the c hibi, the pedatteck and czech i contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis- is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis- suppresses primary antibody response more efficiently in infected mice. some of the fts- infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis- show a high degee of homology between this variant and the prototype f-mulv clone . in this study we have attempted to localize the genomic determinant of fis- which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis- and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd ) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th cd t cell subset. interestingly, when cd responses were observed, cd tcell responses were also detectable. . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex- xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp was strain specific, and the polymerase ( d) was the most immunogenic and cross reactive. d) a construct comprising d and the immunodominant vp epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus (ehv- ; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv- . to generate useful amounts of individual ehv- glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv- infection. au three glycoproteins induced serum (elisa) antihodies to ehv- , and ehv- gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv- , protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv- antigen were observed for each of the ehv- glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv- . molecular pathogenesis of ural infeetiom - enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb , infection in adolescent c h/hesnj mice. antigen presenting cell, -helper cell and -cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo plaque forming units of cvb , at day and with lo srbc's at days , , and post-cvbb, infection. splenocytes were harvested days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb , was associated with . % to . % of cd- positive t-cells and w a % to % of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a % and % under non-stimulated conditions. an average of % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. , in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by % and % of residues, and the nt s genome is shorter by nt. we surveyed reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; ( . %) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california ( ), arizona ( ), new mexico (l), and mexico ( ). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to . %. but amino acid distances were less than %. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd + ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a + patients, htlv i-specific cd + ctl restricted by hla-a recognize a peptide derived from the htlv i tax protein (tax - llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd ' tax - specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag - vl&sdfri) presented by hla-a . these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd + t helper cells in this autoimmune response by treating mice with the cw-specific gk . monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c h mice by showing that depletion of cd + cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b o.br mice than in c h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl (h n ) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues - and . . when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl -immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha - subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha - epitope of influenza strain a i m in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha - specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv- gp and in the n-terminus of hla class i beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = . by fisher two tail analysis)in a second, year-longitudinal study of progre.ssors and stable patients we found: ( ) the production of crab was seen in - % of rapid progresson, while the true stables produce only infrequent low-titers crab. ( ) in rapid pmgressors, production of crab preceded by - years the marked drop in cd counts. ( ) crab production did not correlate with the degree of hyperglobulinemia in these patients. ( ) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn- receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn- and - production, without detectable - , and with decreased - secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i - producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th- -type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord- -gy f oyt authors: shetty, mamatha; brown, thelma alfonse; kotian, mohan; shivananda, p. g. title: viral diarrhoea in a rural coastal region of karnataka india date: - - journal: j trop pediatr doi: . /tropej/ . . sha: doc_id: cord_uid: gy f oyt abstract. a total of children below years of age admitted to the kasturba medical college hospital manipal karnataka (south india) were investigated over a period of months to determine the aetiologkal role of viruses in acute diarrhoea. viral aetiological agents isolated were rotaviruses in ( per cent) cases, adenoviruses in ( per cent) cases, corona virus and astroviruses in two ( per cent) cases each. non-viral isolates were cryptosporidium and salmonella typhimurium in two cases each, and entamoeba histolyticaand and shigella flexneri in one case each. in many developing countries nearly two-thirds of diarrhoea used to be of unknown aetiology. the introduction of electron microscopy for the examination of faecal samples led in the s to the discovery of a number of viruses which may cause diarrhoeal disease in man and animals. " in view of the recent recognition of some viral aetiological agents of acute infantile diarrhoea, we conducted the present study to identify viruses as the causative agents of infantile diarrhoea in manipal, a place in coastal karanataka (south india). one-hundred-and-six children aged below years, suffering from acute watery diarrhoea of less than days' duration who attended the out patient clinic of paediatric dept of the kasturba medical college hospital, karanataka, south india were included in the study. of the children, ( per cent) were less than years old, ( per cent) were below year, and the remaining five ( per cent) were between and years old. the stool specimens were also tested for cryptosporidium; other intestinal parasites, and for bacterial pathogens like salmonella, shigella, and vibrio cholerae by previously described methods. ' the stool samples were frozen and stored at - °c for viral testing by electron microscopy at the liverpool university, london. the stool samples were processed for the detection of cryptosporidium by modified ziehl-neelsen staining, sheathers sugar flotation technique, and also by phenol-auramine staining.* detection of rota virus was done by slide latex agglutination test as per the method advocated by the manufacturers (mercia diagnostic ltd, england). cultures showing typical biochemical reactions favouring salmonella, shigella, and vibrio cholerae were confirmed by agglutination with specific antisera. the specimens were also examined by both direct and concentration method for parasitic ova and for fungal isolation. of the total stool samples received, viruses were detected in samples ( per cent). they were identified as rotavirus in ( per cent) cases, coronavirus in two ( per cent) cases, adenovirus in three ( per cent) cases, and astrovirus in two ( per cent) cases each. fifty control infants, without diarrhoea during last weeks served as controls. among the control group, a single adenovirus was seen in the stool sample of a -year-old child. clinical picture of viral diarrhoea was characterized by a high frequency of vomiting, fever, and respiratory symptoms. in six infants, vomiting was the first symptom preceding diarrhoea (table ) . table shows the enteric pathogens (viral and non-viral) isolated from cases of suspected viral diarrhoea. in our study, rotavirus was isolated in children in the - -month age groups. coronavirus, adenovirus and astrovirus were isolated in children between and years of age group. salmonella typhimurium was isolated from two infants below months and a single isolate of shigella flexneri in a -year-old school-going child. frequency of detection of viruses in stool samples were high in winter months (december to february) or in the cold wet seasons than in the dry. diarrhoeal disease is perhaps one of the most important causes of sickness and death among infants and children in developing countries like india. seasonal characters such as prevalence in winter months supported the diagnosis of viral disease. rotaviruses are reported as the commonest cause of acute non-bacterial gastroenteritis. " rotavirus enteritis is generally a disease of infants and young children and appears to have a worldwide distribution. it is common in children of - months old with a peak incidence at - months. rotavirus is responsible for - per cent of all cases of severe watery diarrhoea in young children. in the past two decades, the importance of rotavirus as a cause of illness and mortality has been clearly documented and substantial progress has been made towards developing vaccines to control this agent. enteric adenoviruses are well established as respiratory viruses and are second to rotavirus as the most common cause of pediatric viral gastroenteritis. it is found to be common below years of age, particularly during the first year of life. serotypes of adenovirus responsible for diarrhoea are , , , , and . diarrhoea is often protracted, but vomiting and fever are less prominent than with rotavirus. astrovirus were first associated with gastroenteritis in in a report by macheley & cosgrove who visualized astroviruses on the electron microscope. astroviruses are found to produce clinical findings similar to those caused by rotavirus infection, but dehydration is uncommon here. the recent development of an enzyme-linked immunoassay using monoclonal antibodies has enabled the rapid detection of antigen common to all five serotypes in the stool. they have not been firmly established as a cause of gastroenteritis in humans because of the lack of controlled studies and the small number of patients studied. astroviral disease is most frequent in children from infancy to years of age." coronavirus have also been associated with diarrhoeal illness with the electron microscopic demonstration in faeces. the virus is seen both in ill and well patients. its causal relationship to ge is still questioned by many investigators. appreciation of role of viruses in childhood diarrhoea should lead to a decrease in the wasteful use of antibiotics and a greater emphasis being placed on oral rehydration in the management of the condition. viral diarrhoea is not uncommon in india, but very few reports have been published so far. this may be attributed to the fact that diagnostic centres possessing an electron microscope for detection of viral aetiological agents are very few in india. despite the large amount of investigative work carried out in viral gastroenteritis, an understanding of the natural history and epidemiology of this disease is still lacking. practically all the patients in our study are from rural areas where the people have been in close contact with nature and animals. moreover, this rural population is exposed to unprotected drinking water obtained from open wells, puddles, and streams. sporadic outbreaks of gastroenteritis and diarrhoea along with other water-borne diseases have been reported in this geographical area. ' as regards viral diarrhoea, no reports have been so far published from coastal karnataka in india, and further studies are indicated. shigellosis is one of the commonest causes of morbidity and mortality due to dysenteric illness in developing countries. it is evident from the epidemic of shigelia dysentry reported from west bengal, india. - the disease is worldwide in distribution and affects all the age groups. the reported incidence of shigellosis varies from - per cent. " sigmoidoscopic and histological features have been studied in adults, but not in the pediatric age group. epidemic viral gastroenteritis viral gastroenteritis identification of enterobacteriaceae manual for identification of medical bacteria comparison of sedimentation and floatation techniques for identification of cryptosporidium species oocytes in a large outbreak of human diarrhoea bacteria, parasitic agents and rotavirus associated with acute diarrhoea in hospital inpatient indonesian children viral diarrhoeas in childhood rotavirus, the first years importance of enteric adenovirus and in acute ge in infants and young children astrovirus as a cause of ge in children astrovirus associated ge in children human viral gastroenteritis viral diarrhoea rotavirus and bacterial enteropathogens causing acute diarrhoea an investigation of cholera outbreak in raipur district key: cord- - vynwk authors: galdiero, stefania; falanga, annarita; vitiello, mariateresa; cantisani, marco; marra, veronica; galdiero, massimiliano title: silver nanoparticles as potential antiviral agents date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: vynwk virus infections pose significant global health challenges, especially in view of the fact that the emergence of resistant viral strains and the adverse side effects associated with prolonged use continue to slow down the application of effective antiviral therapies. this makes imperative the need for the development of safe and potent alternatives to conventional antiviral drugs. in the present scenario, nanoscale materials have emerged as novel antiviral agents for the possibilities offered by their unique chemical and physical properties. silver nanoparticles have mainly been studied for their antimicrobial potential against bacteria, but have also proven to be active against several types of viruses including human imunodeficiency virus, hepatitis b virus, herpes simplex virus, respiratory syncytial virus, and monkey pox virus. the use of metal nanoparticles provides an interesting opportunity for novel antiviral therapies. since metals may attack a broad range of targets in the virus there is a lower possibility to develop resistance as compared to conventional antivirals. the present review focuses on the development of methods for the production of silver nanoparticles and on their use as antiviral therapeutics against pathogenic viruses. viruses represent one of the leading causes of disease and death worldwide. thanks to vaccination programmes, some of the numerous diseases that used to kill many and permanently disable others have been eradicated, such as smallpox in [ ] or have greatly reduced the burden of the disease, such as in the case of the paralytic disease poliomyelitis [ ] . however, for some of today's most pressing viral pathogens, there is still no vaccine available. to realize the huge economic impact that several viral diseases cause to the global community, we need only to think to common colds, influenza, various problems due to herpesviruses (from shingles, genital herpes, chickenpox, infectious mononucleosis, up to herpes keratitis, neonatal disseminated infections, or viral encephalitis). other viruses are also able to cause considerable distress and sometimes persistent infections that may lead to cancer or to acquired immunodeficiencies, such as hepatitis viruses (mainly hbv and hcv) or human immunodeficiency virus (hiv). much effort has been expended in attempts to develop vaccines for these diseases, without appreciable success, at least for some of these viruses, namely, hcv, hiv and some herpesviruses. presently, the development of new vaccines for such viruses seems likely to continue to be elusive. together with the risk of emerging or re-emerging viral agents, the field of antiviral compound discovery is very promising. emerging and re-emerging viruses are to be considered a continuing threat to human health because of their amazing ability to adapt to their current host, to switch to a new host and to evolve strategies to escape antiviral measures [ ] . viruses can emerge because of changes in the host, the environment, or the vector, and new pathogenic viruses can arise in humans from existing human viruses or from animal viruses. several viral diseases that emerged in the last few decades have now become entrenched in human populations worldwide. the best known examples are: sars coronavirus, west nile virus, monkey pox virus, hantavirus, nipah virus, hendravirus, chikungunya virus, and last but not least, the threat of pandemic influenza viruses, most recently of avian or swine origin. unfortunately the methodological advances that led to their detection have not been matched by equal advances in the ability to prevent or control these diseases. there have been improvements in antiviral therapy, but with a wide margin of ineffectiveness, therefore new antiviral agents are urgently needed to continue the battle between invading viruses and host responses. technological advances have led to the discovery and characterization of molecules required for viral replication and to the development of antiviral agents to inhibit them. most viruses are, indeed, provided by an extraordinary genetic adaptability, which has enabled them to escape antiviral inhibition and in certain cases to regain advantage over the host by mutagenesis that create new viral strains with acquired resistance to most of the antiviral compounds available [ ] . the course of viral infections is governed by complex interactions between the virus and the host cellular system. all viruses depend upon a host cell for their protein synthesis. thus, all viruses replicate via a broadly similar sequence of events ( figure ). the virus must first bind to the cell, and then the virus or its genome enters in the cytoplasm. the genome is liberated from the protective capsid and, either in the nucleus or in the cytoplasm, it is transcribed and viral mrna directs protein synthesis, in a generally well regulated fashion. finally, the virus undergoes genome replication and together with viral structural proteins assembles new virions which are then released from the cell. each of the single described phases represents a possible target for inhibition. drugs that target viral attachment or entrance have proved to be very difficult to be discovered. in fact, to date, only one entry inhibitor has been approved by the us food and drug administration (fda). this is enfuvirtide (t- ), a synthetic peptide that targets the hiv gp envelope protein to prevent fusion. targeting the early steps of virus entry is a very attractive strategy for therapeutic intervention since the site of action of the inhibitor is likely to be extracellular and therefore relatively accessible; this could be paired by a concomitant action of the same drug on multiple targets to obtain a more effective therapeutic compound. moreover one could expect, in the future, antiviral agents with a broad-spectrum of action against viruses of different families, to be used as first aid compounds against unforeseen viral epidemics or pandemics. due to the outbreak of the emerging infectious diseases caused by different pathogenic viruses and the development of antiviral resistance to classical antiviral drugs, pharmaceutical companies and numerous researchers are seeking new antiviral agents. in the present scenario, nanoscale materials have emerged as novel "antimicrobial agents" due to their high surface area to volume ratio and their unique chemical and physical properties [ , ] . nanotechnology is an emerging field of applied science and cutting edge technology that utilizes the physico-chemical properties of nanomaterials as a means to control their size, surface area, and shape in order to generate different nanoscale-sized materials. among such materials, metal-based ones seem the most interesting and promising, and represent the subject of the present review. nanotechnology is directly linked with physics, chemistry, biology, material science and medicine. in fact, it finds application in multiple aspects of research and in everyday life such as electronics and new material design. however, its use in medical research is probably one of the fastest growing areas in which the functional mechanisms of nanoparticles and especially metal-based nanoparticles are just beginning to be exploited. nanotechnologies have been used to develop nanoparticle-based targeted drug carriers [ , ] , rapid pathogen detection [ , ] , and biomolecular sensing [ ] , as well as nanoparticle-based cancer therapies [ , ] . the use of nanoparticles can be extended to the development of antivirals that act by interfering with viral infection, particularly during attachment and entry. nanoparticles are properly defined as particles with at least one dimension less than nm, and have attracted much attention because of their unique and interesting properties. their singular physical (e.g., plasmonic resonance, fluorescent enhancement) and chemical (e.g., catalytic activity enhancement) properties derive from the high quantity of surface atoms and the high area/volume relation, in fact, as their diameter decreases, the available surface area of the particle itself increases dramatically and as a consequence there is an increase over the original properties of their bulk materials. considering that biological interactions are generally multivalent, the interplay between microbes and host cells often involves multiple copies of receptors and ligands that bind in a coordinated manner, resulting in enhanced specificities, efficiencies, and strengths of such interactions that allow the microbial agent to take possess of the cell under attack. the attachment and entry of viruses into host cells represent a terrific example of such multivalent interactions between viral surface components and cell membrane receptors [ ] . interfering with these recognition events, and thereby blocking viral entry into the cells, is one of the most promising strategies being pursued in the development of new antiviral drugs and preventive topical microbicides [ ] [ ] [ ] . in recent years, the use of metal nanoparticles, that may or not have been functionalized on their surface for optimising interactions, is seeing increasing success. the idea of exploiting metals against microorganisms can be considered ancient; in fact, the use of silver was a common expedient for cooking procedures and for preserving water from contamination. the importance of silver for its curative properties has been known for centuries, in fact, silver has been the most extensively studied metal for purpose of fighting infections and preventing food spoilage, and notwithstanding the decline of its use as a consequence of the development of antibiotics, prophylaxis against gonococcal ophthalmia neonatorum with silver ions was considered the standard of care in many countries until the end of the th century [ ] . silver's mode of action is presumed to be dependent on ag + ions, which strongly inhibit bacterial growth through suppression of respiratory enzymes and electron transport components and through interference with dna functions [ ] . therefore, the antibacterial, antifungal and antiviral properties of silver ions and silver compounds have been extensively studied. silver has also been found to be non-toxic to humans at very small concentrations. the microorganisms are unlikely to develop resistance against silver as compared to antibiotics as silver attacks a broad range of targets in the microbes. considering the broad literature that describes silver, as a bulk material, effective against a wide range of pathogens, silver nanoparticles have been analysed and found to be extremely appealing. the silver nanoparticles have also found diverse applications in the form of wound dressings, coatings for medical devices, silver nanoparticles impregnated textile fabrics [ ] . the advantage of using silver nanoparticles for impregnation is that there is continuous release of silver ions enhancing its antimicrobial efficacy. the burn wounds treated with silver nanoparticles show better cosmetic appearance and scarless healing [ ] . silver nanoparticles have received considerable attention as antimicrobial agents and have been shown to be effective mainly as antibacterial. antimicrobial effectiveness was shown for both gram-positive and gram-negative bacteria [ , ] . the antibacterial activity of silver nanoparticles was mainly demonstrated by in vitro experiments. activity against methicillin-resistant staphylococcus aureus (mrsa) [ ] , escherichia coli [ , , , ] , pseudomonas aeruginosa [ ] , vibrio cholera [ ] and bacillus subtilis [ ] has been documented. low concentrations of silver nanoparticles were able to consistently inhibit e. coli [ ] while the growth-inhibitory effect on s. aureus was minor. synergistic antimicrobial activity of silver or zinc nanoparticles with ampicillin, penicillin g, amoxicillin, kanamycin, erythromycin, clindamycin, chloramphenicol and vancomycin against s. aureus, e. coli, salmonella typhi and micrococcus luteus was observed [ ] [ ] [ ] . also gold nanoparticles have been exploited as antimicrobial agents, mainly as a tool to deliver other antimicrobials or in order to enhance the photodynamic killing of bacteria [ ] . many studies have shown the antimicrobial effects of metal nanoparticles, but the effects of silver nanoparticles against fungal pathogens are mostly unknown; silver nanoparticles, indeed, showed significant antifungal activity against penicillium citrinum [ ] , aspergillus niger [ ] , trichophyton mentagrophytes [ ] and candida albicans [ ] . different types of nanomaterials like copper, zinc, titanium [ ] , magnesium, gold [ ] , alginate [ ] and silver have come up in recent years and most of them have proven to be effective against diverse microorganisms. the present review aims at a description of the reported antiviral activities of metal nanoparticles and their production methods, with particular regard to silver nanoparticles. metal nanoparticles have been studied for their antimicrobial potential and have proven to be antibacterial agents against both gram-negative and gram-positive bacteria [ , , , , ] . theoretically, any metal could be analysed for antiviral activity, however, little effort has been done to determine the interactions of metal nanoparticles with viruses, and only recently some studies have emerged showing that metal nanoparticles can be effective antiviral agents against hiv- [ ] [ ] [ ] [ ] , hepatitis b virus [ ] , respiratory syncytial virus [ ] , herpes simplex virus type [ , ] , monkeypox virus [ ] , influenza virus [ ] and tacaribe virus [ ] . seen the paucity of viruses that have been investigated and the fact that most of the nanoparticles used were made of silver, this section will be instrumental to analyse the inhibitory effect for each single virus (table ) . acquired immunodeficiency syndrome (aids), the disease caused by hiv, is responsible for over two million deaths per year, among more than million people that are infected. highly active anti-retroviral therapy (haart), a treatment regimen that employs a cocktail of drugs to suppress hiv infection, has significantly improved the quality of life and life expectancy of millions of hiv-infected individuals. numerous hiv-infected individuals are currently treated with haart, and these individuals harbor chronic long-term infection; as a result, hiv eventually develops resistance to these drugs, resulting in a need to change medication regimens and a subsequent increase in the cost of treatment [ ] . the replication cycle of hiv- is a complex multistep process that depends on both viral and host cell factors. entry into target cells is achieved through fusion of the viral lipid envelope and the cellular plasma membrane [ ] . the viral component that acts as a fulcrum for mediating fusion is the trimeric envelope glycoprotein composed of two subunits: gp , which binds to the cellular receptor, and gp , which is the subunit bearing the transmembrane segment, and that executes fusion [ ] . following gp binding to the cellular receptor, cd , and a subsequent interaction with ccr or cxcr co-receptors, a conformational change of gp leads to membrane fusion and delivery of the capsid to the cytoplasm. soon after entry, the rna is reverse-transcribed into a complementary dna which is converted to a double-stranded dna, and integrated into the cellular genome. the integrated proviral dna is transcribed to generate full-length progeny viral rna and a number of spliced mrna transcripts. transcription and translation, performed by the cellular machinery, result in the synthesis of viral proteins that together with the progeny viral rna are transported to the site of virus particle assembly at the plasma membrane, where the virus gains access to the extracellular milieu upon budding events [ ] . elechiguerra et al. [ ] were the first to describe the antiviral activity of metal nanoparticles, in fact, they found that silver nanoparticles undergo size-dependent interactions with hiv- . in their investigations, they explored the possibility that physicochemical properties of nanoparticles may depend on the nanoparticle interactions with a capping agent molecule. for this reason they tested silver nanoparticles with three different surface chemistries: foamy carbon, poly(n-vinyl- -pyrrolidone) (pvp), and bovine serum albumin (bsa). foamy carbon-coated silver nanoparticles were embedded in a foamy carbon matrix needed to preclude coalescence during their synthesis. pvp-coated nanoparticles were synthesized using glycerine as both reducing agent and solvent. in this method, a metal precursor is dissolved in a liquid polyol in the presence of a capping agent such as pvp [ ] . synthesis in aqueous solution was performed for bsa-conjugate silver nanoparticles. interactions of silver nanoparticles with hiv- were probed with the aid of high angle annular dark field (haadf) scanning transmission electron microscopy technology. it was possible to obtain sufficient data to determine that the interaction between hiv particles and silver nanoparticles is clearly due to the size of the silver nanoparticle since only nanoparticles within the range of - nm were able to bind to the virus. in particular, nanoparticles were not randomly attached to the virus, but all the three species of nanoparticles established regular spatial relationships with the viral envelope. the most probable sites for interaction were found to be the sulfur-bearing residues of the gp glycoprotein knobs, which being limited in number, may also explain the inability of larger nanoparticles to bind the virus. the capacity of silver nanoparticles to inhibit infectivity of a laboratory-adapted hiv- strain at non-cytotoxic concentrations was determined by in vitro assays, and a dose-dependant inhibition of viral infectivity was reported. in particular, bsa-and pvp-coated nanoparticles showed to possess a slightly lower inhibition efficacy, probably because the surface of the nanoparticle is directly bound to and encapsulated by the capping agent. in contrast, the silver nanoparticles released from the carbon matrix have a greater inhibitory effect due to their essentially free surface area. these findings, however, only provide indirect evidence for their proposed mode of interaction through the binding to gp , therefore, a panel of different in vitro assays was used to elucidate the silver nanoparticles mode of antiviral action against hiv- [ ] . a luciferase-based assay showed that silver nanoparticles coated with pvp were an effective virucidal agent against cell-free virus (including laboratory strains, clinical isolates, t and m tropic strains, and resistant strains) and cell-associated virus. the concentration of silver nanoparticles at which infectivity was inhibited by % (ic ) ranged from . to . mg/ml. the observed antiviral effect of silver nanoparticles was due to the nanoparticles, rather than just to the silver ions present in the solution. in fact silver salts exerting antibacterial effect through silver ions, inhibited hiv- with a therapeutic index times lower than the one of silver nanoparticles. silver nanoparticles inhibit the initial stages of the hiv- infection cycle by blocking adsorption and infectivity in a cell-fusion assay. the inhibitory activity of silver nanoparticles against the gp -cd interaction was also investigated in a competitive gp -capture elisa, which together with the cell-based fusion assay, showed that silver nanoparticles inhibit hiv- infection by blocking viral entry, particularly the gp -cd interaction. besides, silver nanoparticles inhibit post-entry stages of the hiv- life cycle, in fact, the antiviral activity was maintained also when the metal nanoparticles were added h after the cell had been infected with hiv. since silver ions can form complexes with electron donor groups containing sulfur, oxygen, or nitrogen that are normally present as thiols or phosphates on amino acids and nucleic acids they might inhibit post-entry stages of infection by blocking hiv- proteins other than gp , or reducing reverse transcription or proviral transcription rates by directly binding to the rna or dna molecules. silver nanoparticles proved to be virucidal to cell-free and cell-associated hiv- as judged by viral infectivity assays. hiv infectivity was effectively eliminated following short exposure of isolated virus to silver nanoparticles. these properties make silver nanoparticles a potential broad-spectrum agent not prone to inducing resistance that could be used preventively against a wide variety of circulating hiv- strains. pvp-coated silver nanoparticles, being an interesting virucidal candidate drug, have been further investigated as a potential topical vaginal microbicide to prevent transmission of hiv- infection [ ] using an in vitro human cervical tissue-based organ culture that simulates in vivo conditions [ , ] . when formulated into a non-spermicidal gel (replens) at a concentration of . mg/ml, pvp-coated silver nanoparticles were not toxic to the explant, even when the cervical tissues were exposed continuously to the metal for hours, but one minute of pre-treatment of the cervical explant with . to . mg/ml of pvp-coated silver nanoparticles prevented the transmission of cell-associated hiv- and cell-free hiv- isolates. when pre-treatment was carried on for minutes followed by extensive washing the drug conferred almost total protection against hiv- transmission for hours, indicating a long-lasting protective effect by the pvp-coated silver nanoparticles in the cervical explants. a different group [ ] also reported about the antiviral activity of silver nanoparticles that had been fabricated using hepes buffer. they showed that silver nanoparticles exhibited potent cytoprotective and post-infected anti-hiv- activities (at mm a % reduction was achieved) toward hut/ccr cells in a dose-dependent fashion. similar inhibitory activities were reported for the silver nanoparticles when a citrate solution with nabh as the reducing agent was used, while lower activity was observed for gold nanoparticles ( nm, fabricated in hepes buffer). the herpesvirus family consists of more than double-stranded dna viruses divided into , β and  subgroups. only eight herpesviruses are known to commonly infect humans and the remainder are animal herpesviruses infecting a wide variety of animal species. all members of the herpesvirus family cause life-long latent infections and, structurally, all have a linear, double-stranded dna genome packaged into an icosahedral capsid and covered by a lipid envelope with embedded proteins and glycoproteins [ ] . symptomatic diseases caused by hsv- (prototypic -herpesvirus) are generally limited to cold sores of the mouth and keratitis in the eyes, but hsv- is capable of causing life-threatening diseases in immunocompromised individuals, including newborns, patients with hiv or patients undergoing immunosuppressive treatment. transmission among humans requires physical contact and after the initial infection, the virus remains latent in neurons, a key feature of -herpesviruses [ ] . hsv entry into host cells marks the first and possibly most critical step in viral pathogenesis [ ] . five viral glycoproteins have been implicated in the viral entry process: gb, gc, gd, gh and gl. all but gc are essential for entry. the initial interaction, or binding to cells, is mediated via interactions of gc and/or gb with heparan sulfate (hs) [ ] . the significant reduction of hsv- infection in the absence of either viral gc or cell-surface hs [ ] point to a key role of gc high-affinity binding to heparan sulfate (hs) on the cell surface. following binding, hsv entry is achieved through fusion of the lipid bilayer of the viral envelope with a host cell membrane. the core fusion machinery is composed by gb and gh/gl [ , ] , in fact, the complete fusion is only achieved when the three proteins act together. glycoprotein b may act as the premier fusogen [ ] [ ] [ ] , but it seems to need the cooperation of several membranotropic sequences harboured in gh [ ] [ ] [ ] [ ] [ ] . transcription, replication of viral dna and assembly of progeny capsids take place within the host nucleus, and then there is a complex mechanism for the exit of the newly assembled viruses from the cell [ ] . baram-pinto et al. have described in two consecutive works [ , ] the potential of exploiting metal nanoparticles for viral inhibition. their strategy was proved valid against hsv- , but was probably intended and may prove useful against other viruses, such as papillomaviruses (hpv) and hiv. in fact, their anti-hsv- agents are based on the principle that they mimic hs and may compete for the binding of the virus to the cell. also hiv or hpv use hs as a docking site during infection, therefore the nanoparticles described by baram-pinto et al. may be useful as a broad topical microbicide for sexually-transmitted viral infections. another point of particular interest from their work is the analysis of the significance of the carrier core material, in fact they designed two different metal particles, one made of silver, and the other made with gold, but both with the same coating of mercaptoethane sulfonate (mes) intended to mimic the polysulfonated hs and therefore expected to create a high local concentration of binding molecules for improved inhibitory effect. the silver-(ag-mes) and gold-(au-mes) mes nanoparticles were tested in antiviral assays using the wild-type hsv- mcintyre strain. for the inhibition experiments, vero cells and/or virus solutions were treated with ag-mes and au-mes nanoparticles at different time points to analyse the different stages of the viral infection that may be blocked. taken together, their results indicate that sulfonate-capped silver and gold nanoparticles inhibit hsv- infections by blocking the attachment and thereby the entrance of the virus into the cells and/or by preventing the cell-to-cell spread of the virus. the inability of soluble mes and unmodified metal nanoparticles to control viral infectivity stressed the importance of spatially oriented functional groups anchored on a nanoparticle core for viral inhibition. at the same time, antiviral activity shown by both ag-mes and au-mes nanoparticles suggest the possibility of using alternative carrier core materials as well. while these results suggest the versatility of the idea of effective viral inhibitions using functionalized nanoparticles, it also indicates that other core materials could also be efficient as long as they are not toxic to the host cells. respiratory syncytial virus (rsv) belongs to the family paramyxoviridae and infects the epithelium of the lungs and the respiratory tract causing serious respiratory disease, especially in children and older people. no vaccine or adequate pharmaceutical compounds are available, underlining the need for the development of future rsv treatments. the rsv genome consists of a single rna molecule of negative-sense rna, which encodes, among others, for two surface glycoproteins, which are exposed on the viral envelope. these glycoproteins are the (g) protein, which serve as a receptor binding protein, and the (f) protein, which is responsible for the fusion between the cell membrane and the viral envelope. as within the name itself of the virus, following infection of cells, the f protein is expressed on the surface of cells and fuse adjacent cells, giving rise to syncytia formation, a well characterised cytopathic effect [ ] . sun et al. [ ] have utilized silver nanoparticles conjugated to various proteins to study the inhibition of rsv infection in hep- cell culture. in their study, the capping agents used for the silver nanoparticles were: ( ) poly(n-vinyl- -pyrrolidone) (pvp); ( ) bovine serum albumin (bsa); and ( ) a recombinant f protein from rsv (rf ). the preliminary analysis by transmission electron microscopy yielded interesting results on the interaction between silver nanoparticles with rsv virion particles. bsa-conjugated silver nanoparticles seemed to interact with rsv but without a specific association or spatial arrangement, while rf -conjugated silver nanoparticles appeared to be floating freely with no proof of regular attachment. on the other hand, pvp-coated silver nanoparticles were able to bind to the viral surface with a regular spatial arrangement, suggesting a possible interaction with g proteins that are evenly distributed on the envelope of the rsv virion. the hypothesised interpretation for the interaction of pvp-nanoparticles with g proteins is that their small size and uniformity ( - nm), compared to the other (bsa and rf ) coated nanoparticles ( - nm) may contribute to the effectiveness of the binding. since toxicity is an imperative issue regarding pharmaceutical compounds, the cytotoxicity of each of the nanoparticle conjugates was established using the trypan blue exclusion assay, and revealed that all of them (bsa-, pvp-and rf -silver nanoparticles) showed less than % cytotoxicity up to a concentration of μg/ml. silver nanoparticles have to be regarded as potentially harmful especially when intended for treating a respiratory disease such as rsv infections. sun et al. [ ] have, therefore considered that a saturated surface capping composed of a natural biomolecule (bsa) and a biocompatible chemical (pvp) could be able to mask the pure nano-silver surface and thus would reduce toxicity without hampering efficacy. nevertheless, further studies are needed to validate these in vitro data for the use into the clinical setting, and investigation of the toxic effects and fate of nanoparticles after their deposition in the respiratory tract is mandatory for the future development of anti-rsv silver nanoparticles-based therapeutic compounds. hep- cells were infected with rsv mixed with bsa-, pvp-and rf -coated silver nanoparticles and infectivity inhibition was evaluated by microscopic examination for syncytia formation and by immunofluorescence microscopy. neither bsa-nor rf -coated nanoparticles showed any significant inhibition of rsv infection, while pvp-coated silver nanoparticles inhibited rsv infection by %. these results led the authors to conclude that when the silver nanoparticles are conjugated to the pvp protein and mixed with rsv, they bind to the g protein on the viral surface and interfere with viral attachment to the hep- cells resulting in the inhibition of viral infection. hepatitis b virus (hbv) is a partially double-stranded dna virus provided with a lipidic envelope coat. hbv has a strong tropism for hepatocytes, and once it has entered the cell, viral particles migrate to the nucleus where the viral genome is completed to form a covalently closed circular dna (cccdna) that serves as the template for the subsequent steps of viral mrna transcription and formation of the pre-genomic rna (pgrna). the pgrna forms the template for the reserve transcription by the viral-encoded reverse-transcriptase that produce new viral genomes [ ] . nucleotide (adefovir) and nucleoside (lamivudine, entecavir, and telbivudine) analogue inhibitors, which represent approved pharmaceuticals with direct antiviral activity against hbv, target primarily the viral polymerase reverse-transcriptase. although their effectiveness has been proven, raising drug-resistant hbv strains is fast, therefore limiting the use of these antivirals. lu et al. [ ] have analysed monodisperse silver nanoparticles for their ability to inhibit hbv replication. the nanoparticles used in their study were prepared from agno in hepes and measured dimensions of ~ nm (ag ns), ~ nm (ag ns) and ~ nm (ag ns). silver nanoparticles with particle diameters of nm were too toxic for being evaluated as antiviral compound, but nm and nm particles showed only a minor toxicity at the concentration able to inhibit hbv replication. in fact, nanoparticles of both sizes showed potent anti-hbv activities. the ag ns reached % inhibition at µm and % at µm, while the ag ns were slightly more active with % and % at concentration of respectively µm and µm. in the same paper by lu et al. [ ] , the activity of silver nanoparticles was also compared to nm gold nanoparticles (au ns) and other silver compounds with silver in different oxidation states, and the overall results showed that the anti-hbv effects of silver nanoparticles are undoubtedly much more pronounced. in conclusion, silver nanoparticles were able to inhibit the production of hbv rna and extracellular virions probably via a specific interaction between the nanoparticles and the double-stranded dna of hbv and/or direct binding with viral particles. the influenza virus is a highly contagious pathogen that causes annual epidemics in the human population, and is much feared for its potential to generate new viruses able to jump to humans from different animal species and causing pandemics. recently, papp et al. [ ] have described their studies in which functionalized gold nanoparticles were used to inhibit the influenza virus. this is an orthomyxovirus containing a helical capsid with a genome of eight rna segments. the capsid is covered by a lipid envelope containing mainly two virally-encoded glycoproteins, namely hemoagglutinin (ha) and neuraminidase (na), that forms spiky projections on the surface. the virus binds to the cell plasma membrane through an interaction between ha and sialic acid (sa) residues present on glycoproteins and lipids on the surface of the host cell. this is soon followed by a mechanism of receptor-mediated endocytosis that brings the enveloped virus particle inside the cytoplasm but surrounded by a second lipid bilayer besides the envelope, the endosomal one. inside the late endosome, environment acidification triggers a conformational change of ha, which sets in motion a mechanism of protein (ha) mediated fusion of the endosomal membrane with the viral envelope ending with the release of the nucleoproteins and genome fragments into the cytoplasm [ ] . papp et al. [ ] strategy was to functionalize gold nanoparticles with sialic acid (sa)-terminated glycerol dendron with the objective to inhibit influenza virus binding to the plasma membrane. gold nanoparticles of different size were produced: one of nm and a second of nm. they found that nm had no inhibitory effect on the hemagglutination, used to test the ability of the influenza virus to bind to a target membrane. on the contrary, the nm gold nanoparticles inhibited hemagglutination at concentrations in the nanomolar range, demonstrating that the activity clearly depends on the particle dimension and the spatial distribution of the interacting ligand/receptor molecules. the same trend, with a more pronounced activity was observed in influenza virus inhibition assays where the sialylated particles of nm size were found to be effective for influenza virus inhibition, whereas the nm analogues did not show significant impact. therefore, they proved that sialic-acid-functionalized gold nanoparticles are able to effectively inhibit viral infection. monkeypox virus (mpv), an orthopoxvirus similar to variola virus, is the causative agent of monkeypox in many species of non-human primates, but it is also a human pathogen with a clinical presentation similar to that of smallpox. mpv is considered a big threat to human life and therefore research is being carried out to develop drugs and therapeutic agents against this virus [ ] . different size nanoparticles were produced by plasma gas synthesis and used by rogers et al. [ ] in a plaque reduction assay of mpv. the silver nanoparticles used in this work were (ag-np- ), (ag-np- ) and (ag-np- ) nm, and some nanoparticles were also coated with polysaccharide, (ag-ps- ), (ag-ps- ) and (ag-ps- ) nm nanoparticles. these nanoparticles, at concentrations ranging from . to g/ml, were evaluated for mpv inhibitory efficacy using a plaque reduction assay. the main results showed that the ag-ps- (polysaccharide-coated, nm) and ag-np- (non-coated, nm) exerted a significant dose-dependent inhibition of mpv plaque formation, but the mechanism by which this inhibition occurs has not been further investigated. poxviruses enter cells by endocytosis or direct fusion at the plasma membrane, and at least or envelope proteins are involved in the entry mechanism, this is followed by a regulated sequence of events that allow virus replication. many steps in the virus life cycle are still unknown, and this report on the activity of silver nanoparticles is too preliminary to attempt to give a satisfactory explanation of their mechanism of action. probably the silver nanoparticles may intervene in the early steps of binding and penetration by blocking virus-host cell binding by physical obstruction or, if internalised, they can disrupt intracellular pathways important for virus replication. rogers et al. [ ] also described that agno was active as a mpv inhibitor, but at the concentration of µg/ml its toxic effect on vero cells impeded the evaluation of the antiviral activity. interestingly, some of the nanoparticles analysed in the study promoted an increase in the mean number of mpv plaques/well at the highest concentrations used. a potential explanation for these contrasting results could lie in the fact that nanoparticles may tend to aggregate and consequently create on cells areas available for increased contacts between viral particles and the cell membrane, therefore augmenting internalization and plaque formation. however, these data are preliminary and need a more in-depth analysis to draw more significant conclusions. the family arenaviridae is composed of different species of viruses divided into two antigenic groups, the old world and new world (tacaribe complex) groups. the tacaribe complex, in addition to the tacaribe virus (tcrv), includes the viral hemorrhagic fever-inducing viruses junin, machupo, guanarito, and sabia. considering the high transmissibility by person-to-person via the respiratory route, the lack of diagnostic testing, and therapeutic options limited to ribavirin (not a satisfactory efficacy and easily emergence or resistant strains), the arenaviruses are included in the category a list of potential bio-weapons [ ] . tcrv is not a human pathogen, but exhibits a close antigenic relationship with junin and guanarito viruses [ ] , therefore could serve well as a model virus for arenavirus derived diseases without human pathogenic potential and adequate safety for laboratory manipulation. speshock et al. [ ] have recently analysed the activity of two types of silver nanoparticles against tcrv: uncoated (ag-np) and polysaccharide coated silver nanoparticles (ps-ag). they found that when tcrv was treated with μg/ml, μg/ml and even μg/ml of the nm ag-nps significant reduction in the progeny virus titer or no detectable progeny virus was produced. ps-ag particles showed a similar trend but were not as effective, but toxicity was reduced. therefore the polysaccharide coating may indeed protect the cell from the toxic effects of the ag-nps, but it also appears to interfere with the ag-np interaction with tcrv. silver nanoparticles seem to interact with tcrv prior to cellular exposure resulting in a decrease in viral infectivity with and nm ag-nps, therefore, the authors suggested that the silver nanoparticles may bind to virally-encoded membrane glycoproteins. in fact, tcrv glycoproteins are rich in cysteine residues [ ] and silver nanoparticles have been shown to bind easily to the thiol groups [ ] , which are found in cysteine residues. this interaction can either prevent the internalization of the viral particle by interfering with cellular receptor binding, or may favour the internalization of the silver nanoparticle together with the virus and produce an inhibitory effect on viral replication interfering with the tcrv rna-dependent rna polymerase (l protein). other possible mechanism of action could be related to the fact that the silver nanoparticles bound to the viral glycoproteins may prevent the virus uncoating in the endosome. finally, speshock et al. [ ] proved that pre-treatment of the cells with silver nanoparticles had no effect on viral replication, therefore they concluded that the ag-nps could be inactivating the virus prior to entry into the cell. the removal of viruses from water (and the environment in general) is of paramount important for health safety maintenance of our modern society that profoundly relies on water safety for drinking and leisure activities. pathogenic viruses such as adenovirus, norovirus, rotavirus, and hepatitis a commonly occur in both surface and groundwater sources [ ] [ ] [ ] and must be effectively inactivated to provide safe water. titanium dioxide has attracted much attention as a photocatalyst for water treatment, being resistant to corrosion and non-toxic when ingested. the antibacterial properties of tio have been well documented [ ] [ ] [ ] [ ] and are attributed to the generation of ros, especially hydroxyl free radicals and hydrogen peroxide [ , ] . while few studies have investigated the antiviral properties of tio , its potential for inactivating viruses has been demonstrated [ , , ] . however, the inactivation rates obtained in most of these studies were extremely low. for example, cho et al. [ ] demonstrated only minor removal of bacteriophage ms after h of irradiation using p tio suspended at g/l. the inactivation kinetics needs to be greatly improved in order to provide efficient drinking water disinfection. liga et al. [ ] have hypothesized a possible synergic mechanism occurring between silver and tio when silver doped titanium dioxide is used for inactivating microorganisms under uv radiation, therefore they demonstrated that silver doping tio greatly enhanced the photocatalytic inactivation of viruses primarily by increasing hydroxyl free radicals production in addition to slightly increasing virus adsorption. silver doping significantly enhanced ms inactivation by p tio and the inactivation rate increased with silver content. with silver doped tio nanoparticles a considerable removal of ms could be obtained in seconds, rendering feasible the goal of achieving virus removal from drinking water using photoreactors exploiting metal nanoparticles. although the continuous evolutions in the field of metal-based nanoparticles for drug delivery, medical imaging, diagnostics, therapeutics and engineering technology, there is a serious lack of information about the impact of metal nanoparticles on human health and environment, probably due to the intrinsic complex nature of nanoparticles that have led to different attitudes on their safety. therefore, an important issue in the use of metal nanoparticles is their potential toxicity. for metal nanoparticles to be effective as antiviral pharmaceuticals, it is imperative to gain a better understanding of their biodistribution/accumulation in living systems. the principal characteristic of metal nanoparticles is their size, which falls in between individual atoms or molecules and the corresponding bulk materials. particle size and surface area can modify the physicochemical properties of the metal material, but can also influence the reactivity of nanoparticles with themselves or with the cellular environment, leading to different modes of cellular uptake and further processing, leading to adverse biological effects in living cells that would not otherwise be possible with the same material in larger forms. in fact, as particle size decreases, some metal nanoparticles show increased toxicity, even if the same material is relatively inert in its bulk form (e.g., ag, au, and cu). apart from size, the biological consequences of metal nanoparticles also depend on chemical composition, surface structure, solubility, shape, and aggregation. all of these parameters can modify cellular uptake, protein binding, translocation to the target site, and most of the biological interactions with the possibility of causing tissue injury. therefore, in terms of safety, the effect of silver nanoparticles is a major consideration: even if they inhibit viral infections, it would not be beneficial if there are adverse effects to humans or animals. a commonly used strategy to reduce a possible toxicity is to use various capping agents to prevent the direct contact of the metal with the cells. potential routes of human exposure to metal nanoparticles used as therapeutic compounds include the gastrointestinal tract, the skin, the lungs, and systemic administration. considering the use of metal nanoparticles from the point of view of a potential antiviral therapy, it is straightforward that the safest and easiest results can be obtained with topical use of nanoparticles as microbicide for direct viral particles inactivation and/or inhibition of the early steps of the viral life cycle, attachment and entry. therefore, the dermal route seems the one of major concern. dermal exposure to metal nanoparticles often takes place when using sunscreen lotions, for example, tio and zno nanoparticles. in healthy skin, the epidermis provides excellent protection against particle spread to the dermis. however, in presence of damaged skin micrometer-size particles gain access to the dermis and regional lymph nodes. a further concern should be the potential of nanoparticles translocation to the brain via the olfactory nerve as a consequence of the vicinity of the nasal mucosa to the olfactory bulb. whether nanoparticles in such tissues have any pathological or clinical significance is uncertain, therefore, more data is needed to properly address the safety concern on the use of metal nanoparticles as pharmaceuticals. several studies have demonstrated the cytotoxic effects of metal nanoparticles [ ] [ ] [ ] [ ] , in fact, silver nanoparticles were found to be highly cytotoxic to mammalian cells based on the assessment of mitochondrial function, membrane leakage of lactate dehydrogenase, and abnormal cell morphologies [ ] [ ] [ ] [ ] . at a cellular level, metal nanoparticles interact with biological molecules within mammalian cells and can interfere with the antioxidant defence mechanism leading to the generation of reactive oxygen species (ros). such species, in excess, can cause damage to biological components through oxidation of lipids, proteins, and dna. oxidative stress may have a role in the induction or the enhancement of inflammation through upregulation of redox sensitive transcription factors (e.g., nf-κb), activator protein- , and kinases involved in inflammation [ ] [ ] [ ] [ ] . the generation of reactive oxygen species by cells exposed to silver nanoparticles [ ] has been showed in human lung fibroblast and human glioblastoma cells, and as a consequence dna damage and cell cycle abnormalities have been observed. accumulation of silver nanoparticles in various organs (lungs, kidneys, brain, liver, and testes) has been evidenced in animal studies [ ] . most of the in vitro studies show dose dependence, in fact, higher doses of silver induce a strongher cellular toxicity. nevertheless, should be considered that in vitro concentrations of nanoparticles are often much higher than the ones used in in vivo experiments, therefore such exposures do not represent a replica of the conditions expected for in vivo exposure. a recent study [ ] showed that mice exposed to silver nanoparticles showed minimal pulmonary inflammation or cytotoxicity following subacute exposures, but longer term exposures with higher lung burdens of nanosilver are not investigated, therefore eventual cronic effects may be underscored. this review presents only a brief description of the toxicity derived from the use of metal nanoparticles. a more detailed coverage of the topic is available in recently published reviews [ ] [ ] [ ] [ ] . although significant progress has been made to elucidate the mechanism of silver nanomaterial toxicity, a proved consensus on the immediate impact or long term effect on human health is still missing. further research is required to provide the necessary warranties to allow a safely exploitation of the interesting in vitro antiviral properties of silver nanoparticles and their transfer to the clinical setting. nanoparticles are nanoscale clusters of metallic atoms, engineered for some practical purpose, most typically antimicrobial and sterile applications. different wet chemical methods have been used for the synthesis of metallic nanoparticles dispersions. the early methods to produce nanoparticles of noble-metals are still used today and continue to be the standard by which other synthesis methods are compared. the most common methods involve the use of an excess of reducing agents such as sodium citrate [ ] or nabh [ ] . ayyanppan et al. [ ] obtained ag, au, pd and cu nanoparticles by reducing metallic salts in dry ethanol. longenberger et al. [ ] produced au, ag and pd metal colloids from air-saturated aqueous solutions of poly(ethylene glycol) (peg). reduction methods can also be used for the production of pt, pd, cu, mi etc., although specific protocols depend on the reduction potential of the source ion [ ] . cu and ni are not very stable as the metal particles are easily oxidized requiring strong capping ligands to prevent the oxidation. initially, the reduction of various complexes with metallic ions leads to the formation of atoms, which is followed by agglomeration into oligomeric clusters. controlled synthesis is usually based on a two-step reduction process: in the first step a strong reducing agent is used to produce small particles; in the second step these small particles are enlarged by further reduction with a weaker reducing agent [ ] . strong reductants lead to small monodisperse particles, while the generation of larger size particles can be difficult to control. weaker reductants produce slower reduction reactions, but the nanoparticles obtained tend to be more polydisperse in size. different studies reported the enlargement of particles in the secondary step from about - nm to - nm [ ] . another general method for the production of different metal nanoparticles (au, ag, pt, pd) uses commonly available sugars, e.g., glucose, fructose and sucrose as reducing agents [ ] . this approach has several important features: ( ) sugars (glucose, fructose, and sucrose) are easily available and are used as reducing agents; ( ) upon their exploitation no other stabilizing agent or capping agent is required to stabilize the nanoparticles; ( ) sugars are very cheap and biofriendly ( ) the nanoparticles can be safely preserved in a essiccator for months and redispersed in aqueous solution whenever required instead of being kept in aqueous solution. an array of other physical and chemical methods have been used to produce nanomaterials. in order to synthesize noble metal nanoparticles of particular shape and size specific methodologies have been formulated, such as ultraviolet irradiation, aerosol technologies, lithography, laser ablation, ultrasonic fields, and photochemical reduction techniques, although they remain expensive and involve the use of hazardous chemicals. therefore, there is a growing concern to develop environment-friendly and sustainable methods. biosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, magnetite and uraninite nanoparticles by bacteria, actinomycetes, fungi, yeasts and viruses have been reported. however, despite the stability, biological nanoparticles are not monodispersed and the rate of synthesis is slow. to overcome these problems, several factors such as microbial cultivation methods and extraction techniques have to be optimized and factors such as shape, size and nature can be controlled through just modifying ph, temperature and nutrient media composition. owing to the rich biodiversity of microbes, their potential as biological materials for nanoparticle synthesis is yet to be fully explored. the production of metal nanoparticles involves three main steps, including ( ) selection of solvent medium; ( ) selection of environmentally benign reducing agent; ( ) selection of nontoxic substances for the nanoparticles stability [ ] . biomineralization is also an attractive technique, being the best nature friendly method of nanoparticle synthesis. in one of the biomimetic approaches towards generation of nanocrystals of silver, reduction of silver ions has been carried out using bacteria and unicellular organisms. the reduction is mediated by means of an enzyme and the presence of the enzyme in the organism has been found to be responsible of the synthesis [ , ] . therefore in search of a methodology that could provide safer and easier synthesis of metal nanoparticles, it seems that the biogenic synthesis using the filtrated supernatant of different bacterial and fungal cultures is having a considerable impact, where the reduction of metal ions occurs through the release of reductase enzymes into the solution [ , [ ] [ ] [ ] . for an extensive coverage of the biological synthesis of metal nanoparticles by microbes, refer to the recent review by narayanan and sakthivel [ ] . in the crusade toward the development of drugs for the therapy of viral diseases, the emergence of resistant viral strains and adverse side effects associated with a prolonged use represent huge obstacles that are difficult to circumvent. therefore, multidisciplinary research efforts, integrated with classical epidemiology and clinical approaches, are crucial for the development of improved antivirals through alternative strategies. nanotechnology has emerged giving the opportunity to re-explore biological properties of known antimicrobial compounds, such as metals, by the manipulation of their sizes. metal nanoparticles, especially the ones produced with silver or gold, have proven to exhibit virucidal activity against a broad-spectrum of viruses, and surely to reduce viral infectivity of cultured cells. in most cases, a direct interaction between the nanoparticle and the virus surface proteins could be demonstrated or hypothesized. the intriguing problem to be solved is to understand the exact site of interaction and how to modify the nanoparticle surface characteristics for a broader and more effective use. besides the direct interaction with viral surface glycoproteins, metal nanoparticles may gain access into the cell and exert their antiviral activity through interactions with the viral genome (dna or rna). furthermore, the intracellular compartment of an infected cell is overcrowded by virally encoded and host cellular factors that are needed to allow viral replication and a proper production of progeny virions. the interaction of metal nanoparticles with these factors, which are the key to an efficient viral replication, may also represent a further mechanism of action ( figure ). most of the published literature describes the antiviral activity of silver or gold nanoparticles against enveloped viruses, with both a dna or an rna genome. considering that one of the main arguments toward the efficacy of the analysed nanoparticles is the fact that they in virtue of their shape and size, can interact with virus particles with a well-defined spatial arrangement, the possibility of metal nanoparticles being active against naked viruses seems appealing. moreover, it has been already proven that both silver and gold nanoparticles may be used as a core material. however, no reports are yet available for the use of other metals, but the future holds many surprises, especially considering that the capping molecules that could be investigated are virtually unlimited. nonetheless, for metal nanoparticles to be used in therapeutic or prophylactic treatment regimens, it is critical to understand the in vivo toxicity and 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assessment adsorption and surface-enhanced raman of dyes on silver and gold sols plasma resonance enhancement of raman scattering by pyridine adsorbed on silver or gold sol particles of size comparable to the excitation wavelength nanoparticles of ag, au, pd, and cu produced by alcohol reduction of the salts formation of metal particles in aqueous solutions by reactions of metal complexes with polymers synthesis, characterization, and stability of dendrimer-encapsulated palladium nanoparticles reproducible preparation of silver sols with small particle size using borohydride reduction: for use as nuclei for preparation of larger particles general method of synthesis for metal nanoparticles completely "green" synthesis and stabilization of metal nanoparticles biosynthesis of silver nanocrystals by bacillus licheniformis biosynthesis of silver and gold nanoparticles using brevibacterium casei fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole biosynthesis of silver nanoparticles from staphylococcus aureus and its antimicrobial activity against mrsa and mrse extracellular synthesis of silver bionanoparticles from aspergillus clavatus and its antimicrobial activity against mrsa and mrse biological synthesis of metal nanoparticles by microbes key: cord- -ayek zbo authors: har-noy, michael; or, reuven title: allo-priming as a universal anti-viral vaccine: protecting elderly from current covid- and any future unknown viral outbreak date: - - journal: j transl med doi: . /s - - - sha: doc_id: cord_uid: ayek zbo background: we present the rationale for a novel allo-priming approach to serve the elderly as a universal anti-virus vaccine, as well serving to remodel the aging immune system in order to reverse immunosenescence and inflammaging. this approach has the potential to protect the most vulnerable from disease and provide society an incalculable economic benefit. allo-priming healthy elderly adults is proposed to provide universal protection from progression of any type of viral infection, including protection against progression of the current outbreak of covid- infection, and any future variants of the causative sars-cov- virus or the next ‘disease x’. allo-priming is an alternative approach for the covid- pandemic that provides a back-up in case vaccination strategies to elicit neutralizing antibody protection fails or fails to protect the vulnerable elderly population. the allo-priming is performed using activated, intentionally mismatched, ex vivo differentiated and expanded living th -like cells (allostim(®)) derived from healthy donors currently in clinical use as an experimental cancer vaccine. multiple intradermal injections of allostim(®) creates a dominate titer of allo-specific th /ctl memory cells in circulation, replacing the dominance of exhausted memory cells of the aged immune system. upon viral encounter, by-stander activation of the allo-specific memory cells causes an immediate release of ifn-ϒ, leading to development of an “anti-viral state”, by-stander activation of innate cellular effector cells and activation of cross-reactive allo-specific ctl. in this manner, the non-specific activation of allo-specific th /ctl initiates a cascade of spatial and temporal immune events which act to limit the early viral titer. the release of endogenous heat shock proteins (hsp) and damp from lysed viral-infected cells, in the context of ifn-ϒ, creates of conditions for in situ vaccination leading to viral-specific th /ctl immunity. these viral-specific th /ctl provide sterilizing immunity and memory for protection from disease recurrence, while increasing the pool of th /ctl in circulation capable of responding to the next viral encounter. conclusion: allo-priming has potential to provide universal protection from viral disease and is a strategy to reverse immunosenescence and counter-regulate chronic inflammation (inflammaging). allo-priming can be used as an adjuvant for anti-viral vaccines and as a counter-measure for unknown biological threats and bio-economic terrorism. har-noy and or j transl med ( ) : pneumococcal pneumonia, shingles and hepatitis a/b. successful prophylactic vaccination mechanisms provide protection through eliciting neutralizing antibodies to prevent viral entry into cells. however, this strategy does not provide protection against antigenic shift or drift variants of the original virus [ , ] . currently, there are at least three known variants of the sars-cov- virus [ ] . in addition, pathological viruses are intracellular and not always accessible to antibodies. for this reason, neutralizing antibody vaccines have not been effective against a number of complex viruses, including hiv, hcv, cmv, zika, rsv, dengue and sars/mers. for the same reason, convalescent serum prophylaxis and treatment may not be able to confer sterilizing immunity or memory. these sophisticated viruses may require an effective cellular immune response for sterilizing immunity [ ] [ ] [ ] [ ] [ ] . without sterilizing cellular immunity, there can be viral recurrence as has been reported with covid- [ ] . many efforts are underway to develop anti-viral vaccines which elicit protective cellular immunity [ ] , but these have not yet been successfully translated to demonstrate clinical benefit [ ] . the age-related functional decline in cellular immunity (immunosenescence) makes the elderly less able to mount a cellular immune response to vaccination, making this population more vulnerable to morbidity and mortality associated with viral diseases and less likely to respond to an anti-viral vaccine. in addition, elderly also suffer detrimental effects on their immune function due to chronic inflammation, known as "inflammaging" [ ] . inflammaging is correlated with comorbidities such as cancer, arthrosclerosis, neurodegenerative diseases (e.g., alzheimer's and parkinson's disease) all which increase the likelihood of serious progression of viral infection. in addition, the aging of the structure and function of the lungs contributes to increased incidence of pneumonia, acute respiratory distress syndrome (ards) and sepsis in the elderly after respiratory viral infection. the remodeling of the senescent immune systems of the elderly through allo-priming is proposed as a method to restore cellular immune function in this population. the ability to restore functional cellular immunity to the elderly can increase responsiveness to viral infections, including covid- and any future emergent novel virus. in essence, an elderly immune system modulated by allo-priming would potentially respond to viral infection in a similar manner to the immune response of younger individuals, resulting in less serious disease. the immunomodulation of the elderly immune system to function more like a youthful immune system should also restore responsiveness to any current or future viralspecific vaccines. a more balanced immune system in the elderly can also counter-regulate inflammaging, providing broad ranging health benefits to the elderly and to society [ ] . the allo-priming concept is designed to prime the immune systems of healthy elderly adults to create high titers of allo-specific th /ctl memory cells which can become activated upon encounter with any virus (bystander activation) [ ] . the by-stander activation of allo-specific th /ctl memory cells upon viral encounter causes the release of interferon-gamma (ifn-ϒ), which creates an "anti-viral state" [ ] . ifns create an antiviral state in both virus-infected cells and uninfected, bystander cells, by inducing a program of gene transcription that interferes with multiple stages of viral replication cycles through various mechanisms [ ] . the ifn-ϒ release from by-stander activated allo-specific th /ctl memory cells activates innate effector cells (e.g., nk, nkt and macrophages) which in turn release additional ifn-ϒ, sustaining the anti-viral state. these activated innate effector cells lyse viral infected cells, controlling acute viral burden. by-stander activation of resident allo-specific ctl can also cross-react to lyse viral infected cells [ ] . the lysis of viral infected cells by activated innate effector cells and cross-reactive allo-specific memory ctl releases "danger signals" [ ] and heat shock proteins (hsp) [ ] which chaperone viral antigens (e.g., grp , hsp ) [ , ] into the microenvironment, creating the conditions for "in situ vaccination", which leads to development of viral-specific cellular immunity. antigen presenting cells (apc), such as dendritic cells (dc), engulf and process released hsp-chaperoned viral antigens. processing in the context of danger signals and ifn-ϒ, causes the maturation of immature dc to il- + dc [ , ] . il- production acts to further increases ifn-ϒ, sustaining the anti-viral state and upregulating mhci, mhcii and co-stimulatory molecules on apc [ , ] . the mature dc migrate to regional lymph nodes and display viral antigens on mhc i and mhc ii with co-stimulator cd / expression, leading to viral-specific th /ctl expansion. the ifn-ϒ upregulates mhci on viral infected cells so these cells can be recognized and killed by viral-specific ctl [ ] . these viral-specific ctl can then orchestrate a sterilizing immune response and later differentiate into memory cells that provides protection from re-infection. thus, allo-antigenic priming can modulate the systemic immune balance and provide a pool of non-exhausted allo-specific th /ctl memory cells capable of rapidly responding to viral infection. the non-specific activation of these allo-specific memory cells upon viral encounter causes release of ifn-ϒ. the release of ifn-ϒ orchestrates the sequential activation of immune cells resulting in formation of an antiviral state, innate elimination of invading viruses and development of a viral-specific effector response and memory. each time this cascade is initiated by viral encounter, the virus elimination serves as a booster to the original allo-antigenic priming. the new viral-specific th /ctl memory cells resulting from viral elimination join the resident memory allo-specific th /ctl resulting from the allo-antigenic priming, increasing the titer of th / ctl memory cells in circulation. encounter with each new virus will non-specifically activate all previous nonexhausted memory cells and support the immune cascade that eliminates the new virus. memory th /ctl to each new virus, in turn, increases the titer of th /ctl memory cells primed to respond to a subsequent viral encounter. creating a memory th /ctl immune response to alloantigen thus provides protection against an unrelated viral infection through the immune mechanism known as "heterologous immunity". heterologous immunity occurs when by-stander activation of immune memory cells to one virus alters the immune response to, and the course of infection of, an unrelated virus encountered later [ , ] . in this manner, healthy adults can be provided pan-viral protection against viral infections, including sars-cov- , influenza a/b and any future variants and unknown novel viruses that may emerge. this self-amplifying process can provide long-term disease mitigation and universal protection against known and unknown viral infections for the elderly. in order to effectuate the anti-viral and anti-aging immune mechanisms, the allo-priming must elicit allospecific th /ctl immunity. injection of alloantigen alone can elicit humoral and th responses, and multiple injections can result in tolerance to the allo-antigens. many factors, both intrinsic and extrinsic to the allograft, can influence the nature and magnitude of the allorejection immune response, including the nature of the allograft, the site of the body where it is placed, and the immunological status of the recipient [ ] . to assure consistent differentiation of allo-specific th /ctl memory after allo-antigen priming, regardless of host immune status and age, a bioengineered, patented, immune cell called "allostim ®" is used. allostim ® is living, activated, intentionally mismatched, ex vivo expanded and differentiated allogeneic th -like cells derived from healthy donors. injection of undifferentiated allogeneic immune cells or non-activated allogeneic immune cells were not able to elicit allo-specific th memory, while priming with activated allogeneic th like allostim ® cells elicited dominant th immunity and had both a protective effect and a therapeutic effect in a cancer model [ , ] . allostim ® cells are administered intradermally (id) in an activated state. to assure the injected cells are activated when administered, they are prepared with anti-cd /anti-cd -coated microbeads attached. the activated allostim ® cells are formulated as a frozen dosage form at × cells/ml in plasmalyte a containing % human serum albumin and % dsmo. individual doses of . ml are thawed and administered intradermally every - days for up to five total doses. this priming schedule is sufficient to provide high titers of circulating allo-specific th /ctl. allostim ® has been evaluated in phase i/ii, phase ii and phase iia clinical trials in usa and thailand in chemotherapy-refractory metastatic solid tumor patients. phase iib pre-registration and phase iib/iii randomized, controlled registration clinical trials are currently being prepared for launch in third line metastatic colorectal cancer and advanced/ metastatic hepatocellular carcinoma in usa and asia, respectively. in these human clinical trials, allostim ® has demonstrated ability to down-regulate checkpoint molecules in the tumor microenvironment, cause system-wide infiltration of effector t-cells and nk cells into metastatic lesions (conversion from "cold" to "hot") and has consistently demonstrated a tail of between and % of long-term survivors in chemotherapy-refractory metastatic disease with a good safety profile [ , ] . activated allostim ® express high density cd l and type cytokines, including ifn-ϒ, tnf-α and gm-csf. allostim ® has been shown to modulate the immune systems of heavily pre-treated metastatic cancer patients (which resemble the senescent immune systems of the elderly) and has been used in protocols which were designed to elicit the same anti-tumor mechanism of allogeneic stem cell transplant procedures (graft vs tumor or "gvt") without the need for a matched donor, chemotherapy conditioning or risk of gvhd [ , , [ ] [ ] [ ] [ ] [ ] . allogenic cells are highly immunogenic and are rejected even by immunocompromised hosts [ ] . the rejection of the mis-matched cells eliminates risk of gvhd side-effects of allogeneic cell administration. multiple id injections of allostim ® amplify the titer of allo-specific th /ctl cells in circulation and a portion of these cells differentiate into long lasting memory cells. these allo-specific th /ctl memory cells provide a functional pool of immune cells that are capable of nonspecific (by-stander) activation [ ] upon encounter with virus [ , ] resulting in immediate release of ifn-ϒ. har-noy and or j transl med ( ) : the production of proinflammatory cytokines by allostim ® , including ifn-γ and tnf-α, initially activates host nk cells to reject the allogeneic cells, due to missing self-mhc i on the cd + allostim ® cells. the nk cell compartment is highly stable in terms of function and phenotype in the elderly [ ] and can therefore readily reject mismatched allogeneic cells. in subsequent id allostim ® injections, the production of ifn-ϒ and expression of cd l by allostim ® activates macrophages that reject the allostim ® [ ] . the rejection of the allogeneic cells by nk cells or macrophages in the skin results in release of endogenous hsp and damp danger signals [ ] , which in the context of cd l and ifn-ϒ expressed by allostim ® result in dc maturation to il- + dc phenotype and their migration to draining lymph nodes to interact with cognate t-cells to elicit allo-specific th /ctl immunity [ ] . the current covid- viral pandemic has led to high number of deaths worldwide. this is the third serious coronavirus (cov) outbreak in less than years, following sars in - and mers in . elderly people account disproportionately to the morbidity and mortality associated with cov infection. vaccines have been the main global strategy to minimize the impact of viral infections, but no successful vaccines have yet been developed for these previous highly virulent covs. in any event, elderly adults generally respond poorly to antiviral vaccines [ ] [ ] [ ] . this creates a high unmet medical need for a vaccine to provide protection to the elderly from the current covid- pandemic and protect from any future pandemics. while healthy younger adults generally present with more mild symptoms in response to viral infection, the elderly are slow to respond and are susceptible to higher viral titers and chronic viral infection which leads to progression to severe symptoms, especially in the setting of respiratory viral infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the delayed and poorly effective responsiveness of elderly to viral infection is due, in large part, to compromised cellular immunity [ ] [ ] [ ] related to "immunosenescence" [ ] [ ] [ ] . elderly individuals have a significantly delayed innate immune response to viral infection and the adaptive immune response that follows is often sub-optimal [ ] . the delayed innate immune response results in accumulation of high viral titers which, in turn, serves to elicit a late over-active immune response which is correlated with a more severe and often lethal clinical course [ ] . the acute high viral levels that are achieved in elderly as a result of late innate immune response also increases the virulence of the virus, by increasing chances of humanto-human spread of infection. there is a dysregulation in the th /th -system in the elderly, which is dominated by th -functions [ , ] . memory th /ctl cells in elderly adults are diminished and functionally exhausted [ ] [ ] [ ] [ ] . memory cells of the elderly become exhausted due to lifelong antigenic challenge which can be as a result of chronic viral infection (e.g., cmv, ebv, hbv, hcv) [ , ] . exhausted memory cells are unable to be non-specifically activated upon viral encounter. this defect in cellular immunity in the elderly limits the availability of heterologous immune mechanisms to react to acute viral infection, which contributes to a slower and often inadequate innate response to viral infection. elderly also have limited t-cell repertoires [ , ] which inhibits their ability to develop viral-specific adaptive t-cell immune responses, both prophylactically (by vaccination) [ ] , and in situ (as a result of adaptive immune clearance of infection). this suppressed cellular immunity together with the aging of the respiratory organs makes elderly more susceptible to pathological effects of viral infection, especially effects of respiratory viral infection [ ] . the immune defects of the elderly make this population less likely to benefit from vaccinations as preventative measures against infectious diseases [ ] . the current inability to provide elderly with protection from viral infection has an enormous economic impact on society [ , ] . the medical and economic impact of pandemics which disproportionately affect the elderly will be exacerbated as the proportion of the adult population increases globally. the current projections indicate that by , the group of years and older will represent . % of the population [ ] . therefore, there is an urgent unmet need to develop strategies to reverse immunosenescence and to develop methods which provide protection of the elderly population from viral epidemics. the progressive age-related dysregulation and decline of the immune system is known as "immunosenescence". in the aging immune system, there is an accumulation of memory cells as a result of chronic stimulation with repeated clinical and subclinical infections [ ] . this chronic stimulation causes these memory cells to become exhausted and no longer able to function, contributing to an increased susceptibility of elderly to infectious pathogens [ , ] . the aging immune system is also characterized by a decline in the numbers of naive t cells, an imbalance in th /th cell subsets, and a decrease in t cell receptor (tcr) repertoire [ , , ] . a cellular immune response with release of th (type ) cytokine profile, including ifn-ϒ, tnf-α and il- , is known to be protective against most viral infections [ ] . ifn-ϒ in particular has been shown to play an essential role in clearance of viral infection [ ] . modulating the immune system of elderly individuals through alloantigen priming to provide high titers of non-exhausted th / ctl memory cells that can be non-specifically activated upon encounter with any virus to cause release type cytokines may provide an immediate anti-viral immune response upon viral exposure and could also reinstate responsiveness to viral vaccines [ ] . the delay in ifn responses to virus infection in the elderly is due to active suppression by viral proteins and immunosenescence [ ] , which allows virus to reach high titers early in infection, leading to immunopathology, t-cell apoptosis and progression to lethal pneumonia [ , ] . the immediate release of ifn-ϒ upon acute viral infection corrects the problem of delayed immune responsiveness and creates an early "anti-viral state" [ , ] . establishment of the anti-viral state with early release of ifn-ϒ provides a crucial initial line of defense against viral infection [ ] . the anti-viral state is normally induced through early release of type i/iii ifn from viral infected cells [ ] . however, many viruses, including coronavirus, can suppress production of type i/iii ifn as an immune avoidance mechanism [ ] [ ] [ ] [ ] . the strategy of providing a pool of non-exhausted memory cells which produce type ii ifn (ifn-ϒ) upon viral encounter, overcomes the viral escape. ifn-ϒ creates an anti-viral state even in type i/iii ifn resistant cells [ ] . further, ifn-ϒ mobilizes innate effector cells that can respond rapidly to eliminate viral infection [ ] and also produce ifn-ϒ. thus, early ifn-ϒ response to viral infection through allo-priming provides a rapid means of control over viral infection in the elderly. chronic inflammation in the elderly is known to be associated with increased susceptibility to cancer [ , ] , atherosclerosis [ ] and neurodegenerative disorders [ , ] . the modulation of the elderly immune system that occurs by allo-priming can also have the effect of counter-regulating resident chronic inflammation [ , ] which causes "inflammaging" resulting in reversing of immunosenescence [ ] . thus, allo-priming protocols may not only provide universal anti-viral protection for the elderly, but may also serve as an "antiaging" mechanism to protect from co-morbid diseases of aging. allo-priming is predicted to reduce the severity of clinical symptoms upon encounter with virus. clinical features of respiratory viral infections in humans vary from a first subset that experience mild flu-like symptoms which subside over a few days; to a second subset that experience moderate to severe flu-like symptoms associated with high fever, hypoxemia and progression to pneumonia-like symptoms. these symptomatic patients are at high risk to progress to acute respiratory distress syndrome (ards), sepsis, multiple organ failure and death. the subset with mild flu-like symptoms is associated with younger healthy individuals, while the subset associated with more severe symptoms is mostly associated with elderly adults that are frail and/or present with co-morbidities. the progression to more serious symptoms can occur even when there is a decline in virus titers [ ] connected to a late over-exuberant immune response. the initial delayed immune response leads to high viral titers which is followed by an exaggerated late immune response and a "cytokine storm". this sequence of events is believed to be responsible for the severe symptoms related to respiratory virus infections in the elderly. during pathogenic virus infection, a cytokine storm leads to excessive inflammatory infiltrates and virusinduced tissue destruction which contributes to morbidity and mortality. the timing and source of the ifn in the cytokine storm can affect the clinical course of viral disease. high serum levels of ifn-ϒ late in the course of viral infection is correlated with more severe disease [ ] and immunopathology [ ] , whereas early ifn release results in lower viral titers and less severe disease. these observations support the prediction that early activation and release of ifn-ϒ from non-exhausted allospecific memory th /ctl will correct this problem and avoid progression to severe disease. the type of cytokine storm associated with severe disease is caused by cytokines released by activated monocytes, producing a storm of ifn-α, ccl- , il- , tnf-α, ccl- , ccl- , cxcl- , il- α, and ifn-γ. this monokine cytokine storm is directly correlated with morbidity and mortality [ ] . the majority of fatalities associated with cytokine storm also developed bacterial pneumonia and sepsis. it is recognized that a major cause of respiratory failure is coexistent bacterial pneumonia leading to ards. ards is characterized by damage to the endothelial-epithelial barrier of the alveoli, resulting in fluid leakage and accumulation in the alveolar lumen inhibiting gas exchange. the elderly have a pre-disposition to develop progressive respiratory compromise and sepsis. in fact, sepsis is considered a disease of aging [ ] and is among the top causes of icu admissions of the elderly. the features of sepsis-induced immunosuppression, independent of age, share many of the same characteristics of immunosenescence. during the last decades, there has been a significant increase in incidence of sepsis in patients over years of age [ ] . many respiratory virus patients that progress to develop pneumonia and ards [ , ] later die due to sepsis. the overactive immune response to sepsis, including monokine "cytokine storm" results from over activated m macrophages in response to tissue damage [ ] . the response to sepsis is similar between old and younger patients. however, the mortality is much higher in older patients [ ] . the difference is related to the dysregulated immune systems of the elderly. the elderly are unable to turn down the over-activated monocyte response, leading to disease progression, while regulatory mechanisms in the young counter-act and prevent the consequences of monocyte over-activation. the dysregulated immune system of the elderly is related to the increased activity of myeloid-derived suppressor cells (mdsc) [ , ] . mdscs are potent inducers of immunosenescence [ ] , sepsis-acquired immunodeficiency [ ] as well as cancer metastasis and progression [ ] . mdsc are positively correlated with il- and negatively correlated with ifn-ϒ [ , ] . immunosenescence causes an imbalanced homeostatic regulation of innate and adaptive immune responses, diminishing the host capacity to rapidly restore balanced immune functions. thus, combined effects of ageinduced immunosuppression, delayed innate immune responses [ ] , exaggerated late immune responses due to altered homeostasis [ ] , all combine to make the post-viral infection period in the elderly have a longer duration. as a result, there are increased levels of viral propagation, higher incidences of tissue injury, and increased progression to ards and septic shock in the elderly. accordingly, allo-priming enabling the early release of ifn-ϒ in response to viral infection can prevent the differentiation of mdsc and counter-regulate resident mdsc immunosuppression in the elderly [ ] . this will have the effect of remodeling the elderly immune system in a manner which will prevent the severe symptoms of viral infection and progression to ards and sepsis. highly pathogenic human covs pose a substantial threat to public health. there are basically two groups of patients upon exposure to cov, with the majority developing a short duration of clinical symptoms and the minority experiencing severe disease characterized by pneumonia and ards. the elderly are disproportionately vulnerable to severe disease due to immunosenescence and comorbidities. it is likely that covs will continue to cross species and cause additional outbreaks in the future. therefore, even if a vaccine is developed for prevention of covid- , it would not protect against the next outbreak of a novel virus. therefore, there is a need to develop novel strategies, not only to control the current pandemic, but also to be prepared to prevent the next pandemic. strategies to develop a vaccine to elicit neutralizing antibodies to sars-cov- are technically challenging due to the conformational hiding of the receptor binding domain (rbd) and the ability of this virus to transfer cell-to-cell in syncytia and infect target cells in a ace -and protease-independent manner by pinocytosis, limiting environmental exposure needed for antibody neutralization activity. development of an anti-viral vaccine against complex viruses, such a sars-cov- , is likely to require a potent and broad t-cell response to overcome viral escape mechanisms related to humoral immunity. a vaccine that elicits a robust cellular immune response requires identification of conserved viral epitopes and effective processing and presentation of viral antigens by apc on mhci and mhcii in conjunction with costimulatory signals, as well as a diversity in the naive t cell repertoire. in addition, ctl memory cells resulting from vaccination will require infected cells to present the selected viral antigens in the vaccine on mhci. selection of common viral epitopes which present on mhc i (and mhc ii) for vaccine development is technically difficult and cov infection causes the down-regulation of mhc i on infected cells, making infected cells invisible to ctl. since the elderly lack naïve t-cells that are necessary for development of ctl with broad specificity to viral antigens, elderly would be less likely to respond efficiently to any future vaccine which targets cellular immunity. the rejection of allografts is one of the most conserved and powerful immune mechanisms, making priming with alloantigens an ideal candidate for use in vaccination of the elderly. allostim ® is in use under a us fda cleared ind and has been shown to readily be rejected by heavily pre-treated, immunosuppressed metastatic cancer patients. a phase i/ii clinical trial protocol for use of allostim ® in healthy elderly adults in currently under review by us fda. upon clearance, clinical trials could be initiated to evaluate this allo-priming concept in short order. for proof-of-concept, longitudinal pbmc samples are proposed to be collected pre-and post-allo-priming. the pbmc are to be pulsed with a panel of inactivated viruses or recombinant viral proteins to determine if they will non-specifically activate the allo-specific th /ctl memory cells. the supernatants from the viral-pulsed pbmc are proposed to be evaluated in live virus cytolytic plaque assays to determine if viral propagation is suppressed. elderly persons are particularly susceptible to progressive viral disease and have delayed immune responses to viral infection due, at least in part, to immunosenescence, th immune bias, decreased diversity of naïve t-cells and high frequencies of exhausted memory cells from chronic inflammation (e.g., cmv). this results in a natural loss of the ability to mount effective innate and adaptive cellular immune responses to invading pathogens. the delayed and suppressed cellular immune response in the elderly enables viruses to become widely established and the resulting increased viral titers causes dys-regulated immune responses that can lead to a late, over-active immune response which can progress to pneumonitis, multiple organ failure and death. the key to an effective natural innate immune response that can limit the course of virus infection is the early production of type i/iii interferons (ifn) and subsequent switch to type ii ifn (ifn-ϒ) production as the immune response matures. successful viral infections become established due, in large part, to delayed innate immune responses and viral-mediated suppression of type i/iii ifn production, allowing for rapid viral propagation which eventually results in dys-regulated immune responses which are correlated with tissue destruction, co-infection, multiple organ failure and death. to prevent accumulation of high viral burden in the elderly upon viral infection, allo-priming provides a mechanism whereby a ready pool of de-novo primed t-cells are in circulation that can respond rapidly to viral infection by producing ifn-ϒ. the presence of non-exhausted th /ctl memory immune cells will modify the elderly immune character by providing a th re-balancing mechanism in the memory cell compartment. this is accomplished through the creation of a high titer of polyclonal, allo-specific, non-exhausted, memory th /ctl t-cells through intradermal injections of activated allogeneic th -like cells (allostim ® ). the allo-specific memory cells resulting from the priming are programmed to produce ifn-ϒ upon activation. ifn-ϒ has a direct anti-viral effect on cells infected with virus and can also protect uninfected cells from infection. ifn-ϒ creates the same anti-viral environment as innate release of type i/iii ifn does in an effective natural innate immune response. these allo-specific memory cells are capable of crossreacting with foreign viral antigens and can be readily activated non-specifically by environmental stimuli such as cytokines and foreign pathogens. the memory pool of allo-specific immune cells in primed individuals can also be re-activated by additional intradermal or intravenous infusion of allostim ® allogeneic cells upon first symptoms of viral disease. the by-stander effect of allo-specific th /ctl memory t-cell activation and ifn-ϒ production is predicted to elicit protective effects on cells in the respiratory tract and generate rapid immune-mediated viral clearance as well as condition the microenvironment in infected tissues (e.g., lung epithelial cells) for an in situ vaccination leading to viral-specific immunity and memory that is specific for the invading virus. the heterologous immune effect can amplify the pan-viral protection upon each viral encounter, providing the possibility of long-term protection. the proposed alloantigen priming strategy can also be used in conjunction with any viral-specific vaccines. co-injection of allo-specific and viral specific antigens could accelerate the adaptive cellular immune response to a known virus and serve as a means to enhance response to vaccines in the elderly. subsequent injection of allogeneic cells will stimulate an allo-rejection memory recall response which can non-specifically activate resident viral-specific cells elicited by vaccination. this dual activation provides a mechanism to upregulate mhci on infected targets, making the memory cells elicited by viral-specific vaccination able to identify and kill viral infected targets. the combination approach also has the advantage of overcoming the narrow immunity conferred by a single peptide vaccine by incorporating the in situ vaccination mechanism to elicit broad viral-specific cellular immune responses. this allo-priming mechanism can also be used as a counter-measure to bio-terrorism and bio-economic terrorism. individuals that have been primed with alloantigen could be treated with an emergency injection of alloantigen to activate innate immunity and initiate the cascade of immune events leading to clearance of an unknown pathogen. alloantigen could be provided in syringes for emergency id injection upon concern for bioweapon exposure or first occurrence of symptoms, much in the same manner as epinephrine is provided to prevent anaphylactic shock. while finding methods to treat and prevent covid infection is of urgent priority, it is also important to consider that the current pandemic is the third major outbreak of a novel coronaviral infection in humans within the past years. currently there are no registered vaccines or means of therapeutic protection against the prior sars or mers outbreaks available anywhere in the world. this is despite considerable efforts from experts worldwide to develop vaccines. the same technical obstacles preventing development of specific vaccines and treatments for these past cov outbreaks likely also exist for development of a vaccine for the current covid- viral pandemic. however, even if an 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participation of t lymphocytes and myeloid-derived suppressor cells in aging-related immune response changes mitigating age-related immune dysfunction heightens the efficacy of tumor immunotherapy in aged mice immunosenescence: the potential role of myeloid-derived suppressor cells (mdsc) in age-related immune deficiency myeloid cells in sepsis-acquired immunodeficiency mechanisms overseeing myeloid-derived suppressor cell production in neoplastic disease myeloid-derived suppressor cells are increased and correlated with type immune responses, malnutrition, inflammation, and poor prognosis in patients with breast cancer interferon regulatory factor (irf ) controls myeloid-derived suppressor cell (mdsc) differentiation and function : • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over ready to submit your research ? choose bmc and benefit from ifn-gamma regulates survival and function of tumor-induced cd b+ gr- high myeloid derived suppressor cells by modulating the anti-apoptotic molecule bcl a publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. mhn conceived of the allopriming concept and wrote the manuscript. ro reviewed and edited the manuscript. both authors read and approved the final manuscript. not applicable. not applicable. not applicable. not applicable. mhn is the founder of immunovative therapies, ltd. and mirror biologics, inc which own patent rights to allostim ® . key: cord- - thh syt authors: carlson, colin j.; albery, gregory f.; merow, cory; trisos, christopher h.; zipfel, casey m.; eskew, evan a.; olival, kevin j.; ross, noam; bansal, shweta title: climate change will drive novel cross-species viral transmission date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: thh syt at least , species of mammal virus are estimated to have the potential to spread in human populations, but the vast majority are currently circulating in wildlife, largely undescribed and undetected by disease outbreak surveillance , , . in addition, changing climate and land use are already driving geographic range shifts in wildlife, producing novel species assemblages and opportunities for viral sharing between previously isolated species , . in some cases, this will inevitably facilitate spillover into humans , —a possible mechanistic link between global environmental change and emerging zoonotic disease . here, we map potential hotspots of viral sharing, using a phylogeographic model of the mammal-virus network, and projections of geographic range shifts for , mammal species under climate change and land use scenarios for the year . range-shifting mammal species are predicted to aggregate at high elevations, in biodiversity hotspots, and in areas of high human population density in asia and africa, driving the cross-species transmission of novel viruses at least , times. counter to expectations, holding warming under °c within the century does not reduce new viral sharing, due to greater range expansions—highlighting the need to invest in surveillance even in a low-warming future. most projected viral sharing is driven by diverse hyperreservoirs (rodents and bats) and large-bodied predators (carnivores). because of their unique dispersal capacity, bats account for the majority of novel viral sharing, and are likely to share viruses along evolutionary pathways that could facilitate future emergence in humans. our findings highlight the urgent need to pair viral surveillance and discovery efforts with biodiversity surveys tracking range shifts, especially in tropical countries that harbor the most emerging zoonoses. dispersal limits). even with dispersal limits, these first encounters are predicted to produce al- most one hundred new viral sharing events (rcp . : ± . ; rcp . : ± . ) that might include zebov, and which cover a much broader part of africa than the current zoonotic niche of ebola . human spillover risk aside, this could expose several new wildlife species to a deadly virus historically responsible for sizable primate die-offs . moreover, for zoonoses like emerging threats like ranavirus causing conservation concern, pathogen exchange among am- phibians may be especially important for conservation practitioners to understand . finally, marine mammals are an important target given their exclusion here, especially after a recent study implicating reduced arctic sea ice in novel viral transmission between pinnipeds and sea otters-a result that may be the first proof of concept for our proposed climate-disease link . because hotspots of cross-species transmission are predictable, our study provides the first template for how surveillance could target future hotspots of viral emergence in wildlife. in the next decade alone, billions could be spent on virological work trying to identify and counteract zoonotic threats before they spread from wildlife reservoirs into human populations . these to implement the grubb outlier tests for a given species we defined a distance matrix between each record and the centroid of all records (in both environmental or geographic space, respec- tively) and determined whether the record with the largest distance was an outlier with respect to all other distances, at a given statistical significance (p = e − , in order to exclude only extreme outliers). if an outlier was detected it was removed and the test was repeated until no additional outliers were detected. the worldclim dataset is widely used in ecology, biodiversity, and agricultural projections of potential climate change impacts. worldclim makes data available for current and future for herbivores and omnivores, the maximum is estimated as d = . m . . we used mammalian diet data from the eltontraits database , and used the same cutoff as schloss to identify carnivores as any species with % or less plants in their diet. we used body mass data from eltontraits in the schloss formula to estimate maximum generational dispersal, and converted estimates to annual maximum dispersal rates by dividing by generation length, formula performs notably poorly for bats: for example, it would assign the largest bat in our study, the indian flying fox (pteropus giganteus), a dispersal capacity lower than that of the gray dwarf hamster (cricetulus migratorius). bats were instead given full dispersal in all scenarios: given significant evidence that some bat species regularly cover continental distances , , and that isolation by distance is uncommon within many bats' ranges , we felt this was a defensible assumption for modeling purposes. moving forward, the rapid range shifts already observed in many bat species (see main text) could provide an empirical reference point to fit a new allo- metric scaling curve (after standardizing those results for the studies' many different method- ologies). a different set of functional traits likely govern the scaling of bat dispersal, chiefly the aspect ratio (length:width) of wings, which is a strong predictor of population genetic differ- entiation . migratory status would also be important to include as a predictor although here, we exclude information on long-distance migration for all species (due to a lack of any real framework for adding that information to species distribution models in the literature). . using a linear model, we show that elevation (c), species richness (d), and land use (e) influence the number of new overlaps for bats and non-bats. slopes for the elevation effect were generally steeply positive: a log -increase in elevation was associated with between a . - . log -increase in first encounters. results are averaged across nine global climate models. legends refer to scenarios: cl gives climate and land use change, while cld add adds dispersal limits. a. b. extended data figure : projected viral sharing from suspected ebola reservoirs is dominated by bats. node size is proportional to (left) the number of suspected ebola host species in each order, which connect to (middle) first encounters with potentially naive host species; and (right) the number of projected viral sharing events in each receiving group. (node size denotes proportions out of % within each column total.) while ebola hosts will encounter a much wider taxonomic range of mammal groups than current reservoirs, the vast majority of viral sharing will occur disproportionately in bats. extended data figure : data processing workflow. summary of species inclusion across the modeling pipeline for species distributions and viral sharing models. the final analyses in the main text use , species of placental mammals across all scenarios. extended data figure : species distribution modeling workflow for a single species. a focal species (the sand cat, felis margarita) is displayed as an illustrative example. the present day climate prediction (top left) was clipped to the same continent according to the iucn distribution (top right). this was then clipped according to cervus elaphus land use (second row, left). the known dispersal distance of the red deer was used to buffer the climate distribution (second row, right). the future distribution predictions (rcp . shown as an example) are displayed in the bottom four panels, for each of the four pipelines: only climate (third row, left); climate + dispersal clip (third row, right); climate + land use clip (bottom row, left) and climate + land use + dispersal clip (bottom row, right). the four distributions clearly display the limiting effect of the dispersal filter (bottom right panels) in reducing the probability of novel species interactions (bottom left panels). the land use clip had little effect on this species as the entire distribution area was habitable for the red deer. bats as 'special' reservoirs for emerging zoonotic pathogens a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? are bats really 'special' as viral reservoirs? what we know and need to know mass extinctions, biodiversity and mitochon- drial function: are bats 'special' as reservoirs for emerging viruses? current opinion in virology viral zoonotic risk is homogenous among taxonomic orders of mammalian and avian reservoir hosts virological factors that increase the transmissibility of emerging human viruses transmissibility of emerging viral zoonoses origins of hiv and the aids pandemic. cold spring harbor perspectives in medicine origin and evolution of pathogenic coronaviruses key: cord- - vjqrnwh authors: hraber, peter; o’maille, paul e.; silberfarb, andrew; davis-anderson, katie; generous, nicholas; mcmahon, benjamin h.; fair, jeanne m. title: resources to discover and use short linear motifs in viral proteins date: - - journal: trends biotechnol doi: . /j.tibtech. . . sha: doc_id: cord_uid: vjqrnwh viral proteins evade host immune function by molecular mimicry, often achieved by short linear motifs (slims) of three to ten consecutive amino acids (aas). motif mimicry tolerates mutations, evolves quickly to modify interactions with the host, and enables modular interactions with protein complexes. host cells cannot easily coordinate changes to conserved motif recognition and binding interfaces under selective pressure to maintain critical signaling pathways. slims offer potential for use in synthetic biology, such as better immunogens and therapies, but may also present biosecurity challenges. we survey viral uses of slims to mimic host proteins, and information resources available for motif discovery. as the number of examples continues to grow, knowledge management tools are essential to help organize and compare new findings. viruses exploit host cellular processes to replicate, and have developed myriad ways to subvert host immune defenses. molecular mimicry (see glossary) is a common and effective strategy, enabling a pathogen to usurp host protein function by resemblance [ , ] . molecular mimicry varies over a continuum, from one extreme that includes sequence and structural similarity (i.e., orthologs) of entire proteins, to another extreme of chemical similarity at only a few localized sites, as is the case for short linear motifs (slims). the growing body of literature on slims indicates that some important virushost interactions can be attributed to a few well-chosen aas [ ] [ ] [ ] [ ] [ ] . rather than devote entire proteins to one function, slims enable multifunctional viral proteins. interactions between globular virus and host proteins have picomolar affinities, while slims have micromolar binding affinities with globular host proteins [ ] . moderate binding affinity of slims facilitates disruption of signaling interactions, rather than competing for stable formation of persistent protein complexes. synthetic biology practitioners can benefit from an introduction to how slims enable viral interference with host cell functions and computational resources available for slim analysis. viral slims are potentially useful in synthetic biology, to provide a toolkit for new functions, for example, to modulate immune responses or to complement and interact with newly developed adjuvants in a synergistic manner [ ] . research efforts to develop broad-spectrum antiviral compounds or design broadly cross-protective vaccine immunogens benefit directly from knowledge of gene products, protein functions, and motifs involved with viral immune interference. n-linked glycosylation of the png sequon is a well-known example used by viral glycoproteins as camouflage against immune recognition [ , ] . the distribution of n-linked glycosylation sites has recently been recognized as essential for the design of immunogens to induce broadly cross-reactive immune protection against such challenging viruses as hiv- [ , ] . motifs associated with cellular trafficking (localization, transport, secretion, and sequestration) are readily edited to modify where expression products go, and change interaction profiles with other proteins [ ] . in addition to motifs that stabilize the structure of immunogens, such as trimerization ('foldon') [ ] [ ] [ ] [ ] and dimerization [ , ] domains, motifs that interact with cellular processes for innate antiviral pathways could be used to enhance immunogenicity. while slims in eukaryotic proteins have been discussed extensively, slim involvement in viral immunomodulation remains less thoroughly explored, and suggests new opportunities for use in engineered biotechnology applications. the ability to transfer genetic components across species, or to introduce such components de novo, enables new functions. while such functions are generally well intended, some risk also exists for harmful effects. subject to the technical advances of synthetic biology, such effects are not short linear motifs (slims) are patterns of three to ten consecutive aas used by eukaryotic cells for tasks that include: signaling, localization, degradation, and proteolytic cleavage. viruses use slims to their advantage, including interference with antiviral innate immune pathways. viral slims can tolerate mutations, evolve quickly to modify host interactions, and co-occur in a modular manner or involve multiprotein complexes. slims are useful in synthetic biology, where minor edits can alter target specificity, modulate persistence, reprogram interactions with cell-signaling domains, and alter protein function in myriad other ways. aside from possible beneficial uses, for example, to produce better immunogens and develop therapeutic interventions against infectious disease, slims may help characterize new and emerging threats to global health. necessarily a taxonomically relevant property. it may be necessary to evaluate risks of new functions by other means than taxonomy or even protein functional evaluations. instead, new methods are needed that assess functions at a finer resolution than the gene, whether by computational analysis or functional phenotypic assessments [ ] [ ] [ ] . slim analysis might help with such assessments. viral proteins can modulate immunity in several ways, which include: shutdown of host macromolecular synthesis, inhibiting antigen production or apoptosis, and interference with such processes as antigen presentation by mhc, natural killer (nk) cell function, antiviral cytokines, or interferon responses. each of these processes involves coordination among multiple components in host cells. viral interference with these functions is frequently attributed to entire proteins, but in some important cases has been localized to slims. because of their compact size, slims are modular, rapidly evolvable sequence elements. different instances of a given slim can vary in sequence while maintaining the overall functional profile, that is, the regular expression for the sequence motif, where a few positions are invariant while other positions tolerate numerous substitutions. thus, partial sequence matches are sufficient for transient binding interactions with target domains, for example, signal transduction proteins. this observation led to the proposal of ex nihilo slim evolution -the evolution of a novel slim 'from nothing' -the appearance of a new functional module from a previously nonfunctional region of protein sequence [ ] . because hosts' interaction networks are often conserved, slims represent a significant vulnerability for opportunistic exploitation. these properties enable pathogens to acquire host-like slims rapidly through ex nihilo convergent evolution, to rewire host interaction networks, and to acquire tropism and virulence traits needed for successful adaptation and propagation [ ] . over motifs are known, with , validated instances, and many more motifs may await discovery [ , ] . focus on viral motifs may reveal practical utility, to broaden the repertoire of tools available to reprogram molecular function in synthetic biology. one example of how viral proteins use slims to subvert host cell function is illustrated by epstein-barr virus (ebv), which persists in resting memory b cells of nearly all (> %) individuals throughout their adult lives [ ] . latent membrane protein (lmp ) is central to ebv persistence. the cytoplasmic tail of this membrane-bound protein includes pxxpxp and pxqxt motifs that recruit signaling proteins (jak , that is, janus kinase , and several trafs, tumor necrosis factor receptor-associated factors, respectively) [ ] . together, the motifs mimic the cytoplasmic domain of cd to activate nuclear factor-kb via intermediates, including a third motif yyd$ (where $ denotes the c terminus), the tradd (tumor necrosis factor receptor-associated death domain) binding domain. the overall result is that lmp inhibits apoptosis and infected b cell proliferation, to confer viral persistence [ ] . other examples of viral slim contributions to motif mimicry involved with immune function include: protein degradation, transcription, translation, and transport into and out of the nucleus [ ] . given the continued growth of this field [ ] , established frameworks can manage and exploit this knowledge beyond catalogues of currently known motifs [ , , ] or details on contributions of one viral protein (e.g., [ , ] ). work to use slims in bioengineering can benefit from understanding viral protein function. this information is organized in viral knowledge bases, such as viralzone (box ). ontologies describe systematically the many different functional roles of viral proteins, including immune evasion. by promoting use of standard terms for relationships between concepts, an ontology arranges concepts into a framework that can be updated as knowledge grows. protein function is captured broadly in such a framework, though the nuanced details of interactions with other molecules are not localized to domains or motifs. go is an authoritative resource for annotating functions of gene sequences [ , ] . an example of interest is 'evasion or tolerance by virus of host immune response' [ ] (www.ebi.ac.uk/quickgo/ glossary adjuvant: an additive to a vaccine that promotes nonspecific immune responses. when administered together with an antigen, it induces more potent responses than the antigen alone. autophagy: an evolutionarily conserved degradation system for maintaining cellular homeostasis and innate immunity to clear pathogens from cells. domain: structure-based modular subunit of a protein, often with a specific function. domains are generally larger and associated with structural subunits, while motifs are shorter and associated with intrinsically disordered protein regions. elm: the eukaryotic linear motif resource (elm.eu.org), a repository of known slims; includes annotation from primary literature and information about experimental assays used. glycosylation: post-translational modification by host transferases linking sugar molecules to side chains of either nitrogen (nlinked) in asparagine or oxygen (o-linked) in serine or threonine. viral proteins like hiv- envelope can be heavily glycosylated, providing camouflage against immune recognition, shifting as the png sequon mutates. go: gene ontology, an ontology for genetic products of any and all organisms, providing a framework to annotate gene and protein functions in genetic sequence databases and bioinformatics analysis procedures. go includes multiple dimensions to capture biological complexity in adequate depth: molecular function, cellular component, and biological process. go development is conducted by a consortium of research communities and databases, which regularly solicits input and feedback from the broader research community. immune modulation: interference with an immune-related process by a pathogen. intrinsically disordered protein/ domain: regions of a protein sequence that are predicted or experimentally shown not to form consistent structure, e.g., a helices or b sheets. such regions tend to be more accessible for interactions with other proteins. term/go: , figure ). concepts are hierarchically organized, and include a definition, synonyms, and lists of parents and children. functional annotation in go reflects the diverse effects of viral proteins on immune interference. modulating autophagy is an example of recent advances in this research area [ , ] . a growing number of reports describe how virus proteins and slims therein modulate autophagy to promote various aspects of their life cycle [ , ] . both go and viralzone have developed concepts to detail molecular mimicry: structural similarity that enables repurposing or hijacking of molecular function by pathogens, such as viruses. molecular mimicry varies in extent from entire globular proteins, to localized domains, down to short linear motifs. motif class: a regular expression that summarizes known variants to define a sequence motif. motif instance: a particular motif as found in a protein or translated genetic sequence from a specific organism, strain, or isolate. ontology: a representation of domain-specific knowledge organized as concepts, their properties, and their relationships. png sequon: three aa motif n [^p] [st] , that is, asparagine, then any aa except proline, then serine or threonine, recognized by glycosyltransferase as a potential nlinked glycosylation site. regular expression: a string of characters that concisely represents many alternative sequence variants; may include wildcards to represent any character, groupings of possible characters, repetition, negation, start and end of sequence, etc. short linear motif (slim): also known as minimotifs or morfs (molecular recognition features). frequently represented as regular expressions, typically three to te-naas long. viralzone: a knowledge base (https://viralzone.expasy.org) that documents viral families, genome architectures, proteins, hostprotein interactions, and an ontology for the functions of viral proteins. a bridge that links literature reports to go term annotation, viralzone is an online knowledge base that contains 'textbook' information about viral taxonomy, replication, genome organization, and virion structure, and provides links to viral sequence data [ , ] . importantly, viralzone staff collaborate with the go consortium to define entries for virus-specific molecular functions [ ] [ ] [ ] [ ] . viralzone cross-references its keywords with go consortium terms and uniprot [ ] identifiers. this makes it possible to search for viral proteins by their functional role. viralzone staff have developed go concepts specifically for viruses, to represent the diversity of viral replication and processes involved with viral entry, replication, and egress [ ] . viralzone staff have also developed a detailed listing of virus-host interactions, with entries for functions and go terms [ , ] . each entry ('keyword') has a unique identifier. unlike enzyme commission (ec) numbers [ ] , viralzone ids are arbitrary numbers and do not indicate position in the concept hierarchy. instead, organization of the keyword hierarchy is provided online. the web address https://viralzone.expasy.org/ is an entry point into the viralzone concept space ( figure i) . blue text indicates a link to more detail. shown is the vertebrate host-virus interactions page [ ] . also available are summaries for invertebrate, plant, and bacterial host-virus interactions. adapted from www.ebi.ac.uk/quickgo/term/go: [ ] . most arrows indicate 'is a' relations, where the hierarchy is refined by specialization. blue arrows indicate 'part of' relations, which relate to the symbiont process as parts to a whole. autophagy processes, including positive and negative regulation of xenophagy, the selective autophagy of pathogens [ ] [ ] [ ] . understanding the functional roles of slims can help identify related mechanisms or processes, or possibly identify knowledge gaps where slims may be posited but not yet identified. an overview of slim functions may also help to prioritize which are of greatest potential for use or abuse when artificially added to modify protein function. databases and discovery tools are also useful to identify known and new slims. identification of shared structural features across divergent protein families led to analysis and identification of modular protein domains. protein domains are used to categorize protein function, and the interpro database [ ] (www.ebi.ac.uk/interpro) aggregates information at this within-protein level. the identification of protein domains led to recognition of slims as compact, small-scale functional modules [ ] . elm is a database of eukaryotic motifs (box ), though its representation of viral-host interactions is not fully developed. at present, elm entries map to go terms. most motifs map to multiple go terms; the median is seven go terms per motif and the maximum is (mod_plk_ ). immuneassociated function of the lig_irf _lxis_ motif is involved in signal transduction responses to pathogen-associated molecular patterns; this motif maps to go terms, but none occur in the viralzone vocabulary. in total, only three elm entries utilize go terms from viralzone: lig_bh_bh _ , lig_hcf- _hbm_ , and lig_rb_pabgroove_ . these three motifs all map to the most general (ii) deg, degradation sites, part of polyubiquitination; (iii) doc, docking sites, involved in protein recruitment but not directly targeted by an active site; (iv) lig, ligand binding sites, primarily for protein-protein interactions; (v) mod, post-translational modification sites; and (vi) trg, targeting sites for subcellular localization. elm has also spun-off several specialized databases: phospho.elm for phosphorylation sites with experimental evidence [ ] , switches.elm for conditional molecular switches, such as requiring that a site be modified [ ] , and ielm, with an emphasis on protein-protein interactions [ ] . a detailed tutorial provides orientation for elm use [ ] . elm documents each motif class with a concise description of its function. for example, one type of nuclear localization signal (nls), trg_nls_bipartite_ the 'classic bipartite nls', which binds to importin-a for nuclear pore transfer and is utilized by the pb protein of influenza a, is documented here: elm.eu.org/elms/ trg_nls_bipartite_ . the abstract and functional site descriptions summarize what is known about the motif. go term, go: ('modulation by virus of host morphology or physiology', a synonym of 'virushost interaction'). this underscores the prevalent mode of elm motif discovery and annotation does not emphasize host-virus interactions, but rather systems-level interactions within eukaryotic cells. thus, better integration of viralzone-go-term vocabulary with elm or another domain-level representation of viral slims is needed to promote potential utility for biotechnology. despite not using the viralzone ontology, elm documents other motif classes with go terms that refer to 'viral', 'virus', 'immune', or 'immunity' (table ) . because the focus is motif function in the host cell context, elm does not directly indicate how viral immune interference results. further, the relative lack of viral motifs in elm does not indicate their absence in vivo, but rather the evidence-based requirement for elm inclusion. related to the earlier observation, a review of how viruses use slims to interfere with host cells [ ] lists examples that represent viral mimicry of host slims (table ). only % of these have corresponding elm entries, though the slims are known. the remaining % indicate that elm does not fully capture all known viral motifs. this strong requirement by elm for evidence-based motif classes and instances is not strictly a drawback. indeed, the elm creators are very aware that computational analysis alone is error prone and can yield misleading outcomes. in [ ] , they discuss this issue in depth, and recommend a workflow for slim discovery that culminates in experimental validation, whether in vivo or in vitro. working with viral-host systems adds layers of difficulty to experimental motif validation, so it should not be surprising or to the detriment of available information resources that viral slims are less thoroughly documented. searching arbitrary sequences for motif instances is computationally straightforward. box provides an example of motif searching with elm, which might facilitate comparative analysis of two related proteins from different species of human herpesvirus (hsv). resources such as interpro and uniprot are able to perform similar assessments, but give broader, domain-level representations with less functional detail than the slim searches enabled by elm. reports in the primary literature take a different approach, by marking slims in a protein alignment, which includes orthologues to mark conservation (e.g., figure in [ ] ). the elm-generated report combines predicted slims with information from annotated domains and local disorder predictions, for a perspective that complements the other approaches. clearly, false positives are inevitable among slim search results. this makes it necessary to filter for the most significant and informative outcomes. this leads to consideration of in silico (computational) methods for slim evaluation. a highly recommended, authoritative review of slim discovery techniques, from an author of the slimsuite software package, discusses motif identification techniques in depth [ ] . methods for slim discovery can be divided into two broad classes: (i) de novo discovery of new slims, and (ii) instance prediction to find new occurrences of known slims. there are currently at least eight software packages available to discover new slims and packages ( stand-alone programs or servers and two software suites that consist of multiple tools: slimsuite [ ] , which includes ten utilities, and meme [ ] , which consists of five tools for slim instance detection). though meme was developed for discovery of dna sequence motifs, it generates ungapped, profile-based motifs using the expectation-maximization (em) algorithm. no single method is inherently better than the rest, but the choice of which to use depends on several factors, such as the input sequence data, whether one sequence, an alignment, or a collection of nonhomologous sequences [ ] . to illustrate the diversity of motif discovery methods, this section mentions only a handful of the software tools available (table ) . readers seeking to learn more about the full set of alternatives are strongly encouraged to consult [ ] , particularly tables , , and therein. another helpful resource ( table in [ ] ) lists online motif discovery bioinformatics services. several alternative approaches for discovery of new motifs have been advanced. edwards and palopoli [ ] review the alternatives in depth, discussing their merits and drawbacks. briefly, they can be divided into alignment-based and alignment-free methods. an alignment-based approach looks for conserved sites among homologous sequences, but can be misled by high sequence conservation in globular domains. a program called slimprints works around this with a specialized approach to model substitutions [ ] . slimprints uses a statistical model of relative local conservation, which looks for clusters of overly constrained sites in a window of about aas, using iupred scores (intrinsically unordered prediction; see later) to weigh sites in intrinsically disordered protein regions more heavily than sites in globular (ordered) regimes [ ] . in contrast, alignment-free methods look for enrichment of amino acid patterns in proteins that are expected by other means to perform similar motif-related roles, for example, by go category annotations or protein-protein interaction (ppi) data, that is, via databases that capture experimental evidence for protein colocalization and functional interactions. an important caveat is that to assume such sequences are independent could yield spurious enrichment of shared patterns, so alignment-free methods need to compensate for evolutionary constraints at the domain level, rather than for full-length homologous proteins. the development of such corrections and their relative advantages are detailed in [ ] . some programs (e.g., slimdisc [ ] , slimfinder [ , ] , and dilimot [ , ] ) produce regular expressions that compensate for phylogenetic relatedness, while others (meme suite, glam [ ] , and nestedmica [ , ] ) produce probabilistic profiles. for more discussion of these and issues of concern for computational motif discovery, see [ ] . filtering methods control high false positive rates from slim instance detection. structural information, whether known or predicted, can be used for filtering. box illustrates how elm filters results to hsv- and hsv- virulence factor icp . assists in viral immune evasion by molecular mimicry. hsv- neurovirulence protein icp . , encoded by the g . gene, initiates immune interference by binding and sequestering cellular proteins that would stimulate autophagy, translational arrest, and type i interferon responses. hsv- icp . binds tank-binding kinase (tbk ) to prevent type i interferon induction [ ] , beclin- to prevent autophagy [ ] , and both pp a and eif a to overcome translational arrest [ ] . hsv- g . contains an intron not present in hsv- , and up to four isoforms of hsv- icp . are known [ ] . full-length hsv- icp . has conserved pp a and eif a-binding domains, but lacks tbk and beclin- binding domains [ ] . additional hsv- motifs influence intracellular localization [ ] , virion maturation, and egress [ ] , not yet characterized in hsv- . hsv- is recognized as more virulent than hsv- , but both can cause neuropathology, including viral encephalitis and meningitis [ ] . to attenuate virulence, icp . is routinely deleted or inactivated when making hsv- constructs for oncolytic therapy [ ] . both are the same length and share domain structures, and partially share slim compositions ( figure i ). identifying differences in slims from each could provide clues for more detailed experimental investigations to understand icp . virulence determinants and host protein targets. exclude a region predicted to fold as globular protein. these predictions were made by smart (simple modular architecture research tool) [ ] and pfam [ ] domain matches, corroborated by globplot [ ] . another widely used approach is to identify regions of local disorder, where protein structure is not clearly defined, making that region accessible to interact with other proteins. iupred [ ] is commonly used for this task, though the choice of parameter settings and how to interpret results varies. elm results include an iupred disorder score and a simple cutoff of . to define the disorder transition. above this value, local protein structure is considered accessible for interaction with other proteins. scoring schemes filter for statistical enrichment of motif instances. an approach of filtering by homology [ ] seems inappropriate for use to detect virus interactions with host proteins, as it may exclude nonhomologous regions with motifs that do interact, yielding false negatives. regardless, failure to consider evolutionary relatedness among sequences being searched could introduce bias due to common ancestry, rather than independence, among sequences. a simple approach to instance prediction is a stand-alone program called shettimotif [ ] . it was used to scan protein sequences from poxviridae genomes (an average of proteins per poxvirus) for low-complexity regions and regular expressions defined by prosite. the approach compared numbers of proteins per genome that carry each motif, and doubtlessly includes many motif instances that are not functional as slims. also, shorter motifs occur more frequently than longer motifs [ , ] , partly due to chance alone. regardless, systematic error may be considered a source of background noise across the large number of proteins in viral proteomes, each having different host specificities, to enable somewhat meaningful comparisons, in such a 'statistical genomics' approach [ ] . the comparisons could be more meaningful if false positive motif instances were reduced. becerra et al. developed another approach to instance counting [ ] , which involves comparison with a null distribution from permuting primary sequence and testing for presence of the motif in the permuted sequences. a motif is considered rare and therefore significantly unlikely to occur by chance if it is present at or below some cutoff frequency. restricting the sequence region that is used for permutation testing, such as by use of structural considerations, can further focus the search. indeed, such a hybrid filtering approach was described recently and evaluated on the hiv- proteome [ ] . following methods described in an earlier study [ ] , becerra et al. used iupred with a modified, window-based scoring procedure to identify intrinsically disordered protein regions, and tested for statistical rarity below % of shuffled variants. the approach further considered conservation above % in a set of aligned sequences, though combining three filtering criteria was too stringent and excluded all motif candidates [ ] . while algorithmic approaches seek to identify a broad range of slim types, more specialized resources have emerged to track the distribution of a particular slim in viral proteins. for example, ilir@viral is a web resource dedicated to detecting lir motif-containing proteins in viruses [ ] . lc interacting regions (lir motifs) are slims that mediate protein-protein interactions involved in autophagy, as used by influenza a virus m protein to subvert autophagy and maintain virion stability [ ] . using curated text mining analysis and position-specific scoring matrices, ilir@viral analyzed reviewed viral sequences available from uniprot across individual viral species and found that viral sequences contain lir motifs. while many predicted instances may represent false positives, the enrichment of lir motifs in viral sequences is consistent with viral adaptation to host xenophagy [ ] . curiously, elm currently lists the lir motif as a candidate, rather than an accepted motif class. embedding slims into engineered constructs may enable specific effects on cellular immune processes, for applications that include targeted drug delivery, pathogen-specific adjuvants, potent and broadly effective immunogens, transformational medical countermeasures, and improved design of vectors for gene therapy. slim modularity may enable easy ways to reprogram protein function with a few localized modifications. to realize the potential utility of slims in synthetic biology, more research is needed to expand and integrate our collection of knowledge on viral slims (see outstanding questions). detecting slims in variant sequences may help to identify functional innovation or changes in virulence, in a manner that does not rely strictly on functional assessment at the whole-gene level, to identify how sequence-specific variation may interact with host responses. this may be particularly useful and important to understand new variants and assess the risk that they may spread and cause harmful effects on human health or agricultural interests. such knowledge is needed in an era where synthetic biology may introduce new risks for biological error and biological terror. detecting and understanding slim variants can help to reduce such risks and identify newly emerging threats to global health and security because watch lists for harmful organisms to ensure public safety by preventing access to select known risks may be inadequate [ ] [ ] [ ] . slims in viral proteins can interact in many different ways with host proteins to modulate immune responses. a motif may be necessary but not sufficient for any inferred function. the simplest case is where a viral slim interacts directly with a host protein to yield an immunomodulated phenotype. more elaborate cases are known, such as the multifunctional proteins e a (ebv), nef (hiv- ), and icp . (hsv). computational prediction of slim classes and new instances is a process, which involves experimental confirmation and validation. high-throughput methods for experimental assessment of protein interactions are useful to validate computational predictions [ , ] , and more assays are needed to evaluate functional and phenotypic effects of adding or deleting slims. resolved map of human-virus protein-protein interaction networks. plos pathog. , e what specific constraints limit slim evolvability? what strategies are most effective to advance knowledge of viral immunomodulatory slims in the design of vaccines and therapies to promote global health? for example, can some viral peptides be useful as adjuvants? signatures of pleiotropy, economy and convergent evolution in a domain pathogen mimicry of host protein-protein interfaces modulates immunity use of host-like peptide motifs in viral proteins is a prevalent strategy in host-virus interactions slimsearch . : biological context 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functionality of the eukaryotic linear motif resource elm: a database of phosphorylation sites -update the switches.elm resource: a compendium of conditional regulatory interaction interfaces ielm -a web server to explore short linear motif-mediated interactions exploring short linear motifs using the elm database and tools control of tank-binding kinase -mediated signaling by the g . protein of herpes simplex virus hsv- icp . confers neurovirulence by targeting the beclin autophagy protein a conserved domain of herpes simplex virus icp . regulates protein phosphatase complex in mammalian cells up to four distinct polypeptides are produced from the g . open reading frame of herpes simplex virus herpes simplex virus icp . confers neurovirulence by regulating the type i interferon response an n-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type icp . protein and its ligand, protein phosphatase replication of herpes simplex virus depends on the g . functions that facilitate virus response to interferon and egress in the different stages of productive infection the herpes simplex virus neurovirulence factor g . : revealing virus-host interactions elm: the status of the eukaryotic linear motif resource key: cord- - q yr np authors: fenner, frank; bachmann, peter a.; gibbs, e. paul j.; murphy, frederick a.; studdert, michael j.; white, david o. title: viral replication date: - - journal: veterinary virology doi: . /b - - - - . - sha: doc_id: cord_uid: q yr np viral replication is the central focus of much experimental virology and is a significant part of molecular biology. studies with bacteriophages in their prokaryotic host cells in the s and s provided the first insights into the complexities of viral replication. with the development of mammalian cell culture procedures, the techniques used for the study of bacteriophages were adapted to animal viruses. progress has been such that the basic mechanisms of transcription, translation, and nucleic acid replication have been characterized for all the major families of animal viruses and the strategy of gene expression and its regulation clarified. many important biochemical phenomena such as the splicing and other types of posttranscriptional processing of rna, the posttranslational cleavage and glycosylation of proteins, the replication of rna, reverse transcription, integration, and the transposition of viral genes and cellular oncogenes were first elucidated by virologists and have general application in cell biology. the chapter provides a general overview on viral replication for understanding pathogenesis, immunity, chemotherapy, and the role of viruses in cancer. viral replication is the central focus of much experimental virology and a significant part of molecular biology. studies with bacteriophages in their prokaryotic host cells in the s and s provided the first insights into the complexities of viral replication. with the development of mammalian cell culture procedures (see chapter ), the techniques used for the study of bacteriophages were adapted to animal viruses. progress has been such that the basic mechanisms of transcription, translation, and nucleic acid replication have been characterized for all the major families of animal viruses and the strategy of gene expression and its regulation clarified. many important biochemical phenomena, such as splicing and other types of posttranscriptional processing of rna, posttranslational cleavage and glycosylation of proteins, replica tion of rna, reverse transcription, integration, and transposition of viral genes and cellular oncogenes, were first elucidated by virologists and have general application in cell biology. our knowledge of viral replication is now very detailed and is expand ing rapidly. every viral family has a different strategy of replication, and for each family several reviews have been published since . it is neither possible nor appropriate to deal comprehensively with the sub ject in this book. this chapter provides a general overview; some addi tional information on particular viral families is provided in part ii. an understanding of viral replication provides a basis for understanding pathogenesis, immunity, chemotherapy, and the role of viruses in cancer. following the pattern established in experiments with bacteriophages, studies of the replication of animal viruses began with the onestep growth experiment. in such experiments, all cells in a culture are infected simultaneously, i.e., at high multiplicity of infection. unadsorbed input virus is removed or neutralized, usually after hour, and the increase in infectious virions over time is followed by titrating cell-free and cell-associated infectivity. shortly after infection, the inoculated virus "disappears"; infectious particles cannot be demonstrated, even intracellularly. this eclipse period continues until the first pro geny virions become detectable some hours later. nonenveloped viruses mature within the cell and are detectable for some hours as intracellular virions before they are released by cell lysis. many enveloped viruses, on the other hand, mature by budding from the plasma membrane and are thus immediately released into the medium. the eclipse period ranges from to hours for the various dna viruses and from to hours for rna viruses (see table - ). early studies, relying on quantitative electron microscopy and assay of infectivity, provided a limited amount of information about the early and the late events in the replication cycle (attachment, penetration, intracellular maturation, and budding) but could not tell us anything about what happened during the eclipse period. investigation of the expression and replication of the viral genome became possible only with the development of molecular methods, and during the last two decades all the sophisticated techniques of molecular biology have been applied to this problem. general features of the viral replication cycle, using a nonenveloped icosahedral dna virus as a model. no topographical location for any step is implied. one step grades into the next such that, as the cycle progresses, several of these processes are proceeding simultaneously. release occurs by cell lysis. principally viral structural proteins; some of these are subject to posttranslational modification, such as glycosylation and/or cleavage. as sembly of icosahedral virions occurs in the nucleus or cytoplasm, de pending on the particular family. enveloped viruses are completed by "budding" through cellular membranes. each infected cell yields sever al thousand new virions over a period of several hours. many of the processes that can be investigated by morphological stud ies and infectivity assays differ according to viral family (table - ). there are three principal methods of penetration, and virions may be released by cell lysis or by budding. some viruses acquire an envelope by budding through plasma membrane, others through nuclear mem brane, and still others in the golgi complex or the endoplasmic reticulum. some viruses shut down the synthesis of cellular macromolecules very effectively, whereas others do not. indeed, some viruses are noncytocidal and others actually induce the cell to divide, or even trans form it to a tumor cell (see chapter ). even more significant are the differences in the strategy of expression of the viral genome. under this heading are subsumed the key processes occurring during the eclipse period: transcription and processing of viral mrna ( fig. - , steps and ), translation and processing of viral pro teins (steps and ), and replication of the viral nucleic acid (step ). before discussing them, we will describe the earlier events: attachment (step ), penetration (step ), and uncoating (step ). because virions and cells are both negatively charged at physiological ph, they tend to repel one another, but random collisions do occur and initial (reversible) attachment may be facilitated by cations. firm binding requires the presence of specific receptors for the virus on the plasma membrane, to which specific molecules on the surface of the virion while there is some specificity about the binding of virions to particular cellular receptors, several different viruses may utilize the same receptor. electron microscopic and other data show that virions can enter cells by at least three different mechanisms: endocytosis, fusion, and translocation. the majority of virions entering a cell fail to initiate infection, many virions taken up by endocytosis being degraded by lysosomal enzymes. however, for some viruses this may be the normal route of penetration, leading to uncoating and productive infection. the majority of mammalian cells are continuously engaged in receptormediated endocytosis, a specific process for the uptake of essential macromolecules. viruses may use receptor-mediated endocytosis to initiate infection (plate - ). following attachment to receptors, virions move down into coated pits. these pits, coated with clathrin, fold inward to produce coated vesicles that enter the cytoplasm and fuse with a lysosome to form a phagolysosome. with enveloped viruses, the envelope of endocytosed virions fuses with the lysosomal membrane, releasing the viral nucleocapsid into the cytoplasm. in this way a virion can be uncoated by a lysosome but escape total degradation by the lysosome's hydrolytic enzymes. recent studies with influenza virus have identified a ph -mediated conformational change in the hemagglutinin molecule which enables fusion to occur between the viral envelope and the mem brane of the phagolysosome. the f (fusion) glycoprotein of paramyxoviruses, in its cleaved form, enables the envelope of these viruses to fuse directly with the plasma membrane, even at ph . this may allow the nucleocapsid to be re leased directly into the cytoplasm. although a number of other enve loped viruses display a capacity to fuse cells or to lyse erythrocytes, it is not clear whether this is the normal way in which they infect cells. some nonenveloped icosahedral viruses appear to be capable of pass ing directly through the plasma membrane. [a, from e. fries and a. helenius, eur. j. biochem. , ( ) ; b, from k. simons et al., sci. am. , ( ) , courtesy dr. a. helenius.] in order that at least the early viral genes may become available for transcription, it is necessary that the virion be at least partially uncoated. with viruses that enter the cell by fusion of their envelope with either the plasma membrane or the membrane of a phagolysosome, the nucleocapsid is discharged directly into the cytoplasm. in the case of vi ruses with helical nucleocapsids, transcription begins from viral rna while it is still associated with nucleoprotein. in the case of the icosahedral reoviruses only certain capsid proteins are removed and the viral genome expresses all its functions, even though it is never fully released from the core ("subviral particle"; plate - ). poxviruses are uncoated in two stages: first, to a core, from which half the genome is transcribed; then completely, following the synthesis of a virus-coded uncoating protein. with the picorna viruses, the process of attachment of the virion to the cell leads to a conformational change in the capsid, . reovirus "cores" that have synthesized mrna for minutes at °c were prepared for electron microscopy by the kleinschmidt technique, stained with uranyl acetate, and shadowed at a low angle with platinum-palladium, showing the fine fibrils of mrna being extruded from the cores or occurring free around them. the results of polyacrylamide gel electrophoretic analysis of such mrna molecules at various times during the replication cycle are illustrated in fig. - . [from n. m. bartlett, s. c gillies, s. bullivant, and a. r. bellamy, j. virol. , ( ), courtesy dr. a. r. bellamy.] resulting in the loss of capsid proteins vp and vp and rendering the particle susceptible to proteases; the attachment step itself triggers the process of uncoating. for some viruses that replicate in the nucleus there is evidence that the later stages of uncoating occur there, rather than in the cytoplasm. the key events in viral replication are the synthesis of viral proteins, the replication of the viral genome, and the assembly of the new compo nents into virions. to synthesize viral proteins, viral mrnas must be produced in a form capable of being recognized and translated on cel lular ribosomes. eukaryotic cells synthesize their own mrna in the nucleus by transcription of the cellular dna followed by processing of the transcript. they lack the enzymes necessary for synthesizing mrna off a viral rna genome and they cannot transcribe viral dna located in the cytoplasm. therefore, only those dna viruses that replicate in the nucleus utilize the cellular machinery for transcription. all other viruses provide their own enzymes to produce mrnas. eukaryotic cells have a further constraint, namely, that the protein-synthesizing machinery ap parently cannot recognize internal initiation sites within polycistronic mrnas. hence viruses must synthesize a separate (monocistronic) mrna corresponding to each gene in their genome, or, alternatively, a polycistronic mrna must be translated into a large precursor "polyprotein" which is then cleaved into individual proteins. the diverse strategies followed by viruses of different families for transcription and translation are illustrated diagrammatically in fig. - (for dna viruses) and fig. - (for rna viruses). necessarily, the pro cesses summarized in these figures and the descriptions of them involve major oversimplifications. we will describe in turn transcription, trans lation, and replication of the viral nucleic acid. the viral rna of (+) sense ssrna viruses binds directly to ribosomes and is translated in full or in part without the need for any prior transcriptional step. with all other classes of viral genomes, mrna must be transcribed. in the case of dna viruses that replicate in the nucleus, the cellular dna-dependent rna polymerase ii performs this function. all other viruses require unique and specific transcriptases which are virus-coded and are an integral component of the virion. cytoplasmic dsdna viruses carry a dna-dependent rna polymerase, whereas dsrna vi ruses have dsrna-dependent rna polymerase, and (-) sense ssrna viruses carry a ssrna-dependent rna polymerase (see tables - and - ). for all dna viruses, mrna must be transcribed by a dna-depen dent rna polymerase. transcription of the viral dna is programmed such that not all genes are expressed simultaneously or continuously throughout the replication cycle. particular parts of the genome are tran scribed in sequence, the so-called early genes first, and the late genes later in the cycle. viruses of different families differ according to whether a cellular or a viral transcriptase is employed, correlating with a nuclear or cytoplasmic site of replication. there are four classes of strategy of ex pression of the viral genome ( fig. - a-d), described below. dsdna; cellular transcriptase ( fig. - a) . this group comprises the papovaviruses, adenoviruses, and herpesviruses, and has in one respect the most straightforward strategy: the viral dna is transcribed within the nucleus by a cellular dna-dependent rna polymerase. there are at least two temporally separated cycles for adenoviruses and herpesviruses; in each instance the structural proteins of the virion are made from mrnas produced in the last cycle of transcription. polycistronic but subgenomic rna transcripts (corresponding to several genes but less than the whole genome) undergo cleavage and splicing to produce monocistronic mrnas, introns being removed in the process. dsdna; virion transcriptase ( fig. - b) . the poxviruses and african swine fever virus, which replicate in the cytoplasm, carry their own transcriptase. it appears that monocistronic mrnas are transcribed di rectly from the viral dna. there are at least three cycles of transcription. the transcripts are translated directly into proteins, some of which need to undergo posttranslational cleavage to yield functional molecules. ssdna; cellular transcriptase ( fig. - c) . the (-) sense ssdna of the parvoviruses requires the synthesis of a complementary strand to form dsdna; this is then transcribed in the nucleus and the transcripts are processed to produce mrnas, before export to the cytoplasm for translation. a virion-associated dna polymerase, and the dna then converted into a supercoiled dsdna. transcription of mrna by cellular rna poly merase ii then occurs. expression of a dna genome. analysis of the -bp sequence of the circular dsdna of the papovavirus sv and its transcription pro gram have provided insights into these processes ( fig. - ) . the follow ing points should be noted: . the early genes and the late genes are transcribed by the host cell rna polymerase ii in opposite directions, from different strands of the dna. . certain genes overlap and are translated in the same frame, so that their protein products have some amino acid sequences in common. . some regions of the viral dna are read in overlapping but different reading frames, so that two completely different amino acid sequences are obtained. . at least % of the viral dna consists of intervening sequences (introns), which are transcribed but not translated, because they are excised from the primary transcript. . up to three distinct proteins can be produced from mrnas derived from a primary transcript by different splicing protocols. studies with adenoviruses have eluci dated the mechanisms that regulate the expression of viral genomes, which operate principally, but not exclusively, at the level of transcrip tion. because of the complications arising from posttranscriptional cleav age of mrna and posttranslational cleavage of precursor proteins in eukaryotic cells, it is no longer adequate to talk of a "gene" and its "geneproduct." more appropriate perhaps is to think in terms of a transcription unit, i.e., that region of the genome beginning with the transcription initiation site, extending right through to the transcription termination site, and including all introns and exons in between. "simple" transcrip tion units may be defined as those encoding only a single protein, whereas "complex" transcription units code for more than one. there are many adeno virus transcription units. at different stages of the viral replication cycle-"pre-early," "early," "intermediate," and "late"the various transcription units are transcribed in a given temporal se quence. a product of the early-region eia induces the other early regions including e ib, but following viral dna replication, there is a -fold increase in the rate of transcription from the major late promoter relative to early promoters such as e ib, and a decrease in eia mrna levels. a second control operates at the point of termination of transcription. transcripts that terminate at a particular point early in infection are read through this termination site later in infection to produce a range of longer transcripts with different polyadenylation sites. processing of rna transcripts. primary rna transcripts from eukaryotic dna are subject to a series of posttranscriptional alterations in the nucleus, known as processing, prior to export to the cytoplasm as mrna. first, a cap, consisting of -methylguanosine (m gppp) is added to the ' terminus. the function of this poly (a) tail is uncertain, but it may act as a recognition signal for processing and for transport of mrna from the nucleus to the cytoplasm, and it may stabilize mrna against degradation in the cytoplasm. third, a methyl group is added at the position to about % of the adenylate residues throughout the rna (methylation). fourth, introns are removed from the primary tran script and the exons are linked together in a process known as splicing; the precise mechanism is not known but may involve excision of the introns by endonucleases, followed by ligation. splicing is an important mechanism for regulating gene expression in nuclear dna viruses. a given rna transcript can have two or more splicing sites and be spliced in several different ways to produce a variety of mrna species coding for distinct proteins; both the preferred poly(a) site and the splicing pattern may change in a regulated fashion as infection proceeds. the rate of degradation of mrna provides another level of regulation. not only do different mrna species have different half-lives, but the halflife of a given mrna species may change as the replication cycle pro gresses. transcription is more complicated for the rna viruses than for rhe dna viruses, which is perhaps not surprising, since they are the only forms of life that utilize rna as the repository of genetic information. there are, broadly, three main strategies: ( ) the virion rna of most viruses with (+) sense rna is itself infectious, because it functions as mrna, ( ) viruses with (-) sense ssrna, or with dsrna, carry a virion-associated rna-dependent rna polymerase which transcribes mrna from the viral rna, and ( ) the (+) sense virion rna of retroviruses is transcribed into dna, which serves as a template for tran scription of viral mrnas by a cellular transcriptase. these three general strategies can be further subdivided on the basis of more subtle dif ferences to give seven groups ( fig. - a-g). ssrna; (+) sense ( fig. - a ,b,c). in these groups the (+) sense virion rna is itself infectious. in the picornaviruses and flaviviruses the ge- nome, acting as a single polycistronic mrna, is translated into a single polyprotein which is subsequently cleaved to give the individual viral polypeptides (fig. - a) . togaviruses of the genus alphavirus also con tain a single polycistronic (+) sense ssrna molecule, but only about two-thirds of the viral rna (the ' end) is translated; the resulting polyprotein is cleaved into four nonstructural proteins, two of which form the rna polymerase. this enzyme then copies a full-length (-) sense strand, from which two species of (+) sense strand are copied: full-length virion rna destined for encapsidation, and a one-third length rna, which is colinear with the ' terminus of the viral rna and is translated into a polyprotein from which three or four structural pro teins are produced by cleavage. the caliciviruses have not been so exten sively studied, but also produce both genome-length and subgenomic mrna species. flaviviruses were recently accorded the status of a family separate from the toga viruses. they do not produce subgenomic mrnas, and translation of the (+) sense virion rna initiates with the capsid protein near the ' end of the genome and proceeds sequentially through the genome to produce one precursor polyprotein. this is rapidly cleaved during the process of translation, so that the complete polyprotein is never seen. corona viruses have a unique strategy. initially, in a step about which little is known, part of the virion rna acts as mrna and is translated to produce an rna polymerase, which then synthesizes a genome-length (-) sense strand. from this, a "nested set" of overlapping subgenomic rnas is transcribed, of which only the unique (nonoverlapping) se quence in each is translated (see chapter ). ssrna; (-) sense; virion transcriptase ( fig. - d,e) . primary tran scription from the (-) sense ssrna viruses occurs in the cytoplasm, when the virion rna is still within the helical nucleocapsid, in associa tion with the nucleoprotein as well as the transcriptase. particular se quences of to nucleotides, located at or near the termini of each rna molecule, may serve as recognition signals for transcriptase binding. the paramyxoviruses and rhabdoviruses have similar transcription strategies, as well as similar consensus sequences at the ' and ' termini of their viral rna, suggestive of a common ancestry. the (-) sense virion rna is copied in two distinct ways: the replication mode and the transcription mode. copying in the replication mode produces a fulllength (+) sense strand which is used as a template for the synthesis of new virion rna. in the transcription mode, five subgenomic (+) sense rnas are produced; each is capped and polyadenylated and serves as a monocistronic mrna. it is still not certain what dictates whether the polymerase reads right through from ' to ' end of the (-) sense rna template (replication mode), ignoring internal termination signals which are obeyed in the transcription mode to produce the family of five mono cistronic mrnas. there is some evidence that the polymerase may have only a certain probability of "falling off" its template as it reaches a termination codon; the five mrnas are made in decreasing molar amounts, reading from the ' end of the parental rna. the orthomyxoviruses, bunyaviruses, and arenaviruses have seg mented genomes, and each segment is transcribed to yield an mrna which is translated into one or more proteins (fig. - e ). in the case of the orthomyxoviruses, most of the segments can be regarded as single genes, for they encode single proteins. special mention needs to be made of a phenomenon known colloquially as "cap-snatching," which is required by orthomyxoviruses for the initiation of mrna synthesis. a virion-associated endonuclease enters the nucleus and removes a short segment from the capped ' terminus of cell mrna; this is transported back to the cytoplasm, where it binds to the virion rna and serves as a primer to initiate transcription. in general, each viral rna segment of the genomes of the bunya viruses and arenaviruses codes for more than one protein. furthermore, the s segment, at least, of arenaviruses and the phlebovirus genus of bunyaviruses is ambisense. the replication strategy of ambisense rna viruses, like the sense of their genomes, is mixed, with features of both (+) sense and (-) sense ssrna viruses (see chapters and ). bunyavirus mrnas also carry nonviral sequences at their ' termini, presumably derived from cellular mrna primers. dsrna; virion transcriptase ( fig. - f) . the two families of viruses with dsrna (birnaviridae and reoviridae) have segmented genomes and each segment is separately transcribed in the cytoplasm by a virionassociated rna-dependent rna polymerase. with reoviruses, each of the , , or dsrna segments corresponds to a single gene. monocistronic mrnas are transcribed from each segment within the partly uncoated subviral particle (see plate - ); these rnas complex with a protein before each is copied to produce a dsrna, which serves as the template for further mrna transcription. ssrna; (+) sense; virion reverse transcriptase (fig. - g ). in the retroviruses the viral rna is (+) sense, but instead of functioning as mrna it is transcribed into dna by a viral rna-dependent dna poly merase, and the resulting rna-dna hybrid molecule is converted to dsdna and integrated into the cellular dna. transcription of rna then occurs from the integrated viral dna via the cellular transcriptase, followed by splicing of the rna transcript as well as cleavage of the resulting proteins (see chapters and ). regulation. transcription from rna viral genomes is generally not as rigorously regulated as with dna viruses. in particular, the temporal separation into early genes transcribed before the replication of viral nucleic acid and late genes transcribed thereafter is not nearly so clear. k. joklik, virology , ( ).] increases steadily during the first hours of reovirus infection as more template becomes available, the relative amounts of each of the mrna species remain unchanged. with some viruses, however, a sub tle form of control can modulate the relative abundance of mrnas for different proteins. for instance, in the case of the ( -) sense ssrna rhabdoviruses and paramyxoviruses, where the whole genome is tran scribed into five monocistronic mrna species, each coding for one of the five structural proteins, the "polarity" of the linear transcription by the viral transcriptase, described earlier, results in favored synthesis of mrna for the proteins coded by the ' end of the viral rna. capped, polyadenylated, and processed monocistronic viral mrnas bind to ribosomes and are translated into protein in the same fashion as cell mrnas. the sequence of events has been closely studied for reovirus. each monocistronic mrna molecule binds via its capped ' termi nus to the s ribosomal subunit, which then moves along the mrna molecule until stopped at the initiation codon. the s ribosomal subunit then binds, together with methionyl trna and various initiation factors, after which translation proceeds. despite the fact that mrna is transcribed from each of the monocistronic dsrna reovirus segments in equimolar amounts, there are pronounced differences in the amounts of each protein made, indicating the existence of a regulatory mecha nism at the level of translation. the proteins translated from the early transcripts of dna viruses include enzymes and other proteins required for the replication of viral nucleic acid, as well as proteins that suppress host cell rna and protein synthesis. however, the function of most early viral proteins of the large dna viruses is still unknown. the late viral proteins are translated from late mrna, most of which is transcribed from progeny viral nucleic acid molecules. most of the late proteins are viral structural proteins, and they are often made in consid erable excess. some of them also double as regulatory proteins, modu lating the transcription or translation of cellular or early viral genes. the temporal order and amount of synthesis of particular proteins of dna viruses is regulated mainly at the level of transcription. with rna viruses it is also usual for nonstructural proteins to be made early and structural proteins later, but the control is generally not as rigorous as for the dna viruses and occurs at the level of translation. for instance, in the case of calici viruses, coronaviruses, and toga viruses, only the ' end of the (+) sense viral rna, which codes for the nonstructural pro-teins, including the rna polymerase, is translated early, hence produc tion of complementary (-) sense rna can commence. this then serves as the template for transcription of subgenomic rna corresponding to the ' end of the viral rna, from which are translated the structural proteins required in abundance later in infection. in the picornaviruses, the polycistronic viral rna is translated di rectly into a single polyprotein which carries protease activity. this virus-coded protease cleaves the polyprotein at defined recognition sites into smaller proteins. the first cleavage steps are carried out while the polyprotein is still bound to the polyribosome. some of the larger inter mediates exist only fleetingly; others are functional but are subsequently cleaved to smaller proteins with alternative functions. posttranslational cleavage occurs in several other rna virus families but is a less prominent feature in the overall production of individual proteins. in the case of the toga viruses and calici viruses, polyproteins corresponding to only part, albeit a large part, of the genome are trans lated from polycistronic mrna and then cleaved. with viruses of sever al other families, cleavage of particular proteins late in the replication cycle is essential for the production of infectious virions. newly synthesized viral proteins must migrate to the various sites in the cell where they are needed, e.g., back into the nucleus in the case of viruses that replicate there. the mechanisms controlling such migration are unknown, but presumably resemble those used for cellular proteins and possibly involve the cytoskeleton. migration is doubtless intimately dependent on the structural features of particular proteins. in the case of glycoproteins, the polypeptide is translated on membrane-bound ribosomes, i.e., on rough endoplasmic reticulum; various co-and post translational modifications, including acylation, proteolytic cleavage, and addition and subtraction of sugars, occur sequentially as the protein moves in vesicles to the golgi complex and thence to the plasma mem brane (see below). different mechanisms of dna replication are employed by each fami ly of dna viruses. we can give only a brief overview here. [from e. d. sebring et al., j. virol. , ( ).] papovaviridae. little is known about the replication of papillomavirus dna, but the polyomaviruses, especially sv , have been studied in great detail. the sv genome, with its associated cellular histones, morphologically and functionally resembles cellular dna and utilizes host cell enzymes, including dna polymerase a, for its replication. an early viral protein, large-t, binds to three sites in the regulatory se quence of the viral dna, thereby initiating dna replication. replication of this circular dsdna commences from a unique palindromic sequence and proceeds simultaneously in both directions at the same rate ( fig. - ) . as in the replication of mammalian dna, both continuous and discontinuous dna synthesis occurs (on leading and lagging strands, respectively) at the two growing forks. the discontinuous synthesis of the lagging strand involves repeated synthesis of short oligoribonucleotide primers, which in turn initiate short nascent strands of dna {okazaki fragments), which are then covalently joined to form one of the growing strands. adenoviridae. adenovirus dna is linear, the ' terminus of each strand being a mirror image of the other (terminally repeated inverted sequences), and each is covalently linked to a protein. the primer for adenoviral dna synthesis is not, as is usual, another nucleic acid, but a precursor to this protein, referred to as adenovirus preterminal protein. dna replication proceeds from both ends, continuously but asynchro nously, in a ' to ' direction, using a virus-coded dna polymerase. it does not require the synthesis of okazaki fragments. herpesviridae. unlike other dna viruses that replicate in the nu cleus, herpesviruses specify a large number of enzymes involved in dna synthesis. analysis of herpes virus dna replication is incomplete, but it appears that a rolling-circle mechanism operates, at least in the later stages. the replicating dna initially consists of circles and linear forked forms, which are later replaced by large bodies of tangled dna. there are three origins of replication, two on the s component and one on the l component (see fig. - ) , the latter being near the genes that specify the dna polymerase and the major dna-binding protein. new ly synthesized viral dna appears to be cleaved to unit lengths during the process of packaging into newly formed capsids. poxviridae. the special features of poxvirus dna replication are that it occurs in the cytoplasm and depends entirely on virus-coded proteins; it can occur in enucleated cells. replication appears to begin at each end of the genome and involves a strand displacement mechanism, with the formation of small dna fragments covalently linked to rna primers. the discovery of the loop structure at the ends of the vaccinia virus genome (see fig. - ) suggested a model whereby nicks near the ends of the genome allow self-priming by the ' ends thus generated. parvoviridae. in the autonomous parvoviruses (genus parvovirus), dna replication occurs in close association with cellular chromatin and is dependent on cellular functions provided in the s phase of the cell cycle, i.e., when cellular dna synthesis is occurring, a feature that is correlated with the pathogenic potential of these viruses (see chapter ). the virion (-) sense dna is copied to give a dsdna replicative form. further dna synthesis requires the binding of a virus-coded pro tein to the ' termini. production of viral ssdna appears to occur after nicks at the ' end and repeated rounds of synthesis. hepadnaviridae. replication occurs in the nucleus by a unique pro cess. the viral dna polymerase converts the viral ss/dsdna into a complete circular dsdna. the (-) sense strand of this molecule is then transcribed by the cellular rna polymerase to produce a full-length "pregenome" rna. this (+) sense rna is then encapsidated in viral cores together with newly synthesized dna polymerase, which also carries reverse transcriptase activity. minus-strand dna is then synthe sized by reverse transcription of the pregenome rna; the template is degraded to leave a full-length (-) sense dna strand. a small rna fragment from the ' end of the pregenome is then used to prime the synthesis of the (+) sense dna strand. complete synthesis of this strand is not necessary for maturation of the virus, hence infectious particles contain dsdna with a single-stranded region. the replication of rna is a phenomenon restricted to viruses. tran scription of rna from an rna template requires an rna-dependent rna polymerase, a virus-coded enzyme not normally found in cells. it is not known whether the polymerase required to transcribe (+) sense rna from (-) sense rna is different from that needed to transcribe (-) sense rna from (+) sense rna. both processes are essential because the replication of virion rna requires first the synthesis of complemen tary rna, which then serves as a template for making more virion rna. where virion rna is of (-) sense the complementary rna is of (+) sense and the rna polymerase is the virion-associated transcriptase used for transcription of subgenomic rnas. however, whereas the pri mary transcripts from such (-) sense virion rna are subsequently cleaved (in most cases) to produce mrnas, some must remain uncleaved to serve as a full-length template for virion rna synthesis. in the case of (+) sense virion rna, the complementary rna is of (-) sense. several rna molecules can be transcribed simultaneously from a single complementary rna template, each rna transcript being the product of a separately bound polymerase molecule. the resulting struc ture, known as the replicative intermediate, is therefore partially doublestranded, with single-stranded tails ( fig. - ) . initiation of replication of picornavirus and calicivirus rna, like that of adenovirus dna, requires a protein, rather than a ribonucleoside triphosphate, as primer. this small protein, vpg, is covalently bound to the ' terminus of nascent (+) and (-) rna strands, as well as virion rna, but not to mrna. retroviruses have a genome consist ing of (+) sense ssrna. unlike other rna viruses, they replicate via a dna intermediate. a virion-associated rna-dependent dna poly merase (reverse transcriptase), using a trna molecule as a primer, makes a ssdna copy. the reverse transcriptase, functioning as a ribonuclease, then removes the parental rna molecule from the dna-rna hybrid. the free (-) sense ssdna strand is then converted into linear dsdna, which contains an additional sequence known as the long terminal repeat at each end. this linear dsdna then migrates to the nucleus and becomes integrated into cellular dna. transcription of the viral rna can then occur from this integrated (proviral) dna (see chap ter ). structural proteins of nonenveloped icosahedral viruses associate spontaneously to form capsomers, which self-assemble to form empty procapsids, into which viral nucleic acid is packaged. completion of the virion often involves proteolytic cleavage of one or more species of capsid protein. the best-studied examples among animal viruses are the picornaviruses (fig. - ) . the capsomer precursor protein (noncapsid viral protein, ncvpla) aggregates to form pentamers; each of the ncvpla molecules is then cleaved by virus-specific proteases into vpo, vp , and vp . twelve such pentamers aggregate to form a procapsid. a final proteolytic event, which cleaves the vpo molecule into vp and vp , is required for rna incorporation. the mature virion is a dodeca hedron with capsomers, each of which is made up of one molecule each of vp , , , and . there are also one or two uncleaved molecules of vpo in the virion. x-ray crystallography shows that the assembling units are not just rigid preformed "bricks"; they have extensions that reach across adjacent units to form second-and third-nearest neighbor relationships. such studies have also shown that there is no fixed way in which rna interacts with ordered parts of the protein. the mechanism of packaging viral nucleic acid into a preassembled empty procapsid has been elucidated for adeno virus. one terminus of the viral dna is characterized by a nucleotide sequence referred to as the packaging sequence, which enables the dna to enter the procapsid bound to basic core proteins, after which some of the capsid proteins are cleaved to make the mature virion. all mammalian viruses with helical nucleocapsids, as well as some with icosahedral nucleocapsids, acquire an envelope by budding through cellular membranes. since such envelopes always contain viral glycoproteins, we begin by discussing the mechanism of glycosylation of these proteins. glycosylation of envelope proteins. much of our understanding of the glycosylation of viral proteins comes from studies with vesicular stomatitis virus (a rhabdovirus), semliki forest virus (a togavirus), and the orthomyxoviruses and paramyxoviruses. the essential steps appear much the same for all enveloped viruses, hence a general overview is presented (fig. - ) . viruses exploit existing cellular pathways nor mally used for the synthesis of membrane-inserted and exported se cretory glycoproteins. the amino-terminus of viral envelope proteins initially contains a se quence of to hydrophobic amino acids, known as the signal sequence, which characterizes the protein as one destined for insertion into membrane and/or export from the cell. the hydrophobicity of the signal sequence facilitates binding of the growing polypeptide chain to a recep tor site on the cytoplasmic side of the rough endoplasmic reticulum and its passage through the lipid bilayer to the luminal side. a signal peptidase then removes the signal sequence. oligosaccharides are added to asparagine residues of the nascent polypeptide in the lumen of the rough endoplasmic reticulum by en bloc transfer of a mannose-rich core of preformed oligosaccharides from a lipid-linked intermediate, an oligosaccharide pyrophosphoryldolichol. glucose residues are then re moved by glycosidases, a process known as "trimming" of the core. the viral glycoprotein is then transported from the rough endoplasmic re ticulum to the golgi complex, probably within a coated vesicle. here the core carbohydrate is further modified by the removal of several mannose residues and the addition of further n-acetylglucosamine, galactose, and the terminal sugars, sialic acid and fucose. the completed side chains are a mixture of simple ("high-mannose") and complex oligosac charides. while in the golgi complex the glycoprotein may become acylated, by the covalent attachment of fatty acids such as methyl palmitate to the hydrophobic membrane attachment end of the molecule. another coated vesicle then transports the completed glycoprotein to the cellular membrane from which the particular virus buds. transport of glycoproteins. different viruses bud from different sites in the plasma membrane (orthomyxoviruses, paramyxoviruses, rhabdoviruses, arenaviruses, togaviruses, and retroviruses), some from the apical and others from the basolateral surface. others bud from intracytoplasmic smooth endoplasmic reticulum (flaviviruses, bunyaviruses, corona viruses) or from the nuclear membrane (herpesviruses). presum ably some structural feature of the glycoprotein serves as the "zip code" ensuring delivery to the correct location in the cell. cleavage of envelope proteins. with the orthomyxoviruses and para myxoviruses, which bud through the plasma membrane, a cellular pro tease cleaves the envelope protein at the time of its insertion into the membrane into two polypeptide chains, which remain covalently linked by disulfide bonds. cleavage is not required for viral release and does not occur in certain types of host cells, but it is essential for the produc tion of infectious virions in the orthomyxoviruses (cleavage of the hemagglutinin) and paramyxoviruses (cleavage of both the hemagglutinin-neuraminidase and the fusion protein). following fusion of the coated vesicle with the plasma membrane, the hydrophilic n-terminus of the glycoprotein projects from the external surface of the membrane, while the hydrophobic domain, which is near the c-terminus, remains anchored in the lipid bilayer. budding. budding may be regarded as a nonphysiological form of exocytosis ( fig. - ) . the process begins with the insertion of the com pleted viral glycoprotein into the appropriate cellular membrane. be cause proteins are free to move laterally in the "sea of lipid" that con stitutes the lipid bilayer of the plasma membrane, cellular proteins are displaced from the patch of membrane into which viral glycoproteins are inserted. it is not known whether there is selection of particular lipids for incorporation into the viral envelope, but the ratio of phospholipids to glycolipids and cholesterol is essentially the same as that of the mem brane of the particular host cell. the monomeric, cleaved viral glycoprotein molecules associate into oligomers, to form the typical rod-shaped peplomer with a prominent hydrophilic domain projecting from the external surface of the mem brane; the hydrophobic domain near the c-terminus spans the mem brane and a short hydrophilic domain at the c-terminus projects slightly into the cytoplasm. in the icosahedral togaviruses (plate - a), each protein molecule of the nucleocapsid binds directly to the c-terminus of a glycoprotein oligomer of the envelope. multi valent attachment of nu merous peplomers, each to an underlying molecule on the surface of the icosahedron, molds the envelope around the nucleocapsid, forcing it to bulge progressively outward until finally the nucleocapsid is completely enclosed in a tightly fitting envelope and the new virion buds off. the capsid proteins of most enveloped viruses with helical nucleocapsids do not bind directly to envelope glycoprotein but to a matrix protein which is bound to the cytoplasmic side of the plasma membrane beneath patches of viral glycoprotein (fig. - ) . coronaviruses and bunyaviruses bud from rough endoplasmic reticulum and the golgi complex; orthopoxviruses may acquire an enve lope in the golgi, but enveloped forms are released from the plasma membrane. the envelope of the herpesviruses is acquired by budding through the inner lamella of the nuclear membrane; the enveloped vir ions then pass directly from the space between the two lamellae of the nuclear membrane to the exterior of the cell via the cisternae of the endoplasmic reticulum. there are basically two mechanisms for the release of mature virions from the infected cell. with most nonenveloped viruses that accumulate within the cytoplasm or nucleus, release occurs only when the cell lyses. this may occur shortly after the completion of viral replication; e.g., cells infected with picornaviruses lyse as soon as assembly of virions is completed, with immediate release of the progeny virions. on the other hand, parvoviruses accumulate within the cell nucleus and are not re leased until the cell slowly degenerates and dies. most enveloped vi ruses, on the other hand, are released by budding, a process which can occur over a prolonged period without much damage to the cell, hence many such viruses (e.g., arenaviruses, retroviruses) are noncytopathogenic and are associated with persistent infections. however, some en veloped viruses that are released by budding are cytolytic, e.g., the alphaherpesviruses. orthopoxviruses may be released as enveloped forms by budding from the plasma membrane or as nonenveloped forms, by cell lysis; both forms are infectious. if this had been a book about bacterial diseases of domestic animals, there would have been at least one chapter on antibacterial chemother apy. however, of the hundreds of antibiotics and other antibacterial compounds now available, not one has the slightest effect on any virus, and there are no specifically antiviral chemotherapeutic agents in com mon use. the reason is that viruses are absolutely dependent on the metabolic pathways of the host cell for their replication, hence most agents that interfere with viral replication are toxic to the cell. increased knowledge of the biochemistry of viral replication has led to a more rational approach to the search for antiviral chemotherapeutic agents. several steps in the viral replication cycle represent potential targets for selective attack. theoretically, all virus-coded enzymes are vulnera ble, as are all processes (enzymatic or nonenzymatic) that are more essential to the replication of the virus than to the survival of the cell. obvious examples include: ( ) transcription of viral mrna (or copy dna, in the case of the retroviruses) by the viral transcriptase, ( ) rep lication of viral dna or rna by the virus-coded dna polymerase or rna-dependent rna polymerase, ( ) posttranslational cleavage of protein(s) by (virus-coded) protease(s). less obvious at first sight, but proven points of action of currently known antiviral agents are: ( ) penetration/uncoating, ( ) polyadenylation, methylation, or capping of viral mrna, ( ) translation of viral mrna into protein, and ( ) assem bly/maturation of the virion. a logical approach to the discovery of new antiviral chemotherapeutic agents is to isolate or synthesize substances that might be predicted to serve as an inhibitor of a known virus-coded enzyme. analogs (con geners) of this prototype are then synthesized with a view to enhancing activity and/or selectivity. the discovery of a class of nucleoside analogs which selectively inhibit herpesvirus dna synthesis has led to a realiza tion that virus-coded enzymes with a broader (or different) substrate specificity than their cellular counterparts can be exploited to convert an inactive precursor ("prodrug") to an active antiviral agent. because the viral enzyme occurs only in infected cells, such drugs are nontoxic for uninfected cells. exploitation of this principle may revolutionize anti viral chemotherapy. acycloguanosine, now commonly known as acyclovir, is a guanine derivative with an acyclic side chain, the full chemical name being -( hydroxyethoxymethyl)guanine ( fig. - ). its unique advantage over earlier nucleoside derivatives is that it requires the herpesvirus-specified enzyme, deoxythymidine-deoxycytidine kinase, to phosphorylate it intracellularly to acycloguanosine monophosphate; a cellular gmp kinase then completes the phosphorylation to the active agent, acycloguano sine triphosphate (fig. - ) . acycloguanosine triphosphate inhibits the herpesvirus-specified dna polymerase. since activation of the prodrug needs the viral thymidine kinase, acyclovir is essentially nontoxic to uninfected cells but is powerfully inhibitory to viral dna synthesis in infected cells. acyclovir and various derivatives, as well as other nucleoside analogs dependent on viral enzymes for conversion to the active form, are begin ning to be used in human medicine for the treatment of herpesvirus infections. it is a small start, but it does demonstrate that antiviral che motherapy may have a future. such drugs find limited use in veterinary medicine, e.g., for treatment of feline herpesvirus corneal ulcers. a few other antiviral agents are in use in human medicine. for exam ple, rimantadine and amantadine can prevent the uncoating of influenza virus, and several compounds known to inhibit reverse transcriptase are undergoing clinical trials against aids. in theory at least, interferons are the ideal antiviral antibiotics. they are naturally occurring, relatively nontoxic, and display a broad spec trum of activity against essentially all viruses (see chapters and ). however, clinical trials in humans have been disappointing. currently, it appears that they have a demonstrable effect on infections with papilloma viruses, herpesviruses, and rhino viruses. it is now possible to produce large amounts of various human and other interferons using cloned interferon genes, but it is still uncertain whether they will be of clinical value in humans. their use for therapy in viral diseases of do mestic animals is even further away. overall, in spite of decades of effort and massive expenditure by the pharmaceutical industry, the yield of useful antiviral drugs has been meager. only a handful of marginally effective agents have found a place in human medicine, and very few are used in veterinary practice. nevertheless, it is important to be aware of the continuing research in this field, for antiviral chemotherapy may one day come to constitute an integral part of veterinary medicine. a) togavirus. (b) accumulation of paramyxovirus sv nucleocapsids. (c, d) budding of sv from the plasma membrane, with some filamentous forms (bars = nm) expression of animal virus genomes segmented negative strand viruses: arena viruses, bunya viruses, and orthomyxo viruses cotranslational and posttranslational processing of viral glycoproteins comparison of initiation of protein synthesis in procaryotes, eucaryotes and organelles animal virus receptors. series b: receptors and recognition structure of a human common cold virus and functional rela tionship to other picornaviruses viral replication initiation signals in viral gene expression how an animal virus gets into and out of its host cell replication strategies of the single-stranded rna viruses of eukaryotes antiviral chemotherapy, interferons and vaccines replication of arenaviruses and bunyaviruses rhabdoviruses. in "virology structure and assembly of alphaviruses the adenoviruses autonomous parvo virus dna structure and replication the reoviridae orthomyxo-and paramyxoviruses and their replication the gene structure and replication of influenza virus replication of poxviruses nucleotide sequence of yellow fever virus; implications for flavivirus gene expression and evolution the herpesviruses picornaviruses and their replication the molecular biology of corona viruses the hepatitis b virus dna tumor viruses rna tumor viruses," and "rna tumor viruses supplements african swine fever key: cord- - k tcgfs authors: burnouf, thierry; griffiths, elwyn; padilla, ana; seddik, salwa; stephano, marco antonio; gutiérrez, josé-maría title: assessment of the viral safety of antivenoms fractionated from equine plasma date: - - journal: biologicals doi: . /j.biologicals. . . sha: doc_id: cord_uid: k tcgfs abstract antivenoms are preparations of intact or fragmented (f(ab′) or fab) immunoglobulin g (igg) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. they are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. manufacturing processes usually include pepsin digestion at acid ph, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. this paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. analysis of production parameters indicate that acid ph treatments and caprylic acid precipitations, which have been validated for the manufacture of some human igg products, appear to provide the best potential for viral inactivation of antivenoms. as many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products. antivenoms are important biopharmaceutical products made from the plasma of immunized horses or sheep. they are used in human medicine to treat the potentially severe pathophysiological complications resulting from the bites and stings from various animals, such as snakes, scorpions, spiders, cnidarians, lepidopterans or fishes, as well as from intoxications with plants [ , ] . envenomings are a serious health problem worldwide and are most particularly dreadful in rural areas of the developing world [ ] , where shortage of antivenom products [ ] and lack of sufficient medical facilities explain numerous fatalities [ , ] . snake bites represent the major cause of envenoming. case fatality associated with some snake bites reach % or more but can be reduced to less than % through antivenom therapy, the only available current treatment [ , e ] . global mortality from snake bites may range from , to , per year, but these figures are likely underestimated [ , ] . viperid snake bite envenoming induces local effects, such as swelling, pain, necrosis, hemorrhage and blistering, often accompanied by secondary infection [ ] . systemic viperid envenoming is characterized by a complex pathophysiological profile including coagulopathy, hemorrhage, hypovolaemia, shock and acute renal failure [ , ] . progressive paralysis may be caused by elapid snake venom neurotoxins, and by some viperid venoms displaying neurotoxic effects. some elapid venoms also induce local necrosis and rhabdomyolysis [ ] . snake bite survivors may have major chronic physical and neurological disability. scorpion stings are the second major cause of human fatalities from envenoming (probably amounting to several hundreds per year). scorpion venoms contain toxins which target sodium, potassium, calcium and chloride channels, causing direct effects and release of neutrotransmitters such as acetylcholine and catecholamines, inducing intense local pain and potentially fatal neurotoxic and hemodynamic disturbances [ ] . the role of antivenom in the treatment of scorpion stings and other arachnids remains controversial but several reports support their manufacture and use, particularly in severe cases [ , ] . fatalities have also occurred from envenoming by jellyfish, and venomous fishes. long-term anecdotal experience supports the beneficial effect of stonefish antivenoms [ ] , but those may need to be given very early to fight a rapid onset of cardiotoxicity. stings by cnidarians, lepidoptera, centipedes and coneshells and bites by spiders, ticks and one genus of octopus, probably account for about deaths per year [ ] . finally, envenomings by massive attacks of africanised bees cause some deaths each year in the americas [ ] . antivenoms are most often made by fractionation of plasma of horses (less frequently sheep) that have been immunized with crude venoms [ ] . pooled hyperimmune plasma is processed to purify the horse immunoglobulin g (igg) fraction that may be subjected to enzymatic treatment to obtain f(ab#) and fab antibody fragments, and caprylate or ammonium sulphate precipitation to improve their purity [ ] . to some extent, some manufacturing steps have features similar to those used to prepare human plasma-derived igg. although there is no known transmission of any infectious agent through antivenoms (albeit under circumstances where rigorous clinical patient follow-up is difficult), theoretical concerns about the possibility of transmission of horse/sheep infectious agents to humans do exist. recent natural transmissions of zoonotic diseases highlight the possible exchanges between humans and the natural reservoirs of biologic agents found in animals [ ] , and the inherent risk of emerging diseases [ ] . examples of such infections originating from animal (or avian) pathogens include human immunodeficiency virus, ebola, hantaan, lassa, nipah viruses and other paramyxoviruses, equine morbilli virus, west nile virus, and probably severe acute respiratory syndrome (sars) coronavirus [ , ] . the parenteral transmission of animal viruses to humans is also possible. infectious sv virus of rhesus monkeys was a contaminant of early polio vaccines which were administered to a large number of people. whether this was the way in which this virus was introduced into the human population is unclear and a controversial issue. the risk of contamination of antivenoms by equine virus is, therefore, of theoretical concern and has been debated at a recent world health organization (who) workshop [ ] . recently, the committee for proprietary medicinal product (cpmp) of the european medicine evaluation agency has published a note for guidance on the manufacture and quality control of animal immunoglobulins and immunsera [ ] . however, most manufacturers of antivenoms are located in the developing world and some of them may not be directly exposed to the state-of-the-art process validation concepts, nor have the financial and logistics capability to perform extensive viral validation studies to assess the viral reduction potential of the process they use. therefore, we found it helpful to examine the viral safety of antivenoms by first reviewing the characteristics of equine viruses. we then evaluated the theoretical ability of manufacturing methods of antivenoms to inactivate or remove those viruses, through a comparison with the well validated technologies used to manufacture human plasma-derived igg preparations. finally, we want to emphasize that applying good manufacturing practices in the production of antivenoms, from the control of the animals to that of all production steps, and ensuring traceability in the whole chain of manufacture, represent the current best investment in the quality and safety of these products. horses can harbour enveloped and non-enveloped viruses. the main structural characteristics of those viruses are presented in table ; epidemiological and clinical data and testing methods are described briefly below. eav is a e nm single-stranded (ss)-rna virus of the arteriviridae family [ ] . eav causes an equine viral arteritis, an endotheliotropic viral disease [ ] . transmission occurs via respiratory and reproductive routes. there is a variety of clinical signs, and strains vary in virulence [ ] , but severe infection can lead to abortions in pregnant mares or neonatal foal death. a one-tube real-time taqman rt-pcr assay was developed for detecting eav. the test was validated using seminal plasma and nasal secretions [ ] . a dna vaccine was shown to induce long-term immunization [ ] . bdv is a e nm ss-rna virus belonging to the new bornaviridae family, order mononegavirales. borna disease, known as 'disease of the head', is a sporadically occurring, progressive viral polioencephalomyelitis that primarily affects horses and sheep. after a few weeks to several months incubation, bdv can cause locomotor and sensory dysfunction followed by paralysis and death. bdv exists world-wide in horses, sheep, cattle, cats, dogs and ostriches and can affect a large number of warm-blooded animal species, including humans [ , ] . the infection can be fatal, but the majority of carriers are asymptomatic [ ] . cross-species transmission of this commensal virus has not been proven, but zoonotic aspects of bdv should be considered [ ] . bdv-specific antibodies and viral rna have been found in humans with various psychiatric disorders. diagnosis can be made serologically, but also by antigen markers in peripheral white blood cells, combined with nucleic acid amplification [ ] . these are closely related e nm ss-rna alphaviruses [arbovirus type a] of the togaviridae family that are transmitted by arthropods (usually mosquitoes). symptoms of eeev infections, an important multisystemic zoonotic disease, include anorexia and colic, changes in sensorium, hyperexcitability, and terminal severe depression. organ coagulative necrosis and cns lesions are observed [ ] . weev is not as neuroinvasive as eeev and the encephalitis caused is not as severe as that caused by eeev. a test to detect eee and wee viral rna has recently been developed [ ] . veev is present in both humans and horses but no evidence of transmission from horses to humans by normal routes of contamination has been found. veev has been identified in pharyngeal secretions and is stable when aerosolized; it has been shown to be stable in dried blood and exudates. vaccines have been developed against those three viruses [ ] ; one possible case of viral transmission in horse has been suspected following vaccination [ ] . ecv is a e nm ss-rna virus of the coronaviridae family. it has been isolated from feces of a diarrhoeic foal, and has close antigenic and/or genetic relationships with mammalian group coronaviruses [ ] . efv, also known as spumavirus, is a e nm ss-rna virus of the retroviridae family. it belongs to the nonpathogenic, complex unconventional retroviruses and has been isolated in nonhuman primates, cattle, cats, and more recently in the blood of horses [ ] . ehvs are large e nm double-stranded (ds) dna gamma herpes viruses. ehv and ehv cause much damage to the horse industry and are ubiquitous in the equine population. they are responsible for life-long latent infections in their hosts even in those with natural or vaccine-induced immunity [ ] . ehv strains are associated with respiratory disease, abortion, and paresis/paralysis, whereas ehv strains induce respiratory disease [ ] . ehv and ehv have a less clear pathogenicity and distribution within the equine population. ehv may have an aetiological role in ocular disease [ ] . the prevalence of ehv in adult horses was found to be up to % in sweden and % in the united kingdom. the prevalence of ehv dna was and % in adult horses in hungary and the united kingdom, respectively [ ] . eiav is a e nm ss-rna lentivirus of the same retroviridae family as hiv. most infected horses are asymptomatic, with life-long latent infection of leucocytes until stressed (e.g. by pregnancy, corticosteroids, surgical operation, disease) or until new virus variants arise. viraemia then increases by -fold. red blood cells become coated by the virus particles and are then lysed by complement, causing jaundice, oedema, hemorrhagic diarrhoea, petechial hemorrhages. eiav can be transmitted from horse to horse by blood. recently a reverse-transcriptase polymerase chain reaction assay (rt-pcr) has been described for quantifying eiav rna in equine plasma [ ] . eiv is a e nm ss-rna virus of the orthomyxoviridae. equine influenza is one of the most economically important contagious respiratory diseases of horses [ ] . hev is a nm ss-rna virus [ ] . the virus is probably transmitted from bats to horses, and causes natural disease in humans and horses. it is the reference virus for a proposed new genus within the paramyxoviridae family, which also includes another newly identified zoonotic bat virus (nipah), and the salem virus. hev has been recognized in australia as a new zoonotic disease of horses since / . this lethal zoonotic viral agent is endemic in certain species of fruit bats (flying-foxes) and over % of them in eastern australia are seropositive [ ] . six species of flying-foxes in papua new guinea have tested positive for antibodies to hev [ ] . hev does not appear to transmit readily between horses under natural and experimental conditions, or from horses to humans [ ] . horses can be infected by oronasal routes and can excrete hev in urine and saliva [ ] . the virus appears to be spread by close contact with body fluids, such as froth from infected lungs [ ] , via nasal discharge, saliva and/or urine [ ] . the most important clinical and pathological manifestation of hev infection in horses and humans is severe interstitial pneumonia caused by viral infection of small blood vessels. from to , epizootic and epidemiological episodes of meningoencephalitis and severe acute respiratory syndromes were reported in australia, malaysia and singapore [ , ] . it may also cause nervous disease. three incidents of hev disease in horses have been recorded in australiadtwo in , which caused the death of two humans and horses and one in , which involved the death of a single horse [ ] . infected horses can develop a severe and often fatal respiratory disease characterized by dyspnoea, vascular endothelial damage and pulmonary oedema. nervous signs may also occur. a number of diagnostic methods have been developed [ ] , based on the examination of blood, lung, kidney, spleen, and, if nervous signs are present, also of the brain. pcr assays have been developed. a rapid and sensitive rt-pcr assay using a fluorogenic (taqman) probe was developed to improve the diagnosis of hev infection [ ] . both are closely related, arthropod-born, e nm ss-rna flaviviruses of the flaviviridae family that may affect the central nervous system of horses [ , ] . evidence of infection with niv was found in the brain of one horse in which inflammation of the meningeal blood vessels occur [ ] . niv emerged in malaysia, spread rapidly through the pig population, and caused the deaths of over people [ ] . salv has been recently identified in horses [ ] . the only known isolate was obtained from a horse that was involved in a disease outbreak of undetermined nature [ ] . vsv is a e nm ss-rna virus of the rhabdoviridae family. it may cause stomatitis in horses, and may represent an emerging equine infectious disease [ ] . an immunoglobulin m (igm) capture enzyme-linked immunosorbent assay (mc-elisa) has been developed for the detection of primary infection of vsv in equine and swine sera [ ] . wnv is a e nm ss-rna arbovirus of the flaviviridae family, first identified in the west nile district of uganda in . wnv is found over a broad geographical range and in a wide diversity of vertebrate host and vector species [ ] . until , the disease was found in african, european, and eurasian countries. more recently, there was an increase in outbreaks of illness due to wnv in animals as well as humans and numerous cases are currently reported in canada and the usa. infected humans may be asymptomatic and the transfusion of donated blood from such individuals has resulted in the infection and death of recipients. this has led to the introduction in of strict regulatory measures in canada and the usa which include the testing by a pcr assay of all blood donations. wnv primarily circulates between birds and mosquitoes, while mammalian infections are incidental. mammal biting mosquitoes become infected when they feed on the blood of an infected bird. once this happens, people, horses and other mammals may get wnv. infected horses are, however, unlikely to serve as important amplifying hosts for wnv in nature [ ] . many wnv-infected horses probably never show symptoms, but a study reported mortality rates close to % [ ] . early symptoms are often indistinguishable from other equine encephalitides including rabies, equine herpesvirus- , equine protozoal myeloencephalitis, and eastern, western, or venezuelan equine encephalomyelitis. a vaccine for horses has been developed, but one horse that received two doses died from the disease [ ] . erav and erbv are picornaviruses reclassified as members of the aphtovirus genus because of resemblance to foot-and-mouth disease virus. they are e nm ss-rna viruses. high neutralizing antibody titers develop and appear to correlate with strong reactivity to vp in western blots [ ] . a new serotype of the genus erbovirus, tentatively named erbv has been identified recently and found in % of horses in australia [ ] . eev is a nm ds-rna virus of the reoviridae family. several serotypes have been identified in southern africa. serotype-specific virus-neutralizing antibody in serum samples from horses suggests a widespread occurrence of infection but there seems to be only a low level of cross-protection in horses to natural reinfection [ ] . erv is a e nm double-stranded rna of the reoviridae. rotaviruses are important pathogens associated with diarrhoeal diseases in almost all species of mammals. a pcr test with a detection limit of approximately . ! tcid per gram faeces, with possible increase in the sensibility by one order of magnitude using nested pcr, was developed, representing a possible diagnostic tool [ ] . antivenoms are produced from the plasma or serum collected from immunized horses or sheep. plasma can be obtained by the centrifugation of whole blood or by apheresis. plasma from several animals is typically pooled into e l batches and subjected to a fractionation process to isolate the igg fraction. following the initial use of crude equine immune serum, still reported to be manufactured by one producer [ ] , various methods of igg purification and refinement have been introduced ( table ). most manufacturers use protocols derived from the basic method described by pope [ , ] , modified by harms [ ] , based on pepsin digestion at low ph, to obtain f(ab#) fragments, followed by ammonium sulphate precipitation of antibody fragments [ , , ] . this basic approach is combined with a number of additional steps, aimed at obtaining a purer [ ] preparation, such as heat coagulation [ ] and ion-exchange chromatography [ , ] . the ph at which pepsin digestion is performed by various laboratories ranges between . and . , at temperatures between and c. pepsin digestion is usually performed in undiluted serum or plasma (protein concentration: e g/l). incubation times range from min to h and with varying pepsin concentrations [ , , , ] . thermocoagulation is used by many laboratories and consists of heating at e c for h [ , , ] although not all methods involving pepsin digestion include heat coagulation [ ] . some fractionation protocols include an additional table steps used for the manufacture of antivenoms screening of production animals for adventitious agents plasma collection (whole blood or apheresis) in bags or bottles plasma thawing at room temperature plasma pooling igg/fragments purification process f(ab#) : b pepsin digestion (acid ph) and ammonium sulphate precipitation, or b pepsin digestion (acid ph) and caprylic acid precipitation fab: ammonium sulphate precipitation and papain digestion at ph e whole igg: b caprylic acid precipitation b ammonium sulphate precipitation igg/fragments concentration: (nh ) so /na so precipitation, ultrafiltration polishing: ion exchange (removes fc and further purifies iggs/ fragments) ultrafiltration sterile filtration aseptic filling storage in the liquid state or lyophilisation acidification step to remove some non-igg globulins ('euglobulins') which are unstable at acid phs [ , ] . one producer includes a bulk pasteurisation step in its antivenom production protocol [ ] . other manufacturers produce whole igg antivenoms using either ammonium sulphate precipitation of igg or caprylic acid precipitation of non-igg proteins [ e ], followed by dialysis or ultrafiltration. recently, a simple, one-step methodology based on caprylic acid precipitation has been described [ ] . the conditions used for caprylic acid fractionation of equine plasma are as follows: plasma ph is adjusted to . and caprylic acid is added directly to this plasma to attain a final concentration of % (v/v). the mixture is stirred during caprylic acid addition and then for one additional hour, after which the precipitate is separated by filtration. caprylic acid is then removed by either dialysis or ultrafiltration and the product is formulated before sterile filtration [ ] . some antivenoms are made of fab antibody fragments, obtained by papain digestion of sheep igg, at neutral ph, after sodium sulphate precipitation of igg [ ] . affinity-chromatography or ion-exchange chromatography steps have been described in the production of some fab antivenoms [ , ] . treatment with b-propiolactone has been evaluated to reduce complement activation by horse plasma-derived products [ ] ; however, it does not appear to be used routinely in laboratories producing antivenoms. in addition to the differences in fractionation protocols, antivenoms made of whole igg, f(ab#) and fab molecules greatly differ in their pharmacokinetic profiles [ , ] . most antivenoms come in liquid presentation, but some are lyophilized. the former need to be stored at e c, since higher temperatures reduce their shelf-life and induce protein denaturation and aggregate formation [ ] . in contrast, lyophilized antivenoms can be stored at room temperature and have a more prolonged shelf-life. most antivenoms contain preservatives to prevent bacterial contamination [ , ] . considerable experience has been gained in recent years on the viral safety of human igg preparations and other human plasma-derived products. historical perspectives on these products help in assessing the viral safety of antivenoms. the safety of human plasma products results from the combination of overlapping strategies that include: (a) selection of donors, (b) viral testing procedures on single donations (e.g. serological tests) and plasma pools (e.g. nucleic acid tests) to exclude donations contaminated by viruses, (c) processing and purification steps to inactivate and remove viruses, and (d) implementation of good manufacturing practices [ ] . whenever feasible, similar approaches should be used for the manufacture of animal plasmaderived products. monitoring the health of animals used for antivenom production is important [ ] . colonies of animals should be kept free of contamination through good husbandry or vaccination, where appropriate. surveillance of the source animals should include screening for pathogens, health monitoring, including routine blood chemistry and haematology tests, and post-mortem examination. ideally, animals should be kept under strictly contained conditions, but this is not readily applicable to horses. however, for instance, animals could be kept free of a particular arthropod-borne virus if they are maintained in an area free of the particular arthropod vector. if a particular pathogen is identified, it should be established whether it is present in plasma. some virological screening methods may be performed, if available and epidemiologically relevant to a particular geographic region, to limit risk of virus presence in the animal herd and/or the plasma pool. to some extent the guidelines from the federation of european laboratory animal science association (felasa), although not referring to horses, may serve as a reference [ e ]. nevertheless, not all of these measures are available in many developing countries nor readily applicable to horses used for antivenom production, and pathogens can escape this surveillance program. this illustrates the need for manufacturing techniques to ensure a sufficient margin of safety with regards to potential infectious agents present in the manufacturing plasma pool. this also strongly emphasizes the crucial importance of ensuring an appropriate traceability system from horse donors to human recipients of antivenoms so that any potential infectious events or risks may be identified in a proper and timely manner and relevant counter-measures taken promptly. confidence in the safety of antivenoms must come from evidence that the manufacturing processes used can reproducibly remove or inactivate high potential levels of viruses. comparison with human plasma fractionation suggests that some of the steps of antivenoms include treatments which may inactivate viruses. human plasma fractionation is a complex integrated manufacturing process from which products like albumin, coagulation factors and igg, are obtained. purification processes combine cryoprecipitation, ethanol fractionation, chromatography, and ultrafiltration [ ] . in addition, various types of dedicated viral reduction treatments have been introduced, most often on purpose, such as chemical treatments by solvent-detergent, caprylic acid precipitation, low-ph incubation, heat treatments in the liquid or in the dry states, and nanofiltration [ ] . among those, caprylic acid and low ph treatments, both of which are commonly used also for the purification of antivenom igg, have been shown to contribute to the viral safety of human plasma igg products as described below. some years ago, unsaturated fatty acids were shown experimentally to inactivate more than log of lipidenveloped viruses (vsv, sindbis virus, hiv) in human plasma protein fractions [ ] . however, specific attention has been given to caprylic acid (also called octanoic acid), as it has been used as precipitating agent for human igg. it has been suggested that the non-ionized form of the caprylic acid disrupts the integrity of the lipid bilayer and membrane associated proteins of enveloped viruses. utilizing the dissociation reaction and varying the concentration of the ionized form of caprylate, a specific amount of the nonionized form of caprylate can be maintained over a wide ph range. treatment of plasma protein solution with caprylate at acidic ph also readily inactivates lipid-enveloped viruses like herpes simplex virus type i, vesicular stomatitis virus, vaccinia virus, and sindbis virus [ ] . detailed validation studies of two caprylic acid treatments of human ig have been published recently. the robustness of a treatment applied to three intravenous immunoglobulin preparations (igg, igm-enriched, and igm preparations) has been investigated using hiv, bvdv, sindbis virus and pseudorabies as model viruses [ ] . the routine treatment conditions for these two igg and this igm preparation are indicated in table . kinetics of inactivation was determined over a period of h of treatment. complete inactivation (corresponding to more than . e . log) is achieved within the first minutes. within a certain range, viral inactivation in the igg product was not affected by ph ( . e . ), temperature ( e c), and protein content ( e g/l). above ph , and most specifically at ph , no bvdv inactivation was found. at a content of caprylic acid of . g/kg or less, inactivation of hiv is significantly reduced. under the conditions applied during manufacture, caprylic acid leads to robust inactivation of lipid-enveloped viruses; ph is a particularly critical parameter and should be less than . these conditions have also been found to inactivate o . log eav, an equine virus used as a model (dichtelmu¨ller, personal communication). another study reported the viral inactivation achieved during caprylic acid precipitation of non-igg proteins from human igg product [ ] . at ph . , c, and in the presence of mm caprylate, r . and r . log of hiv and prv, respectively, were inactivated during the h treatment, but only . log for bvdv was inactivated. at mm caprylate, r . log of bvdv was inactivated within this time period. at ph . , c, mm caprylate, and ph . , c, mm caprylate, complete inactivation of bvdv and of hiv and prv was achieved in less than min. the authors also showed that mm caprylate at ph . and c in supernatant iv- , an intermediate in albumin production, inactivated r . log bvdv almost instantaneously [ ] . the virucidal effect of caprylic acid has also been confirmed in human albumin solution, where it is used as a stabilizer for pasteurisation. elevated temperature and low ph were found to be critical parameters to ensuring significant reduction in virus infectivity. rate and extent of inactivation were sensitive to variations in the caprylate to protein ratio and to changes in ph. in the conditions retained, % w/v protein, mm caprylate, ph . and c, more than log inactivation of the lipid-enveloped viruses tested, including bvdv, was achieved [ ] . it should be kept in mind that treatment of whole plasma or crude fractions, as is the case for equine antivenoms production, may lead to lower rate and kinetics of viral inactivation, due to the high endogenous lipid content, as found in a study that evaluated the virucidal effect of sodium oleate [ ] . finally, one should keep in mind that caprylic acid treatment does not inactivate non-enveloped viruses. several human igg preparations are subjected to an acid ph incubation that was historically introduced to reduce immediate adverse reactions subsequent to intravenous injections. in the late s, acid ph treatment was also shown to contribute greatly to the table comparison of conditions for caprylic acid treatment used for human igg preparations ( ) [ , ] . the treatment of human igg generally consists of incubation at ph , with or without traces of pepsin, at temperatures from to c, using a protein content close to g/l, and for more than h. the treatment is intended to eliminate aggregates. pepsin, when present, is maintained at low concentration to avoid cleavage of the igg molecule. several model enveloped viruses were shown to be inactivated under those conditions [ e ]. more than e log inactivation of hiv and other enveloped viruses used as surrogate models, such as semliki forest virus (sfv), hsv, vsv, and cmv is achieved. by contrast, poliovirus, as other non-enveloped virus, seems more resistant. the presence of trace concentrations of pepsin added to reduce anticomplementary activity during this procedure has been shown to contribute little to virus kill of most processes [ ] . virus inactivation by acid ph and pepsin is influenced by several parameters. hiv, bvdv, sfv, and prv are completely inactivated within mine h at c but inactivation rate and extent is less at lower temperatures. increasing the sucrose content from to % reduced the rate of inactivation of prv but not that of sfv. increasing the nacl content from to mm reduced the rate of inactivation of sfv but that of prv was unchanged. an increase in the igg concentration from to % speeds up the inactivation of prv but decreases that of sfv. therefore, temperature is a major parameter in the virucidal efficacy of ph-pepsin treatment and the impact of solute composition is virus-dependent [ ] . some fractionation protocols used in antivenom production include a low ph treatment to induce the precipitation of 'euglobulins', which are plasma globulins unstable under these conditions. this step was shown to remove between . and . log of eiav, sindbis virus, poliovirus and porcine parvovirus [ ] . virus reduction may occur by co-precipitation with the discarded 'euglobulins' rather than by an inactivation process [ ] . pasteurisation is the treatment of a liquid protein fraction for h at c [ ] and is being used in the production of at least one type of equine-derived antivenom [ ] . experience with human plasma products show that liquid heat treatment may inactivate both enveloped and non-enveloped viruses. pasteurisation of albumin solutions is carried out in the final container in the presence of low concentrations of sodium caprylate alone or with n-acetyl tryptophan. inactivation of sindbis and emc model viruses added to % albumin solution was achieved within min treatment [ ] . however, inactivation of some non-enveloped viruses is less [ ] , and the process appears to be virus-specific. various coagulation factors, protease inhibitors, and intravenous igg are pasteurised during the purification process. most often, pasteurisation is performed in the presence of stabilizers, like amino acids, sugars, or citrate [ , ] , to protect protein functionality, and limit molecular alterations and protein aggregation. protein stabilizers are also known to stabilize viruses, however, further highlighting the need for validating the exact conditions of treatment used. pasteurisation can inactivate viruses of different types, enveloped or nonenveloped, including hiv, hbv, hcv, and hav [ , , , ] . there are few published data on the inactivation of resistant model non-enveloped viruses like porcine parvovirus, sv or reovirus type in plasma products [ , ] . in most instances, inactivation of such viruses is limited to less than e log over the h heating period. heat treatment of whole human plasma at c for h, in the presence of a specific combination of stabilizers, inactivates more than log of hiv, and more than log of enveloped and nonenveloped model viruses [ ] . finally, it was demonstrated that heat treatments like pasteurisation and vapour heat treatment (as well as solvent-detergent and nanofiltration on nm membranes) can inactivate/ remove more than log of wnv [ ] . chromatography is primarily used in human plasma fractionation as a downstream polishing step [ ] . ionexchange chromatography has been described for some antivenom products [ , ] . viral partitioning has been shown to occur during affinity and ion-exchange chromatography, but the mechanism of removal is not easy to predict and control [ ] , making it difficult to consider these as robust viral removal steps. storage at ph . , in the presence of a stabilizer, such as % maltose, for a minimum of days at c, yields aggregate-free and in vitro functionally active human igg preparations [ ] . incubating at ph . for days at c caused a , -fold decrease in bvdv infectivity and complete inactivation of chimpanzee infectious doses per millilitre of hcv [ ] . with the exception of a recent report of a fab ovine antivenom formulated at ph . [ ] , to our knowledge, formulation at low ph is not commonly used for antivenoms but may be considered as a viral safety step, if it does not affect the biological activity, the general safety, and the clinical efficacy of the product. viral validation studies are small-scale experiments designed to provide an estimate of the overall viral reduction level achieved across the manufacturing processes and to identify steps and parameters that are critical for viral inactivation or removal. to perform such studies, as described in the guidelines prepared by the cpmp, production intermediates are voluntarily spiked with known amounts of known viruses, using laboratory-scale mimicking of the production process, and the virus reduction factor is calculated by comparing the infectivity level before and after each individual process step evaluated. when appropriate, a cumulative viral reduction factor can be calculated and provides information on the viral safety margin of processes for a range of model viruses. effective and robust individual steps typically achieve more than log reduction in infectivity under a large range of production parameters. viral validation studies are usually conducted with e model viruses differing in their structural features (presence of an envelope, rna or dna genetic material, size, degree of resistance). ''relevant'' viruses, which are known potential contaminant of the starting plasma, should be used when possible. a list of existing laboratory adapted model viruses that could be used for validation studies of antivenoms manufacturing processes is shown in table . vsv and wnv that may contaminate horses appear of special relevance for process validation of equine products. dedicated viral inactivation and removal production steps should be implemented in a way to ensure the reproducibility in viral reduction and the absence of risks of downstream contamination. examples of approaches used for the manufacture of human plasma products are provided in recent who guidelines [ ] , and a cpmp note for guidance provides recommendation on production of immunsera [ ] . both can be used as reference by manufacturers of antivenoms. viral reduction equipment should have adapted specifications. for instance, equipment for bulk in process virus inactivation, such as the acid ph incubation or the caprylic acid treatment, should ideally be fully enclosed. in addition, vessels should have temperature controls and be designed to avoid ''dead points'' where important parameters such as temperature and ph could not be ensured. similarly, liquid heat treatment or heat shock should be conducted in jacketed tanks with the solution stirred throughout the heat cycle to ensure homogeneity and all points in the tank should be within the specified temperature range. during the qualification phases, the equipment and the required services should be shown to conform to the predefined technical specifications and requirements, and to function within the specified limits. when possible, it is recommended to create a dedicated ''viral safety area'' where production steps are arranged in a clear and logical way, so that recontamination of the virally reduced intermediates is avoided. operating procedures should also be written to reduce the likelihood of crosscontamination. bulk ph or caprylic acid virus inactivation could be carried out in two stages, the first stage being located in a normal production room, followed by a second incubation in another tank located in a segregated, contained area. the duration of the first stage should be such that the majority of virus inactivation (as found during the viral validation studies) has occurred. on completion of the first stage, the product should be aseptically transferred (e.g. through sterile coupling) into the second vessel located in a safety zone for completion of the second stage of viral inactivation. product-contacting-equipment used in the safety area should be dedicated or decontaminated to inactivate any remaining virus. a quality assurance system should ensure that the execution of virus reduction methods conforms to the validated conditions. viral inactivation and removal procedures should be described in approved standard operating procedures (sops) that contain critical process limits for the viral inactivation and removal methods applied to antivenoms. to date there is no documented example of viral transmission to humans from the use of antivenoms, whichever type of product (whole igg, f(ab#) or fab) is manufactured [ ] . considering the difficulty in the containment of large animals like horses, and in an exhaustive viral testing of the starting plasma, the viral safety of antivenoms appears therefore largely dependent upon the capacity of manufacturing processes to reduce infectious agents and on the use of good manufacturing practices, in particular in ensuring production traceability. by contrast to human plasma products, most antivenoms are currently not subjected to purposely introduced viral reduction steps like solvent-detergent, pasteurisation, or nanofiltration [ , , ] . however, a comparison with validated manufacturing processes used for human igg clearly indicates that at least two widely used antivenom production steps, caprylic acid treatment and low-ph incubation, are likely to contribute in a robust manner to viral safety, at least against enveloped viruses. the concentration of caprylic acid ( %) used for antivenoms is higher than that used for human igg (table ) , and the ph of treatment is within the effective range (less than ph ) required for optimal inactivation of enveloped viruses. as the treatment is performed on equine plasma itself, or on crude fractions, the viral kill could be reduced by the presence of lipids. the acid incubation of antivenoms which is performed at a ph ( . e . ), less than that used for human igg, and at a similarly warm temperature ( e c) ( table ) should provide a fast rate of inactivation of lipidenveloped viruses, and possibly also non-enveloped viruses. however, the incubation time ( mine h) is for some processes significantly less than for human igg (typically e h), probably reducing the extent of viral kill achieved. preliminary viral kill data obtained with antivenoms confirm that conditions used for peptic cleavage at low ph of f(ab#) achieve robust inactivation of wnv [ ] . we recommend that further representative viral validation studies using a range of well-established virus models are performed to know the rate and extent of viral kill actually achieved during those production steps. the viruses to use for those validations should be discussed with specialized virologists, and, by analogy with most studies done with human plasma derivatives, might include three lipidenveloped viruses (such as bvdv, vsv, psr, and wnv) and one non-enveloped virus (such as emc, poliovirus, or reovirus ). inactivation data could then be compared to viral kills achieved using similar process steps of human igg products, therefore providing some reference on their robustness and allowing manufacturers and national regulatory authorities to assess scientifically the margin of safety of antivenom products. other manufacturing steps, such as heat coagulation, could be validated as well. formulation of the product at low ph, as done for some human igg preparations, is another potential approach to consider for improved viral safety, but evaluating product stability and efficacy under such conditions would be a pre-requisite. in view of their expected beneficial impact on viral safety, appropriate process implementation of caprylic acid treatments and low-ph incubations should be ensured, as well as measures taken to avoid risks of downstream contamination and, more widely, to respect good manufacturing practices and traceability concepts. such conclusions should also be valid for other animal plasma-derived igg products used in human medicine such as anti-lymphocyte/t-cell, antitoxins, and anti-bacterial or viral agents sera [ ] . snake antivenoms marine antivenoms venomous bites and stings a new pan african polyspecific antivenom developed in response to the antivenom crisis in africa report of a who workshop on the standardization and control of antivenoms venoms, antivenoms and immunotherapy 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the federation of european laboratory animal science associations (felasa) working group on animal health report of the federation of european laboratory animal science associations (felasa) working group on animal health accepted by the felasa board of management chromatography in plasma fractionation: benefits and future trends inactivation of lipid-enveloped viruses in labile blood derivatives by unsaturated fatty acids inactivation of lipid-enveloped viruses in proteins by caprylate inactivation of lipid enveloped viruses by octanoic acid treatment of immunoglobulin solution enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates low ph, caprylate incubation as a second viral inactivation step in the manufacture of albumin. parametric and validation studies potential contribution of mild pepsin treatment at ph to the viral safety of human immunoglobulin products the viral safety of intravenous immune globulin virus validation of ph -treated human immunoglobulin products produced by the cohn fractionation process virus inactivation by pepsin treatment at ph of igg solutions: factors affecting the rate of virus inactivation virus inactivation during intravenous immunoglobulin production viral safety of intravenous immunoglobulins g for therapeutic use strategies to produce virus-safe blood derivatives inactivation of viruses in labile blood derivatives. ii. physical methods pasteurization as an efficient method to inactivate blood borne viruses in factor viii concentrates inactivation of hiv, hbv, hcv related viruses and other viruses in human plasma derivatives by pasteurisation a pasteurized therapeutic plasma pasteurization of antihemophilic factor and model virus inactivation studies virucidal heattreatment of single plasma units: a potential approach for developing countries nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data affinity chromatography in the industrial purification of plasma proteins for therapeutic use chromatographic removal of viruses from plasma derivatives a comparative in vitro study of low ph and enzyme treated immunoglobulin preparation for intravenous use inactivation of hepatitis c virus in low ph intravenous immunoglobulin formulation of a liquid ovine fab-based antivenom for the treatment of envenomation by the nigerian carpet viper (echis ocellatus) world health organization expert committee of biological standardization. guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products. www.who.int/biologicals-quality% assur-ance% and% safety nanofiltration of plasma-derived biopharmaceutical products inactivation of west-nile virus during peptic cleavage of horse plasma igg we express our sincere thanks to dr. herbert dichtelmu¨ller for his review of the manuscript and expert suggestions. key: cord- -jld ygxt authors: neidermyer, william j.; whelan, sean p. j. title: global analysis of polysome-associated mrna in vesicular stomatitis virus infected cells date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: jld ygxt infection of mammalian cells with vesicular stomatitis virus (vsv) results in the inhibition of cellular translation while viral translation proceeds efficiently. vsv rna synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces mrnas that are structurally indistinct to cellular mrnas with respect to their ′ cap-structure and ′-polyadenylate tail. using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mrna, we interrogate the impact of vsv infection of hela cells on translation. analysis of sequence reads in the different fractions shows > % of total cytoplasmic and polysome-associated reads map to the viral genes by hours post-infection, a time point at which robust host cell translational shut-off is observed. consistent with an overwhelming abundance of viral mrna in the polysome fraction, the reads mapping to cellular genes were reduced. the cellular mrnas that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more au rich, features that are shared with the viral mrnas. several of those mrnas encode proteins known to positively affect viral replication, and using chemical inhibition and sirna depletion we confirm that the host chaperone heat shock protein (hsp ) and eukaryotic translation initiation factor a (eif a)—encoded by such mrnas—support viral replication. correspondingly, regulated in development and dna damage (redd ) encoded by a host mrna with reduced polysome association inhibits viral infection. these data underscore the importance of viral mrna abundance in the shut-off of host translation in vsv infected cells and link the differential translatability of some cellular mrnas with pro- or antiviral function. introduction infection of mammalian cells by vesicular stomatitis virus (vsv) results in a profound shut-off of host cell gene expression. this host cell shut-off occurs at the level of mrna transcription through inhibition of rna polymerase ii by the viral-encoded matrix protein (m) [ ] [ ] [ ] . the m protein also forms a complex with ribonucleic acid export (rae ) and nucleoporin (nup ) [ ] thus suppressing host cell mrnp export from the nucleus, including that of mature cellular mrnas [ ] [ ] [ ] [ ] . vsv infection also inhibits protein synthesis by manipulation of the host-cell translation machinery, particularly at the level of translation initiation [ , ] . eukaryotic initiation factor e (eif e)-the rate limiting factor for translation initiation-recognizes the m gpppn mrna cap structure as part of the eif f complex, and in concert with other translation initiation factors facilitates the recruitment of the small s ribosomal subunit to the mrna prior to scanning to the initiating methionine where the s subunit joins [ , ] . vsv infection results in the rapid dephosphorylation of eif e itself, for which the functional consequences are unclear, and of its binding protein (eif e-bp ) leading to eif e sequestration and the suppression of translation initiation [ , ] . viral gene expression evades the shut-off mechanisms employed to suppress host gene expression. as vsv rna synthesis occurs entirely within the cytoplasm, viral rna synthesis is not subject to the inhibitory effects of m on rna polymerase ii and mrna export from the nucleus. the vsv rna synthesis machinery comprises a ribonucleoprotein complex of the negative-sense genomic rna completely encased by a nucleocapsid protein (n) sheath and associated with the viral polymerase complex [ ] . the viral transcriptase copies the n-rna template into monocistronic mrnas that are structurally indistinct to those of the host-cell with respect to their cap and polyadenylate tail [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the enzymes necessary for mrna synthesis, namely an rna dependent rna polymerase (rdrp) and a set of capping enzymes, reside within the viral large protein (l) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vsv l protein cannot engage the n-rna template directly, but instead depends on the viral phoshoprotein (p) to facilitate the interaction [ ] [ ] [ ] [ ] [ ] [ ] . messenger rna polyadenylation is also catalyzed by l through reiterative transcription by the rdrp of a u tract that resides at the end of each gene [ ] [ ] [ ] [ ] [ ] . this program of viral transcription results in the cytoplasmic synthesis of mrnas that depend upon the host machinery for their translation, and must therefore avoid the shut-down mechanisms that effectively suppress host mrna translation. metabolic labeling studies demonstrate that hours post vsv infection of baby hamster kidney cells in culture, total translation is suppressed to about % the level of uninfected controls [ ] . extraction of mrna from infected cells coupled with its in vitro translation confirmed that the cellular mrnas remain intact and are competent for translation [ ] . the vsv mrnas are present in an approximately - fold excess of the total cellular mrna, leading to the model that competition between viral and cellular mrnas for ribosomes results in the dominance of viral translation [ , ] . polysome analysis also demonstrates that the cellular mrnas are associated with significantly fewer ribosomes in infected cells [ ] . for example, infection results in the movement of actin mrna from polysomes containing or more ribosomes to those containing [ ] . this movement reflects the competition between viral and cellular mrna for ribosomes and the limited pool of eif e. the competition model predicts that the kinetics of viral mrna synthesis and the levels of viral mrna should correlate closely with host shut-off. tests of this prediction yielded conflicting results. the kinetics of host shut-off and viral mrna accumulation correlate well for many strains of vsv, consistent with the competition model [ , ] . inhibition of host protein synthesis is, however, largely unaffected following coinfection of cells with increasing quantities of defective interfering (di) particles that suppress viral mrna levels up to -fold [ ] . a similar result was obtained for a vsv mutant that is restricted for genome replication at ˚c, and yields only % of the wild type levels of viral mrna [ ] . collectively, these studies suggest additional mechanisms may contribute to the shut-off of host cell protein synthesis. specific features of the viral mrnas that contribute to their efficient translation have not been defined. the untranslated regions of vsv mrnas are short, being - nucleotides for the viral n, p and l mrnas that encode the proteins required for rna replication [ , ] . how such short utrs serve as effective initiators of translation is unclear. evidence for differential translation of viral mrna comes from small interfering rna suppression of eif e, which inhibits host gene expression but has no impact on viral gene expression [ ] . viral translation is also hypersensitive to the loss of ribosomal protein l , suggesting different mrna features facilitate translation of viral versus host mrna [ ] . flanking cellular or reporter genes by the conserved viral -nt gene-start and -nt gene-end sequences, and inserting them into the viral genome is sufficient to mediate their efficient translation [ ] . by contrast, expression of the same genes following transfection of plasmid dna into cells and subsequent vsv infection does not offer this translational advantage [ ] . thus, transcription of the mrnas from the viral genome appears to contribute to their efficient translation. in the present study, we interrogate global mrna translation in vsv infected cells using rnaseq analysis of the cytoplasmic mrna transcriptome, and parallel sequencing of polysome-associated mrnas. we obtain support for the model that an overabundance of viral mrna contributes to host shut-off by leading to a re-distribution of cellular ribosomes onto viral mrna. by combining this rnaseq analysis with examining the distribution of specific viral and cellular mrnas within polysomes, we also demonstrate that mrnas shift to smaller polysomes. analysis of cellular mrnas less-sensitive to this global shut-down of translation identifies several host proteins that promote viral replication. similar analyses revealed the abundance of viral mrna contributes to the host-cell shut-off for other viruses including coronaviruses, influenza and vaccinia [ ] [ ] [ ] . to interrogate the impact of vsv infection on global translation we isolated total cytoplasmic, monosome-and polysome-associated mrna from hela cells at and hpi and compared the relative sequence reads obtained by deep-sequencing ( fig a) . statistical analysis of sequencing reads between biological replicates from each fraction yields a pearson correlation of > . for cytoplasmic, monosome-and polysome-associated mrna pools validating reproducibility between the replicates. as visible in the polysome profiles (fig b) , vsv infection results in a small but reproducible increase in the pool of monosomes and large polysomes at hpi, and a collapse of large polysomes and an increase in monosomes by hpi. mapping of the sequence reads to the viral and host genome highlights that by hpi > % of the total reads in the cytoplasmic and polysome fractions are viral (fig c) . this increase from the < % observed at hpi (fig c) emphasizes the impact of the exponential phase of viral rna replication and secondary transcription of the viral genome on mrna production. the viral sequence reads map to all genes, with clear dips in coverage at gene-junctions (s fig) . consistent with the order of transcription of the viral genome and the localized transcriptional attenuation at gene-junctions [ ] [ ] [ ] [ ] , the relative reads that map to each viral gene generally diminish with distance from the single promoter ( fig d and s fig) . analysis of the sequencing reads that map to cellular genes supports that like the viral mrnas, the level of reads in the polysome fraction mirrors that in the total cytoplasmic fraction at and hpi ( fig c) . this result demonstrates that the majority of mrnas are polysome-associated in proportion to their abundance. the abundance of the viral mrnas at hpi supports the model that viral mrnas outcompete cellular mrnas for ribosomes [ ] . we note that viral mrnas are, however, underrepresented ( %) and cellular mrnas overrepresented ( %) in the monosome fraction at hpi, compared to their cytoplasmic abundance ( fig c) . this finding is consistent with a differential effect on viral versus host mrna translation. to determine how vsv infection affects the distribution of the population of mrnas between total cytoplasmic, monosome or polysome fractions, we plotted the transcript per million (tpm) for each individual mrna mapped to the human and viral genome in all fractions (fig ) . at hpi, reads that map to the viral genes in each fraction are similar in abundance to those reads that map to highly expressed cellular genes (fig a) . the reads that map to any given cellular gene alter within a relatively narrow range, with few genes showing a greater than -fold change in the relative number of sequence reads (fig b) . for the population of mrnas, the relative reads obtained from the polysome fraction mirrored the relative reads in the total cytoplasmic fraction, consistent with the abundance of an mrna being a determinant of its translatability. by hpi, reads that map to each of the viral genes-with the exception of l-exceed the reads that map to any individual cellular gene (fig c, red triangles) . this is concurrent with a decrease in reads that map to the majority of cellular genes in cytoplasmic, monosome and polysome-associated fractions (fig c) . there were, however, some distinctions between the monosome and polysome fractions. for the majority of the population of cellular mrnas, hela cells were infected with vsv at a moi of and cytoplasmic extracts were prepared at and hpi for mrna isolation and polysome profiling. messenger rna was isolated from fractions corresponding to s monosomes, or polysomes containing or more ribosomes, and used for deep sequencing. (b) polysome analysis of uninfected (black) or vsv (red) infected hela cells. cytoplasmic extracts were sedimented through a - % sucrose gradient and . ml fractions were collected while continuously monitoring absorbance at λ = nm. (c) distribution of fragments mapping to the concatenated hg (human) and vsv genomes for cytoplasmic, monosome, and polysome samples at and hpi. trimming and mapping was performed in clc genomics workbench. (d) distribution of reads among the viral genes at and hpi. expression level is presented as transcripts per kilobase million (tpm) to normalize for gene length and library size, error bars denote standard deviation from two biological replicates. reads were most reduced in the polysome fraction compared to the total cytoplasmic fraction ( fig d) . a smaller reduction in reads was observed in the monosome fraction, and some cellular mrnas even showed an increase in reads compared to the total cytoplasmic fraction ( fig c) . this may reflect differences in the movement of cellular mrnas from large polysomes to monosomes or out of the pool of translating ribosomes. we next mined our sequence data for evidence of differential translation of cellular mrnas following vsv infection. for this analysis we divided the polysome tpm by the total cytoplasmic tpm as an indicator of the efficiency with which any given mrna is translated. we also performed a similar analysis for the monosome pool. we are cognizant of the fact that such ratios ignore the movement of any given mrna from larger to smaller polysomes, and will likely represent an underestimate of the extent of any translational regulation. to identify the subset of the population of cellular mrnas with the highest probability for translational regulation in infected cells, we plotted the fold change in tpm at and hpi (fig a- d ). at hpi the monosome or polysome-associated reads changed within a narrow range for the majority of cellular genes ( fig a) . the marked shut-off of host protein synthesis observed by metabolic labeling suggests that at hpi the association of cellular mrna with polyribosomes would alter significantly at the population level. although we observe a global reduction in polysome-associated reads for the bulk of the population of cellular mrnas the reduction is less than - fold. accompanying this global reduction in polysome-associated reads, we also observe an increase in monosome-associated reads with more than half the mrnas within the population exhibiting a > -fold increase (fig b and d ). from the above ratios we selected the subset of cellular mrnas that exhibit the largest changes in relative polysome-associated reads at hpi to determine whether those mrnas shared any common features. for this purpose, we selected those mrnas that change > standard deviations of the mean and thus exceed the % confidence interval. this analysis identified cellular mrnas as candidates for translational upregulation and cellular mrnas as candidates for translational downregulation following vsv infection (fig b and d ). consistent with monosome and polysomeassociated reads at hpi changing within a narrow range, only genes with increased and with decreased polysome association, overlap between and hpi (fig c and d ). within the monosome fraction genes with increased and with decreased monosome association overlap from to hpi. we next determined whether shared functional or sequence elements are present within the specific subsets of mrnas with increased polysome association, or the mrnas with decreased polysome association (fig b and d , blue and green dots). for the genes with significantly increased polysome-associated reads, gene ontology analysis identifies functions in rna binding, helicase and ntpase activities, among others ( fig e and s dataset) . the genes with decreased polysome-associated reads are associated with cellular responses to stimuli and signaling activities ( fig f and s dataset) . this gene ontology analysis reveals that the up and down regulated transcripts comprise distinct functional groups. at hpi the cytoplasmic abundance of cellular mrnas correlates with their polysome association at hpi ( fig a and b ), consistent with mrna abundance being a determinant of translatability. as described above, we use as an indicator of translation efficiency (te) of an mrna the ratio of polysome to total cytoplasmic associated reads. to determine whether there are shared features between the mrnas with evidence of enhanced polysome association or the with reduced polysome association, we extracted mrna sequences and annotations from the ucsc genome browser. assisted by published datasets we examined whether the half-life, size, gc content or poly(a) tail length correlate with increased or decreased polysome association (fig c- f and s fig) [ , ] . cellular mrnas with increased polysome-associated reads tended to have a longer half-life, were typically larger, and were more au-rich ( fig c- e and s fig) . correspondingly, those with decreased polysome-associated reads tended to have shorter half-lives, higher gc content, and were typically smaller. the correlation between higher au content and increased polysome-associated reads was most evident for the coding region and utr (s fig). the effect of length was predominantly a determinant of the orf and not the or utr (s fig). there was no correlation between poly(a) tail length and polysome-associated reads at hpi ( fig f) . this analysis highlights that the cellular mrnas that exceed the % confidence interval for increased polysome-associated reads in response to vsv infection are most similar to the viral mrnas in that they are typically longer and more au rich. we next examined whether cellular mrnas that exhibit increased polysome association encode proteins that are pro-or antiviral. among the cellular mrnas with increased polysome association several encode known proviral factors including the heat shock proteins (hsp) , , and . previous work demonstrated that inhibition of hsp inhibits viral replication, and linked inhibition of those chaperones to defects in l protein folding [ ] [ ] [ ] . we independently verified the proviral function of hsp using the inhibitor -[ -(dimethylamino)ethyl]amino- -desmethoxygeldanamycin ( -dmag) [ , ] . infection of hela cells with vsv that expresses egfp as a marker of infection demonstrates that -dmag has no effect on the fraction of cells infected, but slows the rate of egfp expression ( fig a and s fig) . this was not simply due to defects in egfp folding, as metabolic labeling of viral rna substantiates the defect in gene expression (s fig) . we also found that polysome association of the mrna encoding eukaryotic initiation factor subunit a (eif a), increases after infection. to test whether this reflects a specific proviral function of eif a, we used sirna depletion to reduce eif a and measured viral gene expression using reporter viruses expressing egfp or luciferase. both reporter viruses displayed a sensitivity to the loss of eif a (figs b and s ). as expected, depletion of eif a also reduced cellular translation in uninfected cells, but that reduction was modest as evidenced by expression of a cmv promoter driven renilla luciferase reporter ( fig b) . translation of the cmv driven reporter, however, reflects the accumulated steady-state levels of luciferase mrna. we therefore measured the effect of eif a depletion on viral vs host translation by metabolic incorporation of [ s]-methionine in infected and uninfected cells (fig b) . following eif a depletion we observed a % reduction in viral m protein synthesis over a minute time period, which is similar to the % reduction in host protein synthesis measured in mock infected cells. this analysis supports a proviral role for cellular mrnas that encode proteins with important house-keeping functions that remain polysome-associated following vsv infection. among the cellular mrnas that exhibit reduced polysome association following infection was dna-damage inducible transcript (ddit ) which encodes regulated in development and dna-damage response (redd ) [ ] [ ] [ ] . existing studies demonstrate that ddit /redd restricts the replication of negative-strand rna viruses, including vsv. depletion of ddit /redd by sirna increased viral gene expression as evidenced from infection of cells with vsv-egfp, and by metabolic labeling of viral protein synthesis (fig c, s fig) . consistent with the enhancement of viral gene expression following ddit depletion, we obtained an approximately -fold increase in viral titers ( fig c) . depletion of ddit /redd also increases cellular protein synthesis likely reflecting its role as a negative regulator of mtor ( fig c and s fig) . the above analysis supports that the polysome association of some host mrnas following vsv infection correlates with their pro-or antiviral functions, but does not directly demonstrate that the level of polysome association is associated with a change in synthesis of the corresponding protein. to independently examine whether changes in polysome association of host mrnas affect synthesis of the corresponding protein we selected the heat shock protein (hsp ) and y-box binding protein (ybx ) as representative mrnas with increased and decreased polysome association, respectively. we selected those mrnas based on their highlevels of expression, stability, and availability of antibodies suitable for the selective immunoprecipitation of the corresponding protein. we compared the effect of vsv infection on protein synthesis by selective immunoprecipitation of proteins following metabolic incorporation of [ s]-methionine from - hours post infection (fig d) . synthesis of hsp - hpi is indistinguishable to that synthesized during a h period from mock infected cells (fig d) . by contrast, ybx synthesis decreases more than two-fold ( fig d) . this result confirms for cellular mrnas that the extent of polysome association observed by our rnaseq analysis is reflected in synthesis of the corresponding host proteins. analysis of cellular mrnas with high cytoplasmic abundance (purple) or low cytoplasmic abundance (orange) as compared to mrnas with cytoplasmic abundance within standard deviations of the mean abundance (gray) in uninfected cells. cytoplasmic abundance by tpm is from the data set published with this paper. ��� p< . x − ; �� p< . x − ; � p< . ; all others p> . as determined by the wilcoxon rank sum test compared to mrnas with relative abundance levels within the % confidence interval of the mean. hinges correspond to the th- th percentiles, and whiskers denote . times the inter-quartile range. (b) analysis as in a for cytoplasmic abundance in infected cells. (c-f) mrna characteristics for mrnas with increased polysome association (blue) or decreased polysome association (green) at hpi, as defined in fig . data for rna half-life and poly(a) tail length were from [ , ] . analysis was performed as in a. for our experiments we pooled fractions that contained or more ribosomes prior to sequencing of the polysome-associated mrna. as a result, we do not assess the impact of the redistribution of mrnas toward smaller polysomes. we therefore selected a subset of cellular mrnas (fig a) , and interrogated their distributions across polysomes using reverse transcription and quantitative pcr. as controls, we analyzed the distribution of n and g mrnas as representative viral transcripts translated by soluble and endoplasmic reticulum-associated ribosomes, respectively [ ] . consistent with the robust production of viral proteins at hpi, the vsv n and g mrnas were localized in fractions corresponding to or more ribosomes (fig b) . for two cellular mrnas with increased polysome tpm-collagen type iv alpha (col a ) and alpha-actinin- mrna (actn )-the mrnas remained associated with larger polysomes in infected cells (fig c) . two cellular transcripts that were largely unaltered in their polysome associated tpm-β-actin (actb) and glyceraldehyde -phosphate dehydrogenase (gapdh)-remained polysome-associated, although there was a shift toward smaller polysomes and some gapdh transcripts exited polysomes (fig d) . for two representative cellular mrnas with decreased polysome tpm-transforming growth factor b induced factors (tgif ) and ubiquitin conjugating enzyme e b (ube b)-the mrnas largely exited the polysome fractions, and those that remained were predominantly present on smaller polysomes ( fig e) . in all cases examined, dissociation of polysomes with edta shifted the mrna distribution toward the fractions corresponding to free ribosomal subunits (s fig). these qpcr data highlight the shift towards smaller polysome fractions for some cellular mrnas, which also likely contributes to suppression of host protein synthesis. this shift might also explain our finding that hsp protein synthesis is relatively unaffected by viral infection (fig d) , although the mrna exhibits increased polysome association. the abundance of viral mrna and the suppression of translation initiation through reducing the pool of eif e will both contribute to the movement of mrnas toward smaller polysomes. recognition of the mrna cap-structure by eif e requires that the guanine-n- position of the mgpppnmn cap is methylated [ ] . we previously reported a panel of recombinant vsvs containing amino acid substitutions within the l-encoded mrna cap methylase domain that are defective in viral mrna cap methylation [ ] . mutants vsv-l g a and vsv-l g a contain substitutions in the binding site for the methyl donor s-adenosyl methionine (sam) and ablate all viral mrna cap methylation (vsv-l g a ) or guanine-n but not ribose- -o methylation (vsv-l g a ) [ ] . as vsv mrna is relatively insensitive to the loss of eif e [ ] , we would anticipate that the methylation status of the mrna cap-structure would have little impact on polysome association. analysis of the distribution of vsv n and g mrna within polysomes at hpi revealed a similar distribution in cells infected with wild-cells infected with wild-type vsv. the position of viral proteins is noted to the right. presented is a representative gel from two independent replicates. (c) vsv gene expression and replication in ddit depleted cells. a representative histogram of egfp expression is shown along with the mfi of cells normalized to a non-targeting sirna control. error bars denote the standard deviation from the mean of three independent replicates. for metabolic labeling a representative gel of two independent replicates is presented. kinetics of viral replication were measured by titration of yields at various times post infection of sirna treated hela cells. error bars denote the standard deviation from the mean of independent replicates. (d) immunoprecipitation of cellular proteins synthesized post-infection with vsv wt . shown is a representative gel from two independent replicates. a quantitative analysis of the hsp and ybx bands is shown in the bottom two panels, error bars denote the standard deviation from two independent replicates. https://doi.org/ . /journal.ppat. .g control of vsv protein synthesis type virus as well as those infected with vsv-l g a and vsv-l g a (fig a- c) . correspondingly, the rate of viral protein synthesis in cells infected with vsv-l wt and vsv-l g a measured by a -minute pulse of [ s]-methionine is similar (s fig). these results demonstrate that defects in viral mrna cap methylation do not significantly alter the rate of viral protein synthesis, consistent with a reduced dependence on eif e [ ] . to directly test whether manipulating eif e levels affects viral translation we depleted eif e levels approximately -fold using a peptide-conjugated morpholino (ppmo) and measured the rates of vsv-l wt and vsv-l g a viral protein synthesis by a -minute pulse of [ s]-methionine at various times post-infection (fig d- f) . depletion of eif e decreased the rate of viral protein synthesis in vsv-l a infected cells, but not l wt infected cells ( fig e and f) . this was not due to sequestration of eif e by differential activation of eif e-bp between vsv-l wt and vsv-l g a infected cells, as the kinetics of eif e-bp dephosphorylation are the same during both infections (s fig). we previously reported that although vsv-l a is defective in mrna cap methylation, up to % of the mrna cap-structures are guanine-n methylated. we interpret this finding as indicative of an eif e dependent mechanism of translation early in infection. we obtained two snapshots into the complex battle for control of protein synthesis in cells infected with vesicular stomatitis virus by sequencing of polysome-associated mrnas at and hpi. those snapshots provide further evidence that the abundance of vesicular stomatitis virus mrnas is a determinant of the dominance of viral protein synthesis in infected cells, but highlight several additional attributes of this complex relationship. those include the demonstration that some host mrnas that remain polysome associated encode proteins that support viral replication, and some of those that exhibit reduced polysome association encode proteins that are antiviral. we also obtained further insight into the seemingly paradoxical observations that viral infection results in a reduction of the available pool of eif e -the rate limiting factor for translation initiation-yet viral mrnas contain a cap structure that is indistinct to that of host mrnas. through the use of a viral mutant defective in mrna cap methylation, and suppression of eif e levels we provide evidence consistent with a transition from an eif e dependent phase of viral translation to one less-dependent on eif e. the sequence data reported here provides some support for the model that the vsv mrnas overwhelm the pool of cellular mrna leading to a redistribution of ribosomes onto viral messages [ ] . evidence in support of this model is based on massively parallel sequencing of mrnas associated with polysomes, compared with those present in the cytoplasm. as a fraction of the total cytoplasmic mrna, the vsv mrnas represent~ % by hpi, but more than % by hpi, illustrating the power of exponential amplification of the viral genome. as a result, the viral n, p, m and g mrnas far exceed the abundance of any given cellular mrna, and even the least abundant viral mrna-that encoding the l polymerase-is present at similar levels to the most abundant cellular mrna. thus, one contributor to host cell shut-off in vsv infected cells appears to relate to the synthetic capabilities of the viral polymerase in transcription of viral mrna. similar conclusions have recently been reached for other viruses. infection of cells with mouse hepatitis virus (mhv) a positive-strand rna coronavirus that replicates within the cytoplasm results in - % of the cytoplasmic mrna being viral by hpi [ ] . for influenza a virus, a segmented negative-strand rna virus that replicates in the nucleus, > % of the total mrna in the cytoplasm is viral [ ] . in this case however, the viral endonuclease pa-x degrades cellular mrna which further contributes to the dominance of viral mrna [ , ] . for vaccinia virus, a dna virus that replicates entirely within the cytoplasm, degradation of host mrna through the viral encoded decapping enzymes d and d also helps the viral mrnas overwhelm those of the host cell [ , , ] . collectively these studies indicate that one shared mechanism for host cell shut-off in virus-infected cells is competition for host cell ribosomes through tipping the balance between viral and host mrna. earlier work concludes that viral mrna abundance is not the determinant of host cell shut-off [ ] . when vsv mrna levels were suppressed up to -fold by using defective interfering particles of vsv or a viral mutant defective in transcription, host shut-off was still observed. we did not directly test how suppressing viral mrna levels impacts host shut-off in this study, but rather conclude that abundance is only part of the mechanism by which the virus induces host cell shut-off-as discussed below. we also obtained evidence in support of additional mechanisms that contribute to host cell shut-off in vsv infected cells. we confirmed earlier work that demonstrated a suppression of the pool of the rate limiting factor for initiation, eif e, by altering the phosphorylation status of its negative regulator, eif e-bp , which results in eif e sequestration [ ] . differences in sensitivity to reductions in eif e may contribute to the overrepresentation of cellular mrna we observe in monosome fractions during infection. we also provide new evidence in support of a phase of vsv gene expression that is dependent on eif e through the use of a viral mutant partially defective in guanine-n methylation [ ] and by the suppression of cellular pools of eif e. when eif e levels are suppressed -fold, we unmask a defect in viral protein synthesis in cells infected with vsv-vsv-l g a a mutant with a -fold defect in methylation at the guanine-n -position of the cap-structure. we suspect that this significantly underestimates the eif e dependent phase of viral replication since transformed cell lines, like the hela cells used here, have higher constitutive levels of eif e [ ] . our findings are consistent with a model where viral mrnas initially compete with cellular mrnas and translate in an eif e dependent manner. as infection progresses and the shut-down of host transcription, mrna export and eif e sequestration continue, the process of initiation is increasingly less dependent on eif e. the mechanism by which the viral mrnas become less dependent on eif e remains uncertain, but earlier studies demonstrate that neither the or utr of viral mrnas facilitate this efficient translation. ongoing transcription of viral mrna from the viral genome has also been linked to efficient protein synthesis [ , ] . whether this reflects the fact that the virus is an efficient producer of mrna that supports the competition model, or whether there is a temporal requirement for continued viral mrna synthesis is uncertain. as obligate intracellular parasites, viruses depend upon host cell functions for their replication. our sequence analysis provides evidence that vsv infection differentially impacts the polysome association of cellular mrnas. several host mrnas increased in polysome association include genes with known "proviral" functions for entry and replication including heparan sulfate, clathrin, and hsp [ , [ ] [ ] [ ] . similarly, host mrnas with decreased polysome association included genes with published roles in restricting vsv replication such as interferon regulatory factor (irf ), ddit , and txnip [ , , ] . it is difficult to definitively determine whether this reflects evolution of the virus to contend with the environment in which it finds itself or a bona-fide pro and antiviral effect of a given host protein. our efforts to address this are confounded by the essential house-keeping nature of many of the proteins encoded by host mrnas that remain polysome associated. an example of this is provided by enhanced polysome association of eif a on vsv infection-a protein that is required for assembly of the multisubunit eif complex. that complex also includes eif d which has demonstrated cap-binding ability, and directs eif e-independent translation of select mrnas [ ] [ ] [ ] [ ] . depletion of eif a, however, resulted in an equivalent reduction in the rates of viral and host translation-inconsistent with a specialized need for eif components in vsv protein synthesis. in this study, we validated that the effect of vsv infection on the polysome association of host mrnas-hsp and ybx -had a concordant impact on protein synthesis. although synthesis of hsp did not increase per se, this is likely explained by the shifting of many cellular mrnas towards smaller polysomes. this finding highlights the fact that the designation of rnas as having "increased" or "decreased" polysome association is imprecise, and reflects the complexities of how any given host gene is regulated. nevertheless, the general finding that mrnas with "increased" polysome association on vsv infection are typically larger, have longer half-lives and higher au content-features that are shared with the viral mrna-highlights commonalities among mrnas that remain polysome-associated and thus are more efficient in competing for ribosomes during host shut-off. the cellular mrnas that exhibit reduced translation efficiency were shorter, have shorter half-lives and higher gc content. although we validated changes in translatability and differential impacts on viral gene expression for a few cellular genes, it would be of significant interest to perform stable isotope labelling by amino acids in cell culture (silac) to non-radioactively label newly synthesized cellular proteins, quantify them on a genome-wide scale and correlate those data with the rnaseq results presented here. in addition to suppression of host translation through mrna synthesis and eif e manipulation, the vsv matrix protein inhibits host rna polymerase ii transcription [ , ] , and blocks nuclear export of mature mrnas through complex formation with the nuclear pore components rae and nup [ ] [ ] [ ] [ ] [ ] . a well characterized viral mutant (vsv-m m r ) fails to interact with the nuclear pore complex and exhibits a delayed kinetics of host shut-off [ , , ] . a similar analysis to that described here of cells infected with such a mutant may help delineate the extent to which ongoing synthesis and export of cellular mrna impacts host cell shut-off. we anticipate that over the time course of vsv infection, the contribution of ongoing synthesis and export of host mrna from the nucleus will result in a relatively modest increase in the fraction of the cytoplasmic mrna that is cellular. this study highlights a strategy shared among distinct viruses to commandeer the host translational machinery by outcompeting cellular mrnas. precisely where the tipping point between viral and host mrna levels with respect to this shut-off occurs is uncertain. for vsv, a viral mutant that makes less mrna than the wild type yet still exhibits host cell shut-off suggests that shut-off can be achieved with less than the % of total cytoplasmic mrna observed here [ ] . additional work will be required to define whether a specific tipping point exists and how this is influenced by other viral strategies such as eif e suppression or blocking host gene transcription. hela cells (a gift from james hogle) were maintained in dulbecco's modified eagle medium (dmem; invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs; tissue culture biologicals, tulare, ca). viral stocks were grown on syrian golden hamster kidney bsrt cells (a gift from k. conzelmann), and purified on linear - % sucrose gradients prepared in nte ( mm tris ph . , mm nacl, mm edta). viral titers were determined by plaque assay on african green monkey kidney vero cells (atcc, ccl- ). for infections, cells were first washed with hanks' balanced salt solution (hbss) and incubated with virus for hour at ˚c in serum free medium, washed with hbss and subsequently incubated with medium supplemented with % fbs. for polysome profiling, hela cells were treated with dmem containing μg ml - cycloheximide at ˚c for minutes. cells were washed twice with x ice-cold phosphate buffered saline (pbs) containing μg ml - cycloheximide, and kept on ice or at ˚c. cells were scraped into a . ml microcentrifuge tube in x pbs with μg ml - cycloheximide, and pelleted at × g for minutes. cells were resuspended in μl of a hypotonic buffer of mm tris (ph . ), . mm mgcl , . mm kcl, and rnasin (promega, madison, wi), supplemented with cycloheximide to μg ml - and dl-dithiothreitol (dtt) to μm. the detergents triton x- . % (vol/vol) and sodium deoxycholate . % (wt/vol) were then added sequentially, cells were briefly vortexed, and incubated for minutes on ice and clarified by centrifugation at , × g for min. polysomes were separated on sucrose gradients prepared on a gradient master station (biocomp, fredericton, canada) using % and % (wt/ vol) sucrose dissolved in mm tris (ph . ), mm mgcl , and mm nacl supplemented with rnasin and μg ml - cycloheximide. following centrifugation for hours at , × g in a beckman coulter ultracentrifuge using an sw ti rotor, μl fractions were collected from the top of the gradient while monitoring absorbance at λ = nm on a gradient master station. rna was extracted from total cytoplasmic, polysome, or monosome fractions using ls trizol (invitrogen) according to the manufacturer's protocol. equal amounts of rna as determined by spectrophotometry using absorbance at nm on a nanodrop (thermo fisher, waltham, ma) were subject to library preparation using the illumina truseq vii rna library preparation kit (illumina, san diego, ca), and sequenced at the whitehead institute (cambridge, ma) on an illumina hiseq . reads were trimmed and mapped to the concatenated hg and vsv genomes using clc genomics workbench (qiagen, redwood city, ca). mapping parameters were as follows; mismatch cost , insertion cost , deletion cost , length fraction . , similarity fraction . , and a maximum of hits per read. raw sequence data is available from the ncbi sequence read archive (sra) under the primary accession code srp . transcripts per kilobase million (tpm) was calculated for genes with or more mapped reads in the cytoplasmic fraction of both uninfected and infected cells using the total number of mapped exon reads. to identify cellular mrnas that were potential targets for translational regulation in infected cells, we determined the tpm in the polysome fraction/tpm in the total cytoplasmic fraction for each individual mrna. this ratio was determined for uninfected and infected cells, and presented as the log fold change. gene ontology analysis was performed in r using goseq. utrs and cds sequences were downloaded from the ucsc table browser using "knowncanonical" mrna identifiers. non-protein coding rnas were excluded from the analysis. poly (a) tail length and mrna half-lives were from published data sets [ , ] . graphs and statistical analyses were performed in r using the "wilcox_test" statistical test, the "density" kernel density estimation, and "geom_boxplot" or "geom_density" functions in ggplot and cowplot. total rna was recovered from polysome fractions using ls trizol according to the manufacturer's protocol. rna ( ng) was annealed with oligo d(t) and reverse-transcribed using superscript iii reverse transcriptase (thermo fisher) at ˚c for hour. following digestion of the rna strand with rnasea and rnaseh for min at ˚c, reactions were diluted : for cellular gene-specific qpcr or : for viral gene-specific qpcr. quantitative pcr was performed using power sybr green (thermo fisher) and relative amounts determined by images of rvsv-egfp infected cells were acquired using a × objective on a nikon eclipse te microscope (nikon instruments, melville, ny) equipped with a spot rt se monochrome camera (diagnostic instruments, sterling heights, mi). for cytometry, cells were washed in hbss, trypsinized, fixed in % paraformaldehyde at ˚c for minutes and measured using a facscalibur (cytek development, freemont, ca). cytometry data was analyzed using flowjo (flowjo inc, ashland, or). for mean fluorescence intensity (mfi) we gated on live cells identified by forward and side-scatter. to measure the % infected cells we subtracted those cells that fell within the gate established from uninfected control cells. for luciferase assays, where indicated hela cells were transfected with sirna, and the medium replenished at h. cells were transfected h later with ng prl-cmv (promega), and activity measured h later. for viral driven luciferase reporters sirna transfected cells were infected at h and monitored h later. luciferase expression was measured in a spec-tramax l microplate reader using the appropriate reagents according to the manufacturer's instructions (promega, e and e ). for depletion of host factors by peptide-conjugated morpholinos (ppmos) approximately . x hela cells per well of a well plate were treated h later with μm of the indicated ppmo. at h post treatment, the media was replaced with fresh medium containing μm ppmo, and used for testing h later. cells were washed twice with ice-cold x pbs and lysed in rose lysis buffer consisting of mm tris-hcl (ph . ), mm edta, . % w/v sodium deoxycholate, and % v/v np- on ice for minutes. rose lysis buffer was supplemented with phosphatase inhibitor cocktail (sigma-aldrich, st. louis, mo) and halt protease and phosphatase inhibitor cocktail (thermo fisher) for detection of phospho-eif e-bp . lysates were clarified, protein input was normalized by bradford assay and proteins resolved on polyacrylamide gels- % for eif a and eif e or %, eif e-bp . proteins were transferred to nitrocellulose membranes for minutes, eif e and eif e-bp , or minutes, eif a, at v. membranes were blocked with odyssey blocking buffer in pbs (li-cor, lincoln, ne) for h at room temperature, and incubated overnight at ˚c with the following primary antibodies: rabbit anti-eif a (cell signaling, # ), rabbit anti-eif e (cell signaling, # ), rabbit anti- e-bp (cell signaling, # ), rabbit anti-phospho- e-bp ser (cell signaling, # ), rabbit anti-phospho- e-bp thr / (cell signaling, # ), mouse anti-actin (millipore, #mab ), mouse anti-actin (sigma, #a ). membranes were washed x with x pbs-t for minutes at room temperature, and incubated with the relevant secondary antibodies: goat anti-mouse irdye rd (li-cor, # - ) or goat anti-rabbit irdye cw (li-cor, # - ), for hour at room temperature. membranes were washed again and kept in x pbs, and scanned on an odyssey clx (li-cor). hela cells were starved in dmem lacking l-methionine (corning, # - -cl) for minutes, prior to addition of [ s] express protein labeling mix (perkin elmer, waltham, ma) at . mci ml - . cell lysates were prepared as described above and separated on a low-bis % polyacrylamide gel. the gel was dried for . h at ˚c in a vacuum gel drier and detected using a phosphoimager. quantitative analyses of band intensities was performed in image-quant tl v . (ge healthcare, marlborough, ma). for radioimmunoprecipitations, . x hela cells plated in cm dishes (corning) were starved of methionine for h at h post-plating, and labeled with [ s]-express for h. cells were washed twice with ice-cold x pbs, collected by scraping and subsequent centrifugation for minutes at ˚c and , × g and lysed in ml of mm tris (ph . ), mm nacl , mm edta, % v/v np , mm dtt, supplemented with pierce protease inhibitor (thermo fisher) on ice for minutes. protein input was normalized by bradford assay, sds was added to . %, and μl lysate was pre-cleared for h at ˚c on a nutator with μl prewashed pierce protein a agarose beads. protein a beads were pelleted by centrifugation for minutes at ˚c and , × g and the labeled supernatant incubated with primary antibody at ˚c overnight. the antibodies used for immunoprecipitation were μg anti-yb (abcam, #ab ) and μg anti-hsp (enzo, #adi-spa- ). immune complexes were isolated using μl pre-washed pierce protein a agarose beads, by incubating for hours at ˚c. bead complexes were collected by centrifugation for minutes at ˚c and , × g, washed x with μl ice-cold ip lysis buffer with . % sds, on an orbital shaker for minutes at room temperature. protein-antibody complexes were eluted by boiling in x sds loading buffer, the beads pelleted for minutes at ˚c in a microcentrifuge and the supernatant loaded on a % polyacrylamide gel. after electrophoresis the gel was dried and imaged using a phosphoimager. approximately . x hela cells were plated per well of a -well dish and hours later incubated with phosphate free media (gibco, - ) for h. thirty minutes before infection, actinomycin d-mannitol (sigma, #a ) was added to a final concentration of μg ml - . infections were carried out in phosphate free media supplemented with μg ml - actinomycin d and . μm -dmag. at hpi cells were washed, fresh medium added, and supplemented h later with [ p]-orthophosphoric acid (perkin elmer, #nex h) . μci μl - . cells were harvested at hpi in rose lysis buffer, and total rna was extracted using ls trizol. rna was separated on a m urea-agarose gel as previously described, and detected using a phosphoimager [ ] . vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo the role of vesicular stomatitis virus matrix protein in inhibition of host-directed gene expression is genetically separable from its function in virus assembly effect of vesicular stomatitis virus matrix protein on transcription directed by host rna polymerases i, ii, and iii vesiculoviral matrix (m) protein occupies nucleic acid binding site at nucleoporin pair (rae * nup ) inhibition of ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus the matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup vsv disrupts the rae / mrnp mrna nuclear export pathway vesicular stomatitis virus infection alters the eif f translation initiation complex and causes dephosphorylation of the eif e binding protein e-bp inhibition of host and viral translation during vesicular stomatitis virus infection. eif is responsible for the inhibition of viral but not host translation the mechanism of eukaryotic translation initiation: new insights and challenges quantitative studies of mrna recruitment to the eukaryotic ribosome dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis b and t virions ribonucleic acid synthesis of vesicular stomatitis virus. . multiple complementary messenger rna molecules polysomal ribonucleic acid of vesicular stomatitis virus-infected hela cells translation of vesicular stomatitis messenger rna by extracts from mammalian and plant cells in vitro synthesis of methylated messenger rna by the virionassociated rna polymerase of vesicular stomatitis virus translation and identification of the mrna species synthesized in vitro by the virion-associated rna polymerase of vesicular stomatitis virus methylated and blocked ' termini in vesicular stomatitis virus in vivo mrnas ribonucleic acid synthesis of vesicular stomatitis virus, ii. an rna polymerase in the virion l protein requirement for in vitro rna synthesis by vesicular stomatitis virus transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus rna polymerase amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity a single amino acid change in the l-polymerase protein of vesicular stomatitis virus completely abolishes viral mrna cap methylation a unique strategy for mrna cap methylation used by vesicular stomatitis virus unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping rebinding of transcriptase components (l and ns proteins) to the nucleocapsid template of vesicular stomatitis virus location of the binding domains for the rna polymerase l and the ribonucleocapsid template within different halves of the ns phosphoprotein of vesicular stomatitis virus structure of the vesicular stomatitis virus nucleoprotein-rna complex structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor molecular architecture of the vesicular stomatitis virus rna polymerase critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus synthesis of poly(a) in vitro by purified virions of vesicular stomatitis virus in vitro synthesis of rna that contains polyadenylate by virion-associated rna polymerase of vesicular stomatitis virus site on the vesicular stomatitis virus genome specifying polyadenylation and the end of the l gene mrna aberrant polyadenylation by a vesicular stomatitis virus mutant is due to an altered l protein cis-acting signals involved in termination of vesicular stomatitis virus mrna synthesis include the conserved auac and the u signal for polyadenylation translational control of protein synthesis after infection by vesicular stomatitis virus vesicular stomatitis virus mrna and inhibition of translation of cellular mrna-is there a p function in vesicular stomatitis virus? effect of intracellular vesicular stomatitis virus mrna concentration on the inhibition of host cell protein synthesis complete intergenic and flanking gene sequences from the genome of vesicular stomatitis virus complete sequences of the ribosome recognition sites in vesicular stomatitis virus mrnas: recognition by the s and s complexes translation of mrnas from vesicular stomatitis virus and vaccinia virus is differentially blocked in cells with depletion of eif gi and/or eif gii a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas preferential translation of vesicular stomatitis virus mrnas is conferred by transcription from the viral genome high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling a systematic view on influenza induced host shutoff ribosome profiling reveals translational upregulation of cellular oxidative phosphorylation mrnas during vaccinia virus-induced host shutoff localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription transcriptional mapping of vesicular stomatitis virus in vivo order of transcription of genes of vesicular stomatitis virus determination of molar ratios of vesicular stomatitis virus induced rna species in bhk cells genome-wide determination of rna stability reveals hundreds of short-lived noncoding transcripts in mammals tail-seq: genome-wide determination of poly(a) tail length and ' end modifications antiviral activity and rna polymerase degradation following hsp inhibition in a range of negative strand viruses hsp inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus hsp chaperoning in addition to phosphoprotein required for folding but not for supporting enzymatic activities of measles and nipah virus l polymerases inhibition of heat shock protein hsp -pp v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation crystal structure and molecular modeling of -dmag in complex with human hsp txnip potentiates redd -induced mtor suppression through stabilization of redd chemical inhibition of rna viruses reveals redd as a host defense factor apobec g-regulated host factors interfere with measles virus replication: role of redd and mammalian torc inhibition separate pathways of maturation of the major structural proteins of vesicular stomatitis virus biophysical studies of eif e cap-binding protein: recognition of mrna ' cap structure and synthetic fragments of eif g and e-bp proteins an overlapping protein-coding region in influenza a virus segment modulates the host response selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein characterization of a second vaccinia virus mrna-decapping enzyme conserved in poxviruses vaccinia virus d protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression malignant transformation by a eukaryotic initiation factor subunit that binds to mrna ' cap new mrnas are preferentially translated during vesicular stomatitis virus infection pathway of vesicular stomatitis virus entry leading to infection entry pathway of vesicular stomatitis virus into different host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization systematic identification of type i and type ii interferoninduced antiviral factors eif targets cell-proliferation messenger rnas for translational activation or repression ' utr m( )a promotes cap-independent translation eif d is an mrna cap-binding protein that is required for specialized translation initiation dynamic m( )a mrna methylation directs translational control of heat shock response ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis rna molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination we thank members of the whelan lab for scientific discussions and suggestions; we further thank carl novina and cailin joyce for help with polysome profiling, keith ketterer for assistance with clc genomics workbench, and alos diallo for help with r and statistical analyses. we thank david stein and hong moulton, of oregon state university, for design and production of the ppmos used in this study. key: cord- -scc wee authors: to, kelvin kai-wang; tsang, owen tak-yin; leung, wai-shing; tam, anthony raymond; wu, tak-chiu; lung, david christopher; yip, cyril chik-yan; cai, jian-piao; chan, jacky man-chun; chik, thomas shiu-hong; lau, daphne pui-ling; choi, chris yau-chung; chen, lin-lei; chan, wan-mui; chan, kwok-hung; ip, jonathan daniel; ng, anthony chin-ki; poon, rosana wing-shan; luo, cui-ting; cheng, vincent chi-chung; chan, jasper fuk-woo; hung, ivan fan-ngai; chen, zhiwei; chen, honglin; yuen, kwok-yung title: temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: scc wee background: coronavirus disease (covid- ) causes severe community and nosocomial outbreaks. comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus (sars-cov- ) are not yet available. nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. we aimed to ascertain the serial respiratory viral load of sars-cov- in posterior oropharyngeal (deep throat) saliva samples from patients with covid- , and serum antibody responses. methods: we did a cohort study at two hospitals in hong kong. we included patients with laboratory-confirmed covid- . we obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. serial viral load was ascertained by reverse transcriptase quantitative pcr (rt-qpcr). antibody levels against the sars-cov- internal nucleoprotein (np) and surface spike protein receptor binding domain (rbd) were measured using eia. whole-genome sequencing was done to identify possible mutations arising during infection. findings: between jan , , and feb , , patients were screened for inclusion, of whom were included (median age years [range – ]). the median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was · log( ) copies per ml (iqr · – · ). salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope − · , % ci − · to − · ; r( )= · ). in one patient, viral rna was detected days after symptom onset. older age was correlated with higher viral load (spearman's ρ= · , % ci · – · ; p= · ). for patients with serum samples available days or longer after symptom onset, rates of seropositivity were % for anti-np igg (n= ), % for anti-np igm (n= ), % for anti-rbd igg (n= ), and % for anti-rbd igm (n= ). anti-sars-cov- -np or anti-sars-cov- -rbd igg levels correlated with virus neutralisation titre (r( )> · ). no genome mutations were detected on serial samples. interpretation: posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. unlike severe acute respiratory syndrome, patients with covid- had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. this finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. serological assay can complement rt-qpcr for diagnosis. funding: richard and carol yu, may tam mak mei yin, the shaw foundation hong kong, michael tong, marina lee, government consultancy service, and sanming project of medicine. coronavirus disease , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), was first reported from china in december, . although middle east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome coronavirus (sars-cov) infections have a higher mortality rate than does covid- , sars-cov- spreads much more rapidly than mers-cov and sars-cov. reliable data for profiles of serial viral load and serum antibody responses are needed urgently to guide antiviral treatment, infection control, epidemiological measures, and vaccination. the peak viral load of patients with mers-cov and sars-cov infections occurs at around - days after symptom onset, which could be associa ted with nosocomial outbreaks involving healthcare workers. , clinical studies of antiviral agents for sars showed that the viral load decreased significantly with treatment success. no systematic study of these two important variables with statistical analysis has been done for sars-cov- , although preliminary descriptive studies have been reported. [ ] [ ] [ ] in most studies of respiratory virus infections, serial sampling of nasopharyngeal or throat swabs is used for viral load monitoring. however, collection of nasopharyngeal or throat swab specimens can induce coughing and sneezing, which generates aerosol and is a potential health hazard for health-care workers. collection of throat swabs also requires direct inspection of the patient's posterior pharynx and tonsils. furthermore, collection of nasopharyngeal specimens is a relatively invasive procedure, which is uncomfortable and can even induce bleeding. a patient's reluctance to provide a sample can account for the paucity of timepoints in viral load studies of respiratory virus infections. findings of previous studies have shown high concordance between saliva and nasopharyngeal aspirate as specimens for laboratory diagnosis of respi ratory viruses. we have reported use of posterior oro pharyngeal (deep throat) saliva for diagnosis and viral load monitoring in a cohort of patients with covid- . here, we report use of self-collected posterior oro pharyn geal saliva samples from patients with covid- , which avoids close contact between healthcare workers and patients, for viral load monitoring. we also monitored serial serum antibody levels of patients. we included consecutive patients with laboratory confirmed covid- who were admitted to princess margaret hospital and queen mary hospital in hong kong. in hong kong, patients were tested for sars-cov- based on clinical and epidemiological criteria as outlined and updated by the hospital authority. initial laboratory confirmation was done using nasopharyngeal or sputum specimens at the public health laboratory centre of hong kong. we excluded patients if archived saliva or serum samples were insufficient for testing. this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw - ). since archived specimens were used, written informed consent was waived. of patients included in this study have been reported previously, but their clinical information, viral load by single copy rna-dependent rna polymerasehelicase gene, antibody response, or viral genome analysis has not been reported before. for viral load monitoring, all patients were asked to produce an early morning saliva sample from the posterior oropharynx (ie, coughed up by clearing the throat) before toothbrushing and breakfast, because naso pharyn geal secretions move posteriorly and broncho pulmonary secretions move by ciliary activity to the posterior oro phar yn geal area while the patients are in a supine position during sleep. patients were instructed and supervised by nurses. viral transport medium was added to the saliva specimen. if patients were intubated, we obtained endotracheal aspirate instead of posterior oropharynx saliva. , [ ] [ ] [ ] our initial experience showed that such saliva samples are promising in viral load monitoring in patients with covid- . we also retrieved serum remnant from blood samples taken for routine bio chemical testing, and refrigerated these samples at - °c until antibody testing could be done. we recorded clinical findings in a predesigned database, including a patient's history and physical examination and findings of haematological, biochemical, radiological, and microbiological investigations. we defined severe disease as the need for supplemental oxygen, admission to the intensive care unit (icu), or death. evidence before this study we searched pubmed on feb , , with no limitations by starting date, with the terms "covid- ", "coronavirus", "antibody", and "viral load"; we restricted our search to articles published in english. our search did not retrieve any reports on clinical progression of coronavirus disease (covid- ) with respect to temporal viral load and concomitant serum antibody profiles. we identified one correspondence piece on viral load with no statistical analysis, and another article with a few cases of antibody response. we present findings of an observational cohort study of the temporal profile of viral load of severe acute respiratory syndrome coronavirus (sars-cov- ) from posterior oropharyngeal saliva samples and serum antibody responses, dated by symptom onset and correlated with clinical findings. salivary viral load was highest during the first week after symptom onset and subsequently declined with time. eia of igg and igm against internal viral nucleoprotein (np) and surface spike protein receptor binding domain (rbd) showed correlation between antibody response and neutralising antibody titre. posterior oropharyngeal saliva specimens are non-invasive and acceptable to patients and can be used for initial diagnosis and subsequent viral load monitoring of covid- . the early peaking of viral load has important implications for transmission of sars-cov- in the community and hospital settings. eia of igg and igm against internal viral np and surface spike protein rbd can be used for those with delayed presentation or retrospective diagnosis of mild cases. as the positive eia antibody level correlates well with neutralising antibody titre, further studies on its role in immunopathology or antiviral therapy are warranted. we did in-house reverse transcriptase quanti tative pcr (rt-qpcr) targeting the sars-cov- rna-dependent-rna-polymerase-helicase gene region, as described (appendix p ). we did eias for sars-cov- nucleo protein (np) and spike protein receptor binding domain (rbd), as described but with modifications. recombinant np and spike protein rbd of sars-cov- were used for the eias. we assessed the purity of np and rbd by sodium dodecyl sulphate polyacrylamide gel electro phor esis and western blotting (figure a, b; appendix pp - ). a positive sample was included in each run as a positive control. we used an archived anonymous sample from as a negative control. the cutoff for seropositivity was set as the mean value of anonymous archived serum specimens from , plus sds. we verified the validity of eias by competitive eia (appendix p ) and by western blotting, using patients' serum samples (figure c, d; appendix p ). we did microneutralisation assays and virus culture, as described (appendix pp - ). , we did whole-genome sequencing using the oxford nanopore minion device (oxford nanopore technologies, oxford, uk), as described (appendix p ). we did statistical analyses using spss version . or prism version . . we compared categorical variables using fisher's exact test and continuous variables with the mann-whitney u test. we used spearman's correlation to assess the relation between age and viral load. a p value less than · was judged statistically significant. the funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication. between jan , , and feb , , patients were screened for inclusion, of whom were included ( male and ten female). ten patients had severe covid- , of whom all required oxygen supplementation, and patients had mild disease. the median age of patients was years (range - ). ( %) of patients had chronic medical illnesses, and the most common under lying diseases were hypertension in six ( %) patients and diabetes in four ( %). chronic comorbidities were more common among patients with severe covid- (seven [ %] patients with severe disease had chronic comorbidities vs four [ %] with mild disease), although this difference was not significant (table). five patients were admitted to the icu, including three who required intubation. two patients died. the median interval between symptom onset and hospitalisation was days (range - ). on presentation, the most common symptom was fever in patients ( %), followed by cough in five ( %), chills in four ( %), and dyspnoea in four ( %; table). dyspnoea was significantly more frequent among the ten patients with severe disease than among those with mild disease (four [ %] of ten vs none [ %] of ; p= · ). serum alkaline phosphatase was significantly higher among patients with severe disease than among those with mild disease ( u/l [range - ] vs u/l [ - ]; p= · ). the lymphocyte count was lower among patients with severe disease than among those with mild disease ( · × ⁹ cells per l [range · - · ] vs · [ · - · ]), but this difference was not significant (p= · ). lymphopenia and neutrophilia were present in a higher proportion of patients with severe disease than in those with mild disease, but the differences were not significant (p= · and p= · , respectively). chest radiographic abnormalities were seen in ( %) patients. multifocal ground-glass lung opacities were seen in ( %) patients on ct. sars-cov- rna was detected in blood samples in five ( %) patients and rectal swabs in four ( %), but the detection rate between severe and mild cases did not differ (p= · and p= · , respectively; table). sars-cov- rna was not detected in any urine specimens. lopinavir-ritonavir with or without ribavirin or interferon beta b was given in ( %) patients at different timepoints after symptom onset. in total, respiratory specimens were obtained from pati ents (mean · respiratory specimens per patient). the median viral load at presentation was · log copies per ml (iqr · - · ). sars-cov- rna was not detected in the saliva of three ( %) patients. specimens with undetectable viral load were assigned a value of log copies per ml. no correlation was noted between days after symptom onset and initial viral load (spearman's ρ= · ; p= · ). the viral load in posterior oropharyngeal saliva samples was highest during the first week of symptom onset then gradually declined (slope - · , % ci - · to - · ; r²= · ; figure ). endotracheal aspirate viral load was available from day after symptom onset and showed a non-significant decline (slope - · , % ci - · to · ; r²= · ). of the patients who survived, seven ( %) had viral rna detected for days or longer after symptom onset. no association was seen between prolonged detection of viral rna (≥ days after symptom onset) and severity of illness (p= · ). one patient had viral rna detected for up to days after symptom onset; another patient had undetectable viral load on days and after symptom onset, with rebound of viral load on days and , followed by days of undetectable viral load. a significant positive correlation between age and peak viral load was noted (spearman's ρ= · , % ci · - · ; p= · ; figure a). the median initial (p= · ) and peak (p= · ) viral loads in severe cases were about log higher than those in mild cases, although the difference was not significant (figure b, c). the initial (p= · ) and peak (p= · ) viral loads did not differ between patients without comorbidities and those with comorbidities (figure d, e). for patients with both viral load and antibody results available in week or week , median viral load was · log copies per ml (range · - · ), and the concomitant median optical density (od) for anti-np igg was · (range · - · ) in week , whereas in week , median viral load was · log copies per ml (range · - · ) and the concomitant median od for anti-np igg was · (range · - · ). serum specimens were obtained from patients (mean · serum specimens per patient). an increase was noted in igg or igm antibody levels against np or rbd for most patients at days or later after symptom to assess for host factors that affect the antibody titre, the correlation was analysed between the highest od value during the convalescent period (third week after symptom onset) and age or comorbidities. patients with comorbidities had a lower anti-rbd igg od than did those without comorbidities, although the difference was not significant (median od, · vs · ; p= · ; appendix p ). no association was seen between comorbidity and anti-np igg or igm od values, or between age and anti-np igm or igg or anti-rbd igm or igg od values (appendix p ). specimens with microneutralisation assay titres less than were assigned a value of , and specimens with microneutralisation assay titres greater than were assigned a value of . for one patient, a microneutralisation antibody assay was done with ten serial samples. the correlation between micro neutralisation assay titres and anti-np igg (r²= · ) or anti-rbd igg (r²= · ) was better than those between microneutralisation assay titres and anti-np igm (r²= · ) or anti-rbd igm (r²= · ; figure ). nanopore sequencing was successful for paired samples from four patients. the interval between the first and second specimens was - days. no viral mutations were identified between paired samples from individual patients. we analysed the serial viral load, antibody kinetics, and viral genome of patients with covid- in hong kong. for most patients, the viral load of sars-cov- was very high at presentation and declined steadily. despite develop ment of antibodies against surface and internal proteins of sars-cov- , viral rna could still be detected in posterior oropharyngeal (deep throat) saliva samples from a third of patients for days or longer. peak viral load correlated positively with age. most patients had an antibody response at days or later after onset of symptoms. viral whole-genome sequencing of paired samples from four patients did not identify any single nucleotide poly morphisms. a high viral load on presentation of covid- was recorded in our cohort, even for patients who were hospitalised shortly after symptom onset. using nasal swab and throat swab, zou and colleagues have also reported a high viral load shortly after symptom onset. however, in that study, only cycle threshold values (not exact viral loads) were reported, and no statistical or correlative analysis was done with clinical variables such as age, comorbidities, disease severity, and antibody response. the viral load profile of sars-cov- is similar to that of influenza, which peaks at around the time of symptom onset, but contrasts with that of sars-cov at around days and that of mers-cov at the second week after symptom onset. , , the high viral load on presentation suggests that sars-cov- can be transmitted easily, even when symptoms are relatively mild. this finding could account for the efficient person-to-person transmission noted in community and health-care settings. clusters in families, workplaces, religious gatherings, and food premises have been widely reported. the viral load profile is important for guiding antiviral treatment. since viral load had already peaked around the time of hospital admission, the risk of emergence of antiviral resistance could be similar to that of single-drug treatment of influenza by adamantanes, acid polymerase inhibitors, and neuraminidase inhibitors. however, our previous clinical trial of influenza treatment showed that a triple antiviral combination could significantly improve the clinical outcome and viral load profile and could reduce emergence of resistant virus quasispecies. currently, no standard treatment is available for figure : relation between viral load and age or disease severity correlation between age and peak viral load (a). comparison of initial (b) and peak (c) viral load between severe and mild cases. comparison of initial (d) and peak (e) viral load between patients with comorbidities and those without comorbidities. covid- . for sars-cov infection, our previous treatment study showed that a combination of lopinavirritonavir and ribavirin led to significantly fewer compli cations (eg, acute respiratory distress syndrome) or deaths than reported with historical controls treated with ribavirin. lopinavir-ritonavir or interferon beta b also reduced lung damage and decreased viral load in a nonhuman primate model of mers-cov. lopinavir is a protease inhibitor with in-vitro activity against sars-cov and mers-cov. however, the idea that sars-cov c-like protease was the antiviral target of lopinavir was based purely on binding in computational modelling. other protease inhibitors and nucleotide analogues (eg, remdesivir [gilead sciences, foster city, ca, usa]) are potential candidates for treatment. combination treatment with virus-targeting and host-targeting agents to improve clinical outcome should be investigated. studies for sars-cov have shown that a high initial viral load was associated with death. however, our study only showed that the median viral load was log higher in severe cases than in mild cases, and the difference was not significant. but, older age was associated with a higher peak viral load. in a previous study of patients infected with sars-cov, older age was an independent factor associated with higher viral load, as expected for immunosenescence, which impairs our innate and adaptive immune responses. sars-cov- rna could be detected for days or longer in a third of patients who survived in our cohort, and one patient had sars-cov- rna detected for days. prolonged detection of viral rna of days or longer was also commonly seen for patients with mers-cov or sars-cov infections. , prolonged detection of viral rna represents a challenge for the limited availability of hospital isolation facilities because patients might not be discharged until viral rna is undetectable in respiratory specimens. further studies are warranted to ascertain whether patients are shedding live virus, by viral culture of the prolonged rt-pcr-positive specimens obtained from patients with concomitant seropositivity when shedded virions are coated with host antibodies which render them non-infectious. a criterion for discontinuation of transmission-based precautions is a negative rt-qpcr result from two sets of nasopharyngeal and throat swab specimens. in the current study, one patient with complete symptom resolution tested positive for sars-cov- again after days of negative findings. our results suggest that sars-cov- might be excreted at low levels despite clinical recovery. thus, both serial viral load monitoring and antibody response should be considered when making decisions about infection control measures, because viral load seemed to be related inversely to serum antibody response in this study. the antibody profile is vital for timing requests for serological assays and interpretation of antibody test results. serological diagnosis is important for patients who present late with a very low viral load, below the detection limit of rt-pcr assays. because most patients have rising antibody titres days after symptom onset, collection of serial serum samples in the convalescent phase would be more useful. serum igg amounts can rise at the same time or earlier than those of igm against sars-cov- . by comparison with findings of a study on igm and igg eia, in which more patients were seropositive for igg than igm at day and day of hospital admission, a higher anti-np igg anti-np igm proportion of patients in the current study also had earlier igg than igm seroconversion. however, this finding could also be accounted for by a lower sensitivity of the igm eia, which warrants investi gations with more patients. serum antibody levels were not correlated with clinical severity. notably, one patient with severe disease had an early antibody response days after symptom onset. deceased patients infected with sars-cov developed faster peak anti-spike antibody responses when compared with patients who recovered and had subsequent reduced b-cell immunity with impaired neutralising ability. in a sars-cov macaque model, anti-spike igg stimulated pulmonary proinflammatory responses and caused acute lung injury. the detrimental effect of antispike igg was attributable to the effect on wound-healing macrophages, which was mediated via the fcγ receptor. our findings showing correlation between antibody level detected by eia and virus neutralisation titre are especially important for design of vaccine studies, and use of convalescent plasma or therapeutic monoclonal antibodies, which could improve clinical outcome or paradoxically cause immuno pathological damage to the recipient. whole-genome sequencing on paired samples from four patients was successful and showed no differences in individually paired genome sequences. however, single nucleotide polymorphisms were shown to emerge during hospitalisation for mers-cov infection, using a targeted sequencing approach. further studies in more patients with samples obtained at longer intervals could be more informative. a high viral load in throat wash and saliva (up to ⁸ copies per ml of sars-cov rna) was reported in patients with sars. in a chinese macaque model of sars-cov, salivary gland ducts were early targets of sars-cov and, therefore, were a likely source of the virions found in patients' saliva, particularly early in infection. because of these important findings, our study used posterior oropharyngeal saliva brought up by a throat-clearing manoeuvre to ascertain the temporal viral load profile. the posterior oropharynx is the meeting point between secretions coming from the posterior nasopharynx and the salivary glands and respiratory secretions swept up from the tracheal-bronchial tree. testing of saliva could show viral shedding from both the salivary glands and the upper and lower respiratory tract. moreover, because of greater patient acceptability for posterior oropharyngeal saliva samples than for nasopharyngeal or throat swabs, we obtained · respiratory specimens per patient for testing. thus, our temporal viral load profile can be analysed by statistics, unlike previous clinical studies of viral kinetics of infections by highly pathogenic betacoronaviruses. , further studies are needed to ascertain whether the salivary glands can be infected by sars-cov- . our study has several limitations. first, we could only include a few patients, and viral load and antibody titre data were not available everyday. this limitation is a common problem in studies of emerging infections such as sars-cov and mers-cov. the few patients enrolled does not allow for adjustment for potential confounding factors that could affect viral load or antibody response. second, % of patients enrolled had chronic medical illness, which is a higher proportion than that reported in a large clinical series ( %). although a lower anti-rbd igg level was noted among patients with comorbidities, further studies are warranted with more patients. third, posterior oropharyngeal saliva samples cannot differentiate whether the virus is coming from the nasopharynx or from secretions from the lower respiratory tract; thus, our study cannot indicate whether sars-cov- has a predilection for both upper and lower respiratory tract. moreover, some patients might not clear the throat effectively to cough out saliva from deep in the throat, which could decrease test sensitivity when compared with that of nasopharyngeal swabs, particularly in patients with predominant upper respiratory involvement or mild symptoms. finally, the most abundantly expressed internal np might have some cross-antigenicity between sars-cov- and sars-cov ( % amino acid homology) and, occasionally, oc -cov ( % amino acid homology). thus, the less abundantly expressed surface spike protein rbd, which is specific for sars-cov- and is the direct target for neutralising antibodies, was used to guard the specificity of our dual antibody assays. covid- is an emerging infection with many unknowns. this study has shed light on viral kinetics and antibody response in patients and provides scientific evidence for guiding infection control policies and therapeu tics. further virological and immunological studies are needed to understand sars-cov- infection; infection control measures should be reviewed with the rapidly evolving epidemiology of covid- . kk-wt and k-yy contributed to study design, data collection, data analysis, data interpretation, the literature search, and writing of the report. ot-yt, w-sl, art, t-cw, dcl, jm-cc, ts-hc, dp-ll, cy-cc, vc-cc, jf-wc, and if-nh contributed to patients' recruitment, data collection, and clinical management. cc-yy, j-pc, l-lc, w-mc, k-hc, jdi, ac-kn, rw-sp, c-tl, zc, and hc contributed to the experiments, data collection, data analysis, and data interpretation. all authors reviewed and approved the final version of the report. we declare no competing interests. a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings consistent detection of novel coronavirus in saliva a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov- viral load in upper 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multiple shedding routes antibody responses against sars coronavirus are correlated with disease outcome of infected individuals anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection analysis of intrapatient heterogeneity uncovers the microevolution of middle east respiratory syndrome coronavirus detection of sars-associated coronavirus in throat wash and saliva in early diagnosis epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques clinical characteristics of coronavirus disease in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses e and oc we thank wallace wong, charlotte yee-ki choi, and travis law for technical assistance and data collection. key: cord- -nao qx authors: wargo, andrew r; kurath, gael title: viral fitness: definitions, measurement, and current insights date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: nao qx viral fitness is an active area of research, with recent work involving an expanded number of human, non-human vertebrate, invertebrate, plant, and bacterial viruses. many publications deal with rna viruses associated with major disease emergence events, such as hiv- , influenza virus, and dengue virus. study topics include drug resistance, immune escape, viral emergence, host jumps, mutation effects, quasispecies diversity, and mathematical models of viral fitness. important recent trends include increasing use of in vivo systems to assess vertebrate virus fitness, and a broadening of research beyond replicative fitness to also investigate transmission fitness and epidemiologic fitness. this is essential for a more integrated understanding of overall viral fitness, with implications for disease management in the future. fitness is a complex concept at the foundation of all ecology and evolution. for viruses fitness was originally defined as ''the capacity of a virus to produce infectious progeny in a given environment'' [ ] . this definition is still in wide use today, referred to more specifically as replicative fitness [ ] , measured in cultured cells, tissue explants, or within individual hosts. this standard definition is founded in, but does not exactly match, the more general darwinian definition of overall fitness, which is the amount of genetic material passed on to the next generation. due to host immune clearance of viruses and finite host lifespan, viruses must be transmitted to new hosts to survive. as such, transmission fitness is an important component of overall fitness. ultimately, replication and transmission contribute to the prevalence of viral genetic material at a population level in the field over time. the capacity of a virus (a serotype, clade, or variant) to become dominant in the field, relative to other serotypes, clades, or variants of the same virus has been defined as epidemiologic fitness [ ] . in this review we focus on viral fitness work published from through early . literature searches found nearly publications involving viral fitness during this period, and we have focused on a subset of papers selected to represent the current breadth of work in the field. in the interest of brevity these papers are not all cited here, but they were used to discern the general trends described below. the field of viral fitness was originally developed through studies of a relatively small number of bacteriophage, animal, and plant viruses. with increasing recognition of the importance of viral fitness, there is now a wide array of study systems as detailed in table . the majority of viral fitness study systems are based on rna viruses, and the highest numbers of publications in recent years involves human pathogens associated with major disease emergence events, such as human immunodeficiency virus- (hiv- ), influenza virus, and dengue virus (denv). during development of the viral fitness field most research has assessed replicative fitness of viral variants within individual hosts (in vivo) or in cultured cells (in vitro) . this trend has continued during recent years, likely due to the fact that replicative fitness is most readily measured in the laboratory. typically replicative fitness studies compare the replication of two or more virus isolates that are variants of the same viral species. under the general umbrella of replicative fitness there are many variations that will be described here and in later sections. replicative fitness is sometimes assessed by comparing viral replication in parallel hosts or cell cultures infected with single viral variants. however, it has long been recognized that assessment of fitness in mixed infections of two viral variants is a more sensitive and valid measure of viral fitness differences [ ] . therefore competitive fitness is often examined in growth competition assays, in which cells or hosts are co-infected with mixtures of two viral variants and the replication of each variant is determined in a competitive environment. variables in these assays include the use of different input ratios, use of a standard reference strain or head-to-head competitions between test variants, and varied timing for analysis of progeny populations. for example, replicative fitness is sometimes examined at one time point post-infection, but it is often assessed at multiple time points [ , , , , , , ] there are a variety of methods used to measure replicative fitness. the traditional method quantifies individual viral variants as plaque forming units (pfu). recent advances in molecular techniques have led to the use of methodologies that measure viral dna, rna, or protein levels, such as quantitative pcr, elisa, fluorescent probe labeling, and next generation sequencing. these molecular methods estimate viral load, and have the advantage that they typically allow for higher throughput as well as sensitivity, and can be used to distinguish viral genotypes in mixed infections. however, they do not quantify infectious virus as pfu assays do, and the relationships between pfu and molecular quantifications of viral load remain to be defined in many systems. viral fitness is expressed in various ways, such as comparing viral loads statistically [ ] , using a fitness parameter equation [ , , ] , or determining the slope of a fitness vector in comparison with a constant reference strain after multiple sampling times or serial passage [ ] . in the past fitness data were generally presented as relative fitness values, reported as a fitness ratio between two viral variants. the use of appropriate standards with qrt-pcr quantification has recently made it possible to determine absolute fitness values in terms of average rna copy numbers per mg of host tissue, for each viral variant [ ] . a major variable in replicative fitness work is the in vitro, ex vivo, or in vivo nature of the environments used for viral replication (figure ). for viruses of bacteria, insects, and plants there is a long record of sophisticated in vivo fitness studies using controlled laboratory populations of living hosts, and in vivo work in these systems has remained prolific to date [ , , , , , , , , , , , , ] . viruses of vertebrates have traditionally been studied in cultured cell lines, and this work also continues [ , , , , , , , , , , , , , , ] . however, over the last three years there has been an expansion of vertebrate virus fitness studies in vivo using systems such as influenza, denv, west nile virus (wnv), viral fitness wargo and kurath replicative fitness studies typically measure the average fitness of populations of multiple virus particles in populations of in vitro cultured cells or in vivo host tissues. an exciting recent advance has been analysis of the replicative fitness of individual virus particles, defined as the total number of virus progeny produced when one virus infects an individual susceptible cell [ ] . work with vesicular stomatitis virus (vsv) has revealed dramatic variation ranging from to progeny virus particles per host cell, with additional experiments indicating the host cell cycle stage as a major influence on this variability. another component of competitive co-infection fitness is superinfection fitness, in which infection with one viral variant is established before exposure to the second variant. despite the clear relevance of superinfection to natural viral infections in the field, there are few studies of controlled superinfections [ ] . in general, viruses isolated from natural superinfections have been analyzed in simultaneous co-infection assays, often indicating that the superinfecting strains have higher fitness [ ] . due to the major role of transmission in overall viral fitness, transmission fitness is a research area that is currently expanding. the majority of work on transmission fitness has been conducted in plant viruses in many cases the ultimate goal of replicative and transmission fitness studies is to understand the epidemiology virus evolution and population level processes governing viral evolution, emergence, and displacement in the field. as such there has been an increased effort in recent years to quantify epidemiologic fitness. quantification of epidemiologic fitness is based largely on observational data and examines changes in distribution, prevalence, and composition of viral genotypes over time to infer their relative fitness. this is particularly valuable for systems where a wealth of epidemiological data is available, and the ability to conduct in vivo experimental studies is limited. it is of no surprise therefore that most recent work in this area was conducted in human virus systems such as hiv, in recent years there has been an increased effort to mathematically quantify viral fitness. this includes standard statistical package approaches as well as systemspecific mathematical model development. , and the evolutionary impact of incorporating new traits into the viral genome such as photosynthesis genes in a cyanophage [ ] . parameters in mathematical models are typically defined with in vivo laboratory and field data, and recent growth in these areas has made modeling approaches more tractable. exciting advances in the field of viral fitness have largely been driven by the rapid expansion in molecular technologies and computing power. for example molecular barcoding microarray technologies have been employed to create quasispecies swarms in the laboratory and simultaneously characterize the fitness of numerous mutants of poliovirus [ ] . next generation sequencing is also being used to rapidly sequence entire viral genomes and determine how genome wide mutation accumulation impacts fitness [ ] . likewise, large scale site directed mutagenesis has recently been used as a tool to determine the molecular interactions that regulate fitness [ ] . new bioinformatics tools are being employed to explore large hiv field sequence databases and determine the fitness landscapes [ ] , and we have entered the age of purely in silico studies that use advanced agent based mathematical models parameterized from published literature to make inferences about viral evolution [ ] . ultimately these advances have made understanding the genetic regulators of fitness within arms grasp, and not surprisingly, revealed that the story is likely to be more complex than previously assumed [ , ]. for vertebrate viruses the recent increase of in vivo virus fitness research is encouraging, but the majority of studies still remain in vitro. this is likely due to the ethical and practical constraints of conducting in vivo research in many vertebrate systems, particularly human viruses. furthermore in vitro work has numerous advantages, most notably it allows for a higher level of control of variables than in vivo work and is useful for understanding phenomena at a molecular or cellular level [ ] . the drawback is that it is unclear how well in vitro results reflect natural phenomena in vivo. the relationship between viral fitness and virulence has been of interest for decades, but surprisingly little has been published recently. many recent publications assume that viral replicative fitness and virulence are positively correlated and thus use the terms interchangeably. for example, the term 'attenuation' is often used to describe a virus with reduced replicative fitness [ ] . in actuality, the relationship between viral fitness and virulence remains poorly characterized in many systems, and both agreement and exception to this assumption have been reported [ , , , , , , ] . some of the discrepancies in this area may come from the multitude of ways virulence is defined or measured, often beyond the standard definition of morbidity and mortality caused to the host due to infection [ , , - , , , ] . more focus on virulence, and definition of system-specific correlations of virulence with fitness would be a benefit to future work. viral fitness continues to be an active area of research [ ] . given the increase in issues such as drug resistance evolution, vaccine escape, virulence evolution, viral emergence, and host jumps, the understanding of viral fitness has become essential. for decades viral fitness has been primarily defined by replication capacity in the host. this definition is now broadening as researchers attempt to understand the population level evolutionary implications of overall viral fitness in natural infections. making inferences about population level processes requires integration of replicative, competitive, transmission, and epidemiologic fitness measures (figure ). perhaps the most critical need for the field of viral fitness is the further development of integrative approaches, with the ultimate goals of making accurate predictive inferences and informing long-term management or control of disease. many the tools to achieve this goal are now available, and we are collectively faced with the task of putting them together. a mathematical framework for estimating pathogen transmission fitness and inoculum size using data from a competitive mixtures animal model. plos computational biology , : . the authors introduce a mathematical framework for quantitative estimation of the relative transmissibility of two viral types in co-infection in vivo, and the inoculum size associated with transmission events. the model is tested using data from in vivo influenza virus co-infection studies in ferrets, providing one of the first rigorous investigations of transmission fitness for a virus that is directly transmitted between vertebrate hosts (i.e. non-arbovirus). nguyen ah, molineux ij, springman r, bull jj: multiple genetic pathways to similar fitness limits during viral adaptation to a new host. evolution , : - . this study investigates the existence of fitness limits using a bacteriophage of salmonella being adapted to an e. coli host under varying conditions. although the nucleotide changes associated with adaptation differed dramatically, four independent lines achieved similar absolute fitness increases, demonstrating a fitness limit that could be attained by multiple genetic pathways. genomic evolution of vesicular stomatitis virus strains with differences in adaptability gag determinants of fitness and drug susceptibility in protease inhibitor-resistant human immunodeficiency virus type abiotic heterogeneity drives parasite local adaptation in coevolving bacteria and phages in vivo fitness correlates with host-specific virulence of infectious hematopoietic necrosis virus (ihnv) in sockeye salmon and rainbow trout distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f impact of mutations at residue i of the neuraminidase protein on the resistance profile, replication level, and virulence of the pandemic influenza virus fitness and virulence of different strains of white spot syndrome virus escape from human monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus high replication fitness and transmission efficiency of hiv- subtype c from india: implications for subtype c predominance oseltamivir-resistant variants of the pandemic h n influenza a virus are not attenuated in the guinea pig and ferret transmission models incongruent fitness landscapes, not tradeoffs, dominate the adaptation of vesicular stomatitis virus to novel host types mutation t s in hiv- subtype b and c proteases resensitizes them to ritonavir and indinavir and confers fitness advantage the role of evolutionary intermediates in the host adaptation of canine parvovirus the authors explored the mechanism of a documented host switch in canine parvovirus (cpv) by constructing viruses with all possible intermediate genomes and assessing their fitness in feline cells. they show that host adaptation involves complex interactions between mutations and most transition intermediates have lower fitness fitness-related traits of entomopoxviruses isolated from adoxophyes honmai (lepidoptera: tortricidae) at three localities in japan divergent evolution in reverse transcriptase (rt) of hiv- group o and m lineages: impact on structure, fitness, and sensitivity to nonnucleoside rt inhibitors estimating the individualized hiv- genetic barrier to resistance using a nelfinavir fitness landscape variable fitness impact of hiv- escape mutations to cytotoxic t lymphocyte (ctl) response high-throughput analysis of growth differences among phage strains analysis of infectious virus clones from two hiv- superinfection cases suggests that the primary strains have lower fitness point mutations in the west nile virus (flaviviridae; flavivirus) rna-dependent rna polymerase alter viral fitness in a hostdependent manner in vitro and in vivo in vitro characterization of viral fitness of therapy-resistant hepatitis b variants this is the first study to quantify how viral infection cycle traits correlate with viral fitness and virulence, using a fish rhabdovirus in rainbow trout as an in vivo system. although within-host replication had the largest impact on fitness, host entry, competitive fitness, and shedding also contributed a cooperative interaction between nontranslated rna sequences and ns a protein promotes in vivo fitness of a chimeric hepatitis c/gb virus b macaque long-term nonprogressors resist superinfection with multiple cd (+) t cell escape variants of simian immunodeficiency virus decreased infectivity of a neutralization-resistant equine infectious anemia virus variant can be overcome by efficient cell-to-cell spread growth of an rna virus in single cells reveals a broad fitness distribution mixed infections and the competitive fitness of faster-acting genetically modified viruses papers of particular interest, published within the period of review, have been highlighted as:of special interest of outstanding interest . abraha a, nankya il, gibson r, demers k, tebit : - . this paper provides an example of the sophistication of plant virus fitness work, using two groups of ten plant viral satellite rnas that differ in fitness and virulence on a tomato host. on a melon host these satellite rnas differ in multiple measures of viral fitness including replication in single or mixed infections and aphid transmissibility, but they do not differ in virulence, demonstrating both host-specific fitness traits and a lack of correlation between replicative fitness and virulence. da silva j, coetzer m, nedellec r, pastore c, mosier de: fitness epistasis and constraints on adaptation in a human immunodeficiency virus type protein region. genetics , -u . fitness epistasis was investigated here by creating seven mutations, singly and in combination, in hiv- glycoprotein and testing effects on viral infectivity. epistatic effects were found to be common, complex, and often very strong, providing insights into the barriers and probable pathways of evolution of co-receptor usage. key: cord- -smmhhroe authors: de armas‐rillo, laura; valera, maría‐soledad; marrero‐hernández, sara; valenzuela‐fernández, agustín title: membrane dynamics associated with viral infection date: - - journal: rev med virol doi: . /rmv. sha: doc_id: cord_uid: smmhhroe viral replication and spreading are fundamental events in the viral life cycle, accounting for the assembly and egression of nascent virions, events that are directly associated with viral pathogenesis in target hosts. these processes occur in cellular compartments that are modified by specialized viral proteins, causing a rearrangement of different cell membranes in infected cells and affecting the er, mitochondria, golgi apparatus, vesicles and endosomes, as well as processes such as autophagic membrane flux. in fact, the activation or inhibition of membrane trafficking and other related activities are fundamental to ensure the adequate replication and spreading of certain viruses. in this review, data will be presented that support the key role of membrane dynamics in the viral cycle, especially in terms of the assembly, egression and infection processes. by defining how viruses orchestrate these events it will be possible to understand how they successfully complete their route of infection, establishing viral pathogenesis and provoking disease. © the authors reviews in medical virology published by john wiley & sons, ltd. viruses are small structures that lack the metabolic pathways and structures necessary to ensure their own survival, relying on their host's machinery to replicate their genome and spread their progeny. accordingly, viruses have developed strategies to enter cells and exploit their structures to replicate. these strategies also serve to evade immune responses, such as those involving toll-like receptors and autophagic-mediated antigen presentation [ ] [ ] [ ] [ ] . similarly, viruses use the target cell's main trafficking pathways to ensure their propagation, exploiting the endosome or vesicular compartments by recruiting the clathrin, coatomer protein complex (copi) i and ii (figure ), the endosomal-sorting complex required for transport (escrt) and their accessory proteins (reviewed by [ , , ] : figure ), as well as small guanosine triphosphatases (gtpases) [ ] . this is evident during neutrophil-mediated phagocytosis, where microorganisms can be cleared by granule and vesicle secretion [ ] . therefore, determining how viruses use and rearrange intracellular organelles during their biological cycle is an important goal that will aid the development of new antiviral strategies, and our understanding figure . viral factories and virus-triggered autophagic membrane flux for replication and egression. some viruses achieve replication by exploiting the cell's membrane transport pathways, thereby generating membrane organelles named viral factories (vfs). these vfs are organised by different viral proteins, and they represent specialized compartments for viral-gene replication, morphogenesis, export, maturation and release. moreover, these compartments also serve to override or evade the immune responses directed against viral genomes. viral proteins can enter secretory pathways by co-translational translocation into the er in order for them to be further transported to the golgi complex, either in vesicles or in a coatomer protein complex (cop) ii-dependent manner. viral complexes formed inside the vfs communicate with vesicles, mitochondria, golgi cisternae and er membranes. this interaction allows viral complexes to be transported through the golgi network to the plasma membrane and it promotes their final release as viral particles. alternatively, some viruses take advantage of the host's autophagic machinery for their own replication and pathogenesis. viruses first initiate the formation of vesicles that bear key autophagic proteins, such as beclin- and lc , capturing portions of membranes from the er and other cytoplasmic elements. this assembly evolves toward an immature double-membrane vesicle (dmv) that serves as an aggresome compartment to recruit viruses or newly formed viral replication complexes. several rna viruses induce the formation of these autophagosome-like vesicles (also referred to as dmvs) to enhance viral replication and non-lytic egression, such as poliovirus and cvb , hiv- and hcv. how these viruses trigger the accumulation of autophagosome-like vesicles and dmvs remains unclear. some theories involve blocking the fusion of nascent autophagosomes with late endosomes and lysosomes, as in the case of hiv- nef, which appears to cause autophagosome accumulation by inhibiting their progression towards more mature stages. indeed, autophagosome-like vesicles may represent a trafficking pathway for these viruses, connecting to multivesicular bodies (mvbs), and assuring virus assembly and budding at the cell surface while protecting them from intrinsic antiviral factors and immune responses. the morphogenesis and release of mature and infectious hbv particles also require tsg and depend on the escrt-mvb system. under standard conditions the lumen of autophagosomes acidifies after fusion with endosomes that carry vacuolar (h + )-atpase (v-atpase) to form amphisomes. the autophagic membrane flux progresses by fusing with lysosomes in order to form the autolysosome that contains the former's proteinases. poliovirus inhibition of autophagosome formation attenuates viral replication while inhibiting autolysosome formation, and thus, catalytic activity does not affect the virus. however, degradation of cellular triglycerides by autophagy benefits denv replication and autolysosome degradation dampens ifn activation following hcv infection of these pathologies. indeed, there is growing evidence that cell's modify their membranes to defend themselves against pathogens and infection, altering their spatial reorganization and vesicle trafficking. in this review, we focus on the importance of the membrane flux triggered by viruses to achieve replication and egression, and to ensure their propagation. several of the cell's organelles and membrane structures are involved in viral replication and in fact, many viruses use specific cellular compartments to replicate, referred to as viral factories (vfs: figure ). these vfs provide a physical scaffold that brings together elements required for genome replication and morphogenesis [ , ] . vfs are usually formed by rearranging the host's cell membranes, reorganizing the cytoskeleton and recruiting specific organelles, like mitochondria (reviewed in [ ] ). these viral driven events involve the association of replication complexes (rcs) with er derived membranes to form a vf. hence, intracellular membrane dynamics appear to be crucial for viral replication and survival. a well-known example of a vf is that used by vaccinia virus, an enveloped pathogen of the poxvirus family that replicates in the cytoplasm by assembling small rough er-derived cisternae into a microenvironment that resembles a cytoplasmic mini-nucleus for viral replication [ ] . similarly, the rcs of togaviruses associate with endocytic membranes, while nodavirus rcs associate with mitochondrial membranes (reviewed in [ ] ). thus, specific membrane compartments can be used as vfs by rna viruses to concentrate viral replicases and key cofactors, and ensure efficient viral genome replication [ ] . in this context, both rubella virus (rubv), a relevant human teratogenic togaviridae virus [ ] , and semliki forest virus (sfv), a member , some viruses attach structural polyproteins to pip -rich membrane regions of the infected cell for further budding and release into the intercellular space. pip confers fluidity to the cell membrane and favours virus-cell fusion. these virions then bind to specific receptors in order to infect the neighbouring target cell at the vs, fusing with its plasma membrane directly or after surfing on actin-structured filopodia, or being internalized by endocytosis as is believed to occur with hiv- . the vs represents an efficient environment for viral budding. it typically arises in pip -enriched plasma membrane domains, where the membrane of the infected cell is polarized towards the synaptic junction through the movement of vesicles governed by the escrt/alix-tsg machinery or by mvbs coordinating the translocation of the mtoc. this scaffolding facilitates subsequent viral infection and spread from the infected to the nearby uninfected cell. in addition, long membrane nanotubes may also form between neighbouring cells, promoting viral protein trafficking. other dynamic membrane events involved in viral infection and spreading are trogocytosis, arf /pip -mediated membrane dynamics and exosomal transport. trogocytosis involves the exchange of cell surface membrane patches that may contain receptor clusters associated to viral particles, while exosomes are vesicles formed from mvbs that could participate in viral infection and spreading between cells of the alphavirus group of this family [ ] , couple their rna synthesis to endosome and lysosome membranes modified by the association of virus specific components. the subsequent fusion of these late endosomes and lysosomes generates cytopathic vacuoles (cpvs) [ , ] that are lined with small vesicular invaginations or spherules (viral rna replication sites) [ , ] . cpvs establish complex and reversible contact with endocytotic vesicles through internal membranes interconnected with transport endosomes [ ] . for example rubv forms vfs around cpvs via the recruitment of membrane structures from the er cisternae, golgi stacks and mitochondria [ ] (figure ). the golgi apparatus is a highly dynamic organelle with a sustained, functional flux of membrane proteins [ ] , and it can serve as a morphogenic mould for rubiviruses, coronaviruses, arteriviruses and bunyaviruses [ , , , ] . these rubv factories connect viral replication with the assembly and maturation of nascent virions at golgi membranes, contributing to the virus escaping from the host cell's defences [ ] . some viruses induce the formation of doublemembrane vesicles (dmvs) and/or autophagosomes for replication [ ] [ ] [ ] [ ] , ] , such as the positive rna viruses of the flaviviridae family and nidovirales order [ , ] (figure ). the rna polymerase of the human poliovirus, a picornaviridae family member responsible for poliomyelitis, can also assemble dmvs [ ] . infection by this virus triggers the modification of different intracellular membrane structures and organelles (but not mitochondria), converting them into virus replication vesicles. in fact, poliovirusassociated dmvs resemble autophagosomes, as also described for another picornaviridae family member, coxsackievirus b (cvb [ , ] : figure ). autophagosomes are dmvs generated by membrane trafficking and they are related to the catabolic process of autophagy, which involves the degradation of cytoplasmic components within lysosomes [ , ] . autophagy maintains the organism's homeostasis by sequestering undesired intracellular elements for lysosomal degradation and recycling [ , ] . viruses often use autophagy to complete their lifecycle and evade immune responses, even though it is based on catalytic pathways [ , , ] . poliovirus, like other positive rna viruses, has evolved the capacity to convert autophagy into a key cellular motor for replication [ , , ] . during autophagy, a cytosolic form of the microtubule-associated protein a/ b light chain (lc -i) conjugates with phosphatidylethanolamine to form lc -ii and associate with autophagosomal membranes, ultimately producing the degradation of lc -ii during the late steps of autophagy [ ] . conversely, the p protein (or sequestosome- , sqstm ) interacts with ubiquitinated proteins, lc and other proteins to ensure the correct degradation of undesired material. lc -ii augments during active autophagy when p is degraded [ , ] . in this context, poliovirus or cvb infection triggers the generation of autophagosomes with a higher lc -ii/lc -i ratio and with lc foci, structures that support the rna rc without promoting lysosome degradation (evident through p stabilization [ , ] : figure ). however, it is unclear whether these viruses block autophagosome maturation into amphisomes, avoiding autophagosome fusion with endosomes [ ] . such events override the appearance of degradative autolysosomes [ ] or they may provoke the formation of autophagosome-like structures disconnected from catalytic pathways. it is also thought that these autophagosomes may ultimately serve as a membrane scaffold to permit the egression of nascent virions from infected cells, preventing cell lysis [ ] (figure ). all hcv viral genotypes ( a, b and a), positive rna flaviviruses that are a major cause of chronic liver disease [ ], induce autophagosome accumulation [ , ] . this involves regulation of the unfolded protein response (upr), which relieves er stress and prevents the formation of catalytic autolysosomes by suppressing their fusion with lysosomes [ , ] ( figure ). apparently, the success of viral replication relies on the recruitment of membrane-trafficking proteins to er-derived membrane scaffolds [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hence, domain of the non-structural a (ns a) protein and the helicase domain of ns are sufficient to achieve efficient dmv formation, which also depends on tightly regulated cis cleavage of the hcv-polyprotein precursor [ ] and requires cyclophilin a isomerase activity [ ] . ns a associates with ns b, a rnadependent rna polymerase, a complex that interacts with vamp (vesicle-associated membrane protein)-associated proteins (vaps) [ , ] and recruits ras-like small gtpases (e.g. rab , rab and rab ), enlarging the viral replication compartment by docking membrane vesicles [ ] [ ] [ ] . this process also regulates autophagy [ ], given that hcv-induced autophagosomes support viral replication and the delivery of incoming viral rna to the translation apparatus, and/or the recruitment of cellular factors for translation. however, some controversy still surrounds this issue, autophagosomes can mature into acidic amphisomes in hcv-infected cells [ , ] , and subsequently fuse with late endosomes or lysosomes [ ] (figure ). autophagic membrane flux appears to be necessary to translate the hcv genome, yet it appears to be dispensable once viral infection has begun [ ] . moreover, no changes in either p or the degradation of long-lived proteins are observed [ ], despite the enhanced autolysosome formation in cells expressing hcv replicons [ ] . while specific silencing of autophagy genes does not affect viral translation and rna replication, it does apparently alter hcv morphogenesis [ ] . however, the silencing of factors critical for autophagosomes formation, like lc or atg , appears to suppress hcv rna replication [ ], while hcv replication is apparently potentiated when the upr promotes autophagy [ ] . conversely, hcv infection seems to promote autophagy without concomitant stimulation of the upr and autophagy does not appear to be required as a platform for hcv rna replication [ ]. thus, doubts remain about the role of autophagy and the upr in hcv replication, although the distinct interactions between autophagy and hcv replication suggest that such membrane flux promotes viral replication. the dengue virus (denv) is a mosquito-borne single positive-stranded rna virus of the flaviviridae family that causes dengue fever [ ] . there are five antigenically related but distinct denv virus genotypes (denv- to denv- ) [ , ]. like hcv, there is evidence that autophagy may be implicated in denv replication. following cell entry and nucleocapsid uncoating, denv rna is translated into a single polyprotein that passes into the er lumen where the different viral proteins are processed. in fact, denv- proteins involved in translation and replication are found in or in close proximity to autophagosomes during viral infection [ , ] . accordingly, inhibition of autophagosome formation dampens the production of infectious denv- particles [ ], while stabilizing autophagosomes and/or amphisomes by impeding their fusion with lysosomes enhances viral egression [ ] . indeed, denv- seems to promote autophagy during early infection [ ] , while inhibition of autophagosome formation also dampens the production of infectious denv- [ ]. hence, denv- and - appear to interact with the autophagy machinery in a different manner, and while it is conceivable that amphisomes or autophagosomes represent the site of denv- translation/replication [ , ], autophagolysosomes could be the crucial site for denv- viral replication [ ] . the distribution of ns or denv- and denv- double-stranded rna (dsrna) in the different autophagy-associated membrane structures confirms these observations ( figure ). moreover, nascent viral particles are formed and mature in these structures, then travelling through the trans-golgi network (tgn) to egress [ , ] . remarkably, the precursor membrane (prm) protein of the denv- - genotypes behaves similarly and it is cleaved by the tgn-protease furin in the secretory pathway [ ], assuring viral assembly and the infectivity of nascent viral particles [ ] . hiv is a single-stranded rna virus (lentivirus genus of the retroviridae family) that causes aids. hiv type (hiv- ) alters the autophagic membrane flux of the host cell's organelles, thereby modulating the intracellular milieu in favour of viral replication and propagation [ ] (figure ). when cd + t cells, monocytes and dendritic cells (dcs) are infected with hiv- , autophagic vacuole formation is blocked and the expression of autophagy proteins down-regulated (e.g. lc and beclin [ , ] : figure ). remarkably, the hiv- protein nef (negative factor) blocks the autophagic flux of membranes, especially during the autolysosome stage of autophagy, resulting in an accumulation of autophagosomes and lc in macrophages ( figure ). thus, nef prevents autophagic degradation of hiv- biosynthetic intermediates of virions by targeting the lipid class iii phosphatidylinositol -kinase (c -pi k) complex and by associating with beclin (atg -autophagy-related protein -in yeast). significantly, beclin is actually part of the c -pi k complex, together with the vacuolar protein sorting-associated proteins (vps ) and (p ). nef therefore alters the sub-cellular distribution of vps , potentially ensuring the survival of the viral progeny [ , ] . indeed, nef is thought to promote the appropriate hiv- gag membrane localization and processing, thereby facilitating viral cell-to-cell transfer [ ] . although the catalytic activity of autophagy appears to be impeded by hiv- , autophagosome formation or accumulation is still promoted. hence, the hiv- gag protein promotes early stages of autophagosome formation by directly interacting with lc in macrophages, enhancing hiv- yields and gag processing, a critical step in virion assembly and release [ ] (figure ) . notably, newly identified components of the ubiquitin-like conjugation system all seem to be involved in hiv- replication (e.g. atg , atg -lc is its best characterized mammalian homologue-atg and atg l -responsible for vesicle elongation) [ ] . however, it remains unclear how these factors actually affect hiv- replication, which occurs in the nucleus of infected cells. moreover, while autophagic vacuoles would appear to be fundamental for hiv- morphogenesis and egression, how hiv- overrides or uses autophagy to persist remains poorly understood. hence, the infectious capacity of nascent hiv- virions depends on the uptake of the viral infectivity factor (vif) during viral budding, a process influenced by histone deacetylase (hdac ), which promotes autophagic clearance of vif [ ] . other positive rna viruses exploit the formation of er-derived membrane scaffolds and membrane autophagic flux to replicate (e.g. the norwalk virus), because the membranebound nsp protein also binds to vap-a [ ] . rna replication may occur in endosomes, lysosomes (togaviruses), peroxisomes and chloroplasts (tombusvirus), or mitochondria (nodaviruses), shielded from immune responses. all positive rna viruses transform cytoplasmic membranes into specialized viral replication sites [ ] . the antiviral effect of brefeldin a (bfa), an inhibitor of anterograde er-golgi network membrane dynamics, suggests that membrane trafficking must be active for enterovirus replication, as reported for picornaviruses, poliovirus and coxsackievirus [ , ] . bfa prevents the membrane flux required to form replication compartments, blocking virus secretion from infected cells [ ] by inhibiting adp (adenosine diphosphate)ribosylation factor (arf)-gtp exchange proteins (arf-gefs). this blockade negatively affects copi coat generation at the golgi by diminishing and sequestering arf -gtp [ ] . for several picornaviruses, copii-coated vesicles may provide membranes suitable for replication [ ] , although autophagosomes may also contribute at this point [ ] (figure ). reovirus and sfv also promote coated-pit formation [ ] . moreover, the small gtpase rab is soon recruited for sfv internalization when associated to intermediate endosomes [ ] , which in turn induces the formation of cpvs that is an important event for viral rna synthesis in target cells [ ] . an important biological process common to the recently proposed megavirales order is viral replication within cytoplasmic vfs [ ] . giant viruses (also called nucleocytoplasmic large dna viruses-cldvs) belonging to this order are double-stranded dna (dsdna) viruses with a genome and particle size comparable to those of small bacteria [ ] . african swine fever virus (asfv; from the asfarviridae family), poxviruses and iridoviruses are the three families of ncldvs that terminate or undergo their entire replication cycle in the cytoplasm [ ] [ ] [ ] . this feature is not observed in herpes viruses or baculoviruses, other large dna viruses of eukaryotes that undergo nuclear dna replication and transcription [ ] . giant viruses provoke vf formation in the cytoplasm of infected cells to permit genome replication and morphogenesis [ , ] . asfv factories are similar to the aggresomes formed at the mtoc (microtubule organizing centre) [ ] , and they provoke a rearrangement of the intermediate vimentin cytoskeleton at the mtoc into a star shaped structure that resembles the microtubule aster formed during mitosis, a structure required for late gene expression [ ] . together with an asfv chaperone, the hsp cell chaperone is recruited to asfv factories, along with mitochondria, facilitating the folding of viral structural proteins like the major capsid protein p [ , ] . nascent asfv virions are formed from vf membranes through the assembly and recruitment of viral proteins in vfs. thus, the viral membranes in vfs may be connected to cellular organelles, particularly given that resident er markers are detected with the viral p , p and pb l proteins in new viral particles [ ] [ ] [ ] [ ] . asfvs are thought to reorganize cell membranes through viral proteins that contain a kde motif, inducing the redistribution of erassociated proteins [ ] and the viral p protein. the latter is required for the correct vf localization of the membranes and the collapse of the er-derived cisternae [ ] . asfv infection is achieved by redistributing membranes from the secretory pathway and tgn [ ] . therefore, these common biological features of giant virus replication and virion architecture could reflect a common origin, and the sharing of a large set of ancestral genes [ , ] . membrane dynamics during viral assembly and budding and infectiousness. during budding, successful infection is achieved by adjustment and distortion of the target cell's plasma membrane [ ] . the structural gag polyprotein is common to several retroviruses, like hiv- and the murine leukaemia virus (mlv), representing the minimal plasma membrane component required for viral assembly [ ] . hiv- gag localizes to phosphatidylinositol- , -bisphosphate (pip ) rich plasma membrane regions, where pip plays a critical role in hiv- virion assembly [ ] (figures and ) . in fact, the matrix viral protein (ma) within the unprocessed hiv- gag polypeptide drives gag towards these pip membrane domains [ , ] in a myristoylation-dependent manner [ ] , raft domains where hiv- buds [ ] [ ] [ ] . phosphate hydrolysis by polyphosphoinositide phosphatase iv ( ptaseiv) diminishes the plasma membrane pip [ ] , causing the gag polypeptide to translocate from hiv- budding sites at the membrane to cd rich compartments, thereby inhibiting viral release [ ] . similarly, arf /q l expression, a gtp-bound mutant of arf , alters the trafficking of arf /pip -associated vesicles, provoking their accumulation in the cytoplasm to where gag is redirected. these complexes lie far from the budding sites at the membrane, thereby dampening virus release [ ] . although the assembly of hiv- at the cell surface is only partially understood, several key steps in the membrane trafficking of viral proteins have been defined, shedding light on both the viral assembly and budding processes [ , ] . enveloped viruses like hiv- , vesicular stomatitis virus (vsv), ebola virus (ebov) and hepatitis b virus (hbv), and other rna and dna viruses, mainly emerge from cells by co-opting the host's escrt machinery [ , ] , which plays a vital role in cellular abscission and in multivesicular body (mvb) biogenesis (a process by which ubiquitinated misfolded or damaged proteins enter endosomes to be destroyed). in addition, mvbs are important intermediates in endolysosomal transport [ ] (figures and ) . gag activity drives escrt-iii complex formation at the budding site of hiv- , which binds to and recruits the escrt-i complex and the alg- (apoptosis-linked gene )interacting protein x (alix). this escrt-iii complex promotes the excision of nascent virions at the cell surface, an event potentially equivalent to the cleavage of intraluminal vesicles from mvbs [ , ] (figures and ) . moreover, the tumour susceptibility gene (tsg ) is a subunit of the escrt-i complex that drives viral rna transport and envelope fusion to late endosomes, processes required for infection and rna release [ ] ( figure ) . however, the interaction of viral gag protein with the escrt machinery appears not to be absolutely required for hiv- viral budding [ ] [ ] [ ] . nevertheless, interferon-stimulated gene protein (isg- ) inhibits hiv- egression by interfering with escrt-iii protein membrane flux during budding [ , ] . remarkably, morphogenesis and the release of hbv particles also require tsg [ ] , although this dna virus lacks a viral protein bearing the late (l) domain necessary to interact with the escrtmachinery [ ] . however, α-taxilin interacts directly with tsg and with the large hbv surface protein (lhbs), thereby recruiting the viral capsids to escrt complexes, thus permitting correct viral formation and egression [ ] . therefore, hbv maturation and egression depends on the escrt-mvb system. notably, hbv infected cells also produce large amounts of non-infectious spherical or filamentous envelope particles (svps). these svps are a mixture of lipids and viral surface proteins that accumulate in an er-golgi intermediate compartment (ergic), budding into the lumen and provoking release through the general secretory pathway [ ] . many other enveloped rna viruses bud in an escrt-dependent manner [ , , ] , as do most negative-strand non-segmented single-stranded rna (ssrna) viruses, such as rhabdoviruses, filoviruses and most paramyxoviruses, all of which recruit escrts for viral egression [ , ] . even the budding of negative-strand segmented-ssrna arena viruses involves an escrt-dependent pathway [ , ] . however, no evidence for the participation of escrts has yet been reported in nipah, measles, hrsv or bornaviridae budding ( [ ] ). indeed, the enveloped influenza virus buds in an escrt-independent manner as its matrix protein is devoid of an escrt-binding domain [ , ] . other viruses are also released from the host's plasma membrane through their mas, such as the newcastle disease virus or vsv. in these cases, bud formation and excision from the membrane are matrix-dependent processes [ , ] , as for influenza virus. however, much work is still required to determine how membrane dynamics affect the trafficking and assembly of these viruses, particularly in terms of the cellular factors that control the [ , ] . given all of these findings, membrane dynamics has a crucial influence on the assembly and budding of numerous viruses, and it may represent an important and complex target to limit the viral life cycle. viruses use various cell communication pathways to achieve effective cell-to-cell dissemination [ ] . first described for type htlv (htlv- ) [ ] , the virological synapse (vs) is a complex structure found at the interface of infected:uninfected cells. viral receptors and the egression machinery accumulate at the vs [ ] , making the infection and spread of htlv- through t lymphocytes cell-cell dependent. direct cell-to-cell transmission facilitated by the formation of stable cellular junctions has several advantages, including faster replication rates [ ] , successful transmigration of infected cells across mucosal barriers [ ] and viral protection from host responses. however, such transmission is still to be confirmed for hiv [ , ] . cell-to-cell spreading of hiv- ( figure ) is considered to involve microtubule-mediated polarization and substantial budding, followed by the entry of free viral particles into target cells [ ] . thus, it involves pathways that regulate cell-free virus entry by modifying membrane dynamics. in this regard, most hiv- -infected galt (gut-associated lymphoid tissue) cells in intestinal crypts are infected by concentrated pools of free hiv- viral particles in hiv- -infected humanized mice. fewer infected cells are found in the mucosal regions and the lamina propria, where vs presumably occur [ ] , explaining why infection of permissive cells by free viral particles is crucial for hiv- replication and pathogenesis in vivo. this is consistent with the recent identification of the key cell signals required for efficient early hiv- infection and the establishment of latency in cd + t cells [ ] [ ] [ ] [ ] [ ] [ ] . interference with retroviral cell-tocell transmission is not only produced by blocking cytoskeletal motility [ ] and depleting membranecholesterol [ ] but also, by interfering with arf governed plasma membrane dynamics. moreover, restricting plasma membrane fluidity caused by altering early hiv- -triggered phosphatidylinositol- -phosphate -kinase (pi p -k) iα activation and the ensuing detrimental effects pip production on hiv- transmission [ , , ] . in fact, arf -coordinated membrane trafficking is required for efficient hiv- fusion, entry and infection of cd + t lymphocytes [ ] (figures and ) . the flux and turnover of pip -enriched vesicles from the plasma membrane, driven and coordinated by the arf -gtp/gdp cycle, ensures cell surface membrane regeneration and it allows membrane exchange between the viral and target cell surfaces. this type of membrane trafficking, coupled with enhanced fluidity, is in strong synergy with the key hiv- /receptor (cd and c-x-c or c-c chemokine receptor type or -cxcr or ccr ), interactions that promote fusion pore formation in target cells. these interactions take place between the non-regenerative hiv- viral membrane and the dynamic cell surface membrane, favouring efficient virus-cell fusion, entry and infection, both for the free virus and in the context of the vs [ , , ] (figures and ) . despite these similarities, some contact-specific events that affect membrane flux should also be considered. during cell-cell hiv transmission, intense viral endocytosis drives entry into neighbouring cells even if they are in contact [ ] (figure ). remarkably, biofilm-like structures at the surface of infected cells concentrate htlv- viruses for their efficient transmission to target cells [ ] and cellular projection is used to transmit pseudo-rabies virus. retroviruses also travel along membrane protrusions that contact adjacent cells and mlv surfs on the filopodia of fibroblasts before entering cells [ ] (figure ). hiv- also takes advantages of filopodia for cell-tocell transmission [ ] , similarly surfing on the narrower membranous nanotubes that connect cells separated no more than μm [ ] and facilitating the transfer of viral proteins to the inner side of the membrane. these actin structures extend from hivinfected cells to target cells irrespective of receptorenvelope interactions [ ] (figure ) . the take up of larger membrane invaginations at the vs of connected cells [ ] is known as trogocytosis, an event that may also control the extent and stability of the synapse, regulating its duration [ ] . hiv particles, like cd molecules and other membrane components, are transferred by trogocytosis from uninfected to infected cells in a manner triggered by the hiv- envelope (env)/ cd [ ] (figure ). this mechanism could be very significant and render cells permissive to hiv infection, as recently proposed [ ] . the cell-cell contacts and signalling induced by the hiv- env complex that occur at the vs can activate autophagic membrane flux, leading to apoptotic cell death of uninfected cd + t cells [ , ] . this lethal autophagy may provoke or enhance immunodeficiency, as observed in vivo where the majority of cd + t cells undergoing apoptosis, as well as the peripheral blood and lymph nodes of hiv patients, remain uninfected [ ] . simultaneously, autophagy can be suppressed in infected cd + t cells [ ] , thereby antagonizing env-mediated apoptosis and allowing viral replication to occur in infected cd + t cells. in this context, hiv- evades immune responses in an hiv- env-cd dependent manner by efficiently impairing autophagy in dcs when early contacts are established [ ] . taking into account the role autophagy plays in viral replication, hiv- can enhance or suppress autophagy at different stages of its viral cell cycle, favouring persistence and the evasion of immune responses, and therefore, its pathogenesis [ ] ( figure ). finally, like other viruses (e.g. cmv), hiv- stably associates with professional apcs during infection (such as dcs) to further infect lymphocytes during t-cell scanning or antigen presentation [ ] . in fact, hiv- enters dcs by exploiting exosomal trafficking during antigen presentation [ ] (figure ). this review examines the intracellular trafficking of viruses that occurs in association with cellmembrane structures, some of which may be newly assembled by viruses to ensure their replication and budding. membranes derived from the er, mitochondria, lysosomes and endosomes are sculpted by viruses to generate vfs, acquiring their own functional morphology. these structures help ensure rna replication is accomplished without alerting the host's defence mechanisms. although the importance of membrane dynamics during viral infection has been established, several questions remain unanswered. it remains unclear how proteins from distinct viruses and host cells use the same intracellular membrane compartments or events (e.g. autophagy) to achieve viral replication, without affecting important cellular processes. conversely, it is not clear why viruses replicate in different subcellular membrane compartments, how they move across membranes and which host factors are involved in these events. similarly, we still do not know how these changes in membrane dynamics enable viruses to avoid immune responses. indeed, it remains unclear whether rearranging intracellular organelles enables viruses to escape the anti-replicative activity of natural restriction factors, such as apolipoprotein b mrna-editing enzyme-catalytic, polypeptide-like (apobec ) proteins, tetherin (bst- /cd / hm . ) or samhd (sterile alpha motif (sam) and histidine-aspartate (hd) domain-containing protein ) for hiv- [ ] . resolving these issues will help decipher how viruses rearrange membranes during their infection cycle, thereby aiding the design of new antiviral strategies that target these dynamic viral-cell interactions and combat viral infection. these findings may also produce innovations in non-viral gene delivery systems to tackle tumours and immune diseases. new technical developments, such as more powerful microscopy systems [ , , , ] , will allow dynamic viral trafficking and egression to be studied in cells with better spatial and temporal resolution. such information will further our understanding of the viral infection process and of how viruses succeed in deceiving the host's immune responses. respectively, spain), unll - e- (erdf) and fundación cajacanarias, and by the project rd / / integrated in the "plan nacional i + d + i" and co-funded by isciii-"subdirección general de evaluación" and erdf (ris-retic) grants. m-s.v and l.a-r are supported by rd / / -(ris-retic) and saf - -fpi-associated grants and fellowships, respectively. we thank dr mark sefton (biomedred sl) for his linguistic revision of the manuscript. we apologize for all research studies and reviews that we have not discussed or cited in this review. we have tried to avoid any and all such omissions but space limitations have surely made this an impossible endeavour. modification of 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lymphocytes from human immunodeficiency virus- -infected children hiv and mature dendritic cells: trojan exosomes riding the trojan horse? exosomes and retroviruses: the chicken or the egg? host factors and hiv- replication: clinical evidence and potential therapeutic approaches virus trafficking-learning from single-virus tracking this work and a.v.-f. are supported by the european regional development fund (erdf), saf - and saf - (micinn and mineco, the authors have no competing interests to declare. key: cord- -fw w u authors: cillóniz, catia; dominedò, cristina; magdaleno, daniel; ferrer, miquel; gabarrús, albert; torres, antoni title: pure viral sepsis secondary to community-acquired pneumonia in adults: risk and prognostic factors date: - - journal: j infect dis doi: . /infdis/jiz sha: doc_id: cord_uid: fw w u we investigated the risk and prognostic factors of pure viral sepsis in adult patients with community-acquired pneumonia (cap), using the sepsis- definition. pure viral sepsis was found in % of all patients ( of ) admitted to the emergency department with a diagnosis of cap, % of those with cap ( of ) admitted to the intensive care unit, and % of those ( of ) with a diagnosis of viral cap. our data indicate that males and patients aged ≥ years are at increased risk of viral sepsis. we investigated the risk and prognostic factors of pure viral sepsis in adult patients with community-acquired pneumonia (cap), using the sepsis- definition. pure viral sepsis was found in % of all patients ( of ) admitted to the emergency department with a diagnosis of cap, % of those with cap ( of ) admitted to the intensive care unit, and % of those ( of ) with a diagnosis of viral cap. our data indicate that males and patients aged ≥ years are at increased risk of viral sepsis. keywords. sepsis; viral sepsis; virus; community-acquired pneumonia. improved molecular diagnostic techniques have increasingly revealed a high prevalence of viral pneumonia over recent years. globally, it is now estimated that million cases of viral pneumonia occur annually, with the incidence varying by seasonality, geographic location, and age group [ ] . respiratory viruses are detected as etiological agents in almost one third of cases of community-acquired pneumonia (cap) [ ] [ ] [ ] [ ] and in %- % of patients with severe cap with a defined microbial etiology [ , ] . recently, jain et al [ ] analyzed cases of pneumonia detected by intensive microbiological diagnosis, including viral molecular techniques. a microbial etiology was identified in cases ( %). the main causes were respiratory viruses ( %), bacteria ( %), and coinfections ( %), indicating the clear prominence of a viral etiology. cap is often complicated by sepsis, which is a multifactorial process for which staging is necessary to provide personalized treatments that target individual needs [ ] . viral sepsis has been defined as a severe inflammatory response to viral infection [ ] , and unlike bacterial sepsis, its prevalence in adults with cap is unknown. we aimed to investigate the prevalence, risks, and prognostic factors associated with pure viral sepsis in adult patients with cap, using the third international consensus definitions for sepsis and septic shock (sepsis- ) criteria [ ] . we performed a retrospective observational study of consecutive adult patients with a diagnosis of cap admitted to the hospital clinic of barcelona from the emergency department between and . we excluded nonhospitalized patients and those with severe immunosuppression, active tuberculosis, viral bacterial coinfection, and unavailable data. we selected patients with pure viral cap and compared those with and without sepsis. severe cap was defined according to the american thoracic society/infectious diseases society of america guidelines [ ] . sepsis was defined as the presence of pneumonia and an increase of ≥ points in the sequential organ failure assessment score [ ] . diagnosis of respiratory virus infection was made on the basis of results of serologic analysis, immunofluorescence assay, and cell cultures from to . however, diagnosis was based on results of polymerase chain reaction (pcr) and/or culture of nasopharyngeal swab samples from to . two independent nested multiplex real-time pcr tests were used to detect human influenza viruses (a, b, and c), respiratory syncytial virus, adenoviruses, parainfluenza viruses ( - ), coronaviruses ( e and oc ), enteroviruses, and rhinoviruses (a, b, and c). the criteria for etiological diagnosis are available in a previous report [ ] . the main clinical outcome was in-hospital mortality. secondary outcomes included length of hospital stay, intensive care unit (icu) admission, mortality among patients admitted to the icu, length of icu stay, need for mechanical ventilation, -day mortality, and -year mortality. patients were followed for one year. for publication purposes, the study was approved by the ethics committee of our institution (comité Ètic d'investigació clínica; registration no. / ). the need for written informed consent was waived because of the noninterventional study design. logistic regression analyses were used to examine the association between sepsis and risk factors. first, each risk factor was tested individually. then, all risk factors that showed an association in the univariate model (p < . ) were added to the multivariable model. finally, backward stepwise selection (p in < . and p out > . ) was used to determine factors associated with sepsis. generalized linear model analyses were performed to determine the influence of the risk factors on in-hospital mortality. models were defined using a binomial probability distribution and a logit link function, using inverse probability of treatment weights (iptws) to account for biases due to observed confounders. first, each risk factor was tested individually. second, a propensity score for patients with sepsis was developed. iptw used the propensity score to form a weight. finally, the weight and the year of admission were incorporated in the multivariable weighted logistic regression model for in-hospital mortality, which included all risk factors and showed an association in the univariate analyses (p < . ), and backward stepwise elimination was performed to detect the factors associated with in-hospital mortality. we used the multiple imputation method for missing data in the multivariable analyses. the level of significance was set at . ( -tailed). all analyses were performed using ibm spss statistics, version . (armonk, ny). we identified consecutive patients admitted to the emergency department with a diagnosis of cap during the study period. a total of patients ( %) were hospitalized, of whom ( %) were found to have a pure viral cap. thirty-six patients ( %) had severe cap. among the cases of pure viral cap, the most common respiratory viruses were influenza a virus ( [ ] ), and coronavirus ( % [ ] ). we did not observe any change in the prevalence of viral cap over the study period (p = . ). the mean age (±sd) was ± years, and the sex of ( %) was male. most patients ( % [ ]) had ≥ comorbidity, with chronic respiratory disease (in %) and diabetes mellitus (in %) being the most frequent. despite bacterial pathogens were not isolated, patients received empirical antibiotic therapy. monotherapy was reported for patients ( %); fluoroquinolones and β-lactams were the most common agents administered. a total of patients ( %) received combination therapy, with the most frequent combinations comprising a β-lactam plus a macrolide ( % [ patients]) and a β-lactam plus a fluoroquinolone ( % [ ]). the median length of hospital stay was days (interquartile range, - days); in-hospital mortality was % ( patients). a total of patients ( %) were admitted to the icu, of whom ( %) required mechanical ventilation; the median length of icu stay was days (interquartile range, - days), and icu mortality was % ( patients). thirty-day mortality was % ( patients), and -year mortality was % ( ). among all patients with a diagnosis of pure viral cap, ( %) presented with sepsis, and ( %) presented with septic shock at admission. table summarizes the main clinical characteristics. the sepsis group had a greater mean age, a greater proportion of males, and a greater prevalence of comorbidities (especially chronic respiratory diseases), compared with the nonsepsis group. there was no statistically significant difference in symptoms (fever, cough, pleuritic pain, purulent expectoration, or dyspnea) between the groups. at admission, a greater proportion of patients in the sepsis group presented with an elevated respiratory rate and lower lymphocyte levels, compared with patients in the nonsepsis group. there was no statistically significant difference in the distribution of respiratory viruses between the groups. thus, we did not find any association between the type of virus and the presence or absence of sepsis (in the nonsepsis group, influenza virus was found in % [ patients] and non-influenza virus in % [ ], compared with % [ ] and % [ ], respectively, in the sepsis group; p > . ). more patients in the sepsis group were classified as having a pneumonia severity index of iv-v, indicating severe cap. overall, patients ( %) received antiviral therapy with oseltamivir. the percentage of patients who received antiviral therapy was similar between the groups ( % vs %; p = . ). forty-four patients ( %) with sepsis were treated empirically with antibiotic monotherapy. the sepsis group received fluoroquinolone-based monotherapy less frequently than the nonsepsis group ( % vs %; p = . ). antimicrobial therapy was inappropriate (ie, nonconcordant with published guidelines) in cases ( %) in the sepsis group, but there was no significant difference from the nonsepsis group ( %). among the variables associated with viral sepsis in the univariate logistic regression analysis, age ≥ years and male sex remained independent risks factors for viral sepsis in the multivariable analysis (table ). internal validation of the logistic regression model by using bootstrapping with samples demonstrated robust results for all variables included in the model, with small % confidence intervals (cis) around the original coefficients. no statistically significant difference was observed between the two groups in terms of in-hospital mortality, icu mortality, length of icu stay, -day mortality, and -year mortality (table ) . however, patients with sepsis showed longer length of hospital stay, were more frequently admitted to icu and needed ( ) . more frequently invasive mechanical ventilation than patients without sepsis. in the propensity-adjusted logistic regression multivariable analysis of in-hospital mortality using the weighted data, after exclusion of patients with septic shock at admission and those with do-not-resuscitate orders, pure viral sepsis was not associated with in-hospital mortality (odds ratio, . ; % ci, . - . ). all variables remained significant after the bootstrapping procedure, with a small % cis around the original coefficients. this study has main findings. first, pure viral sepsis defined according to the sepsis- criteria was found in % of all patients admitted with a diagnosis of cap, % of those admitted to abbreviations: ci, confidence interval; copd, chronic obstructive pulmonary disease; or, odds ratio. a the variables analyzed in the univariate analysis were as follows: age, sex, smoking status, alcohol consumption, influenza vaccination, pneumococcal vaccination, previous inhaled corticosteroid therapy, previous systemic corticosteroid therapy, previous antibiotic therapy in last week, chronic pulmonary disease, chronic cardiovascular disease, chronic renal disease, chronic liver disease, diabetes mellitus, chronic neurologic disease, and nursing home admission (p = . ). defined as the probability of being in the sepsis group divided by the probability of being in the nonsepsis group. c based on the null hypothesis that all ors relating to an explanatory variable equal unity (ie, that there was no effect). patients who initially received noninvasive ventilation but subsequently needed intubation were included in the invasive mechanical ventilation group. the icu, and % of those with a diagnosis of pure viral cap. second, male sex and age ≥ years were shown to be risk factors for pure viral sepsis. third, pure viral sepsis was not found to be a risk factor for in-hospital mortality. sepsis is a life-threatening organ dysfunction due to the host's overwhelming response to infection. although respiratory viruses are reported to be important causative agents of severe cap [ ] , the prevalence of pure viral sepsis is not fully known. a recently published study investigated the role of virus detection by multiplex pcr of nasopharyngeal samples from clinically septic patients during a winter season [ ] . the authors reported that respiratory viruses, including influenza a virus, human metapneumovirus, coronavirus, and respiratory syncytial virus were detected in % of adult patients with sepsis. in another study, montull et al [ ] investigated the predictors of severe sepsis in patients with cap and found that % of patients presented with severe sepsis and that . % were identified to have respiratory viruses as casual agents. the proportion of patients with pure viral sepsis was slightly higher in our study population, but we think that this was due to our use of the new sepsis- definition. montull et al also highlighted the association between older age and development of viral sepsis, which was in line with our finding that viral sepsis affected % of patients ( ) aged ≥ years. these results are consistent with data showing that, because of the increased prevalence of chronic conditions and age-related changes in the immune system, elderly patients are more susceptible to infectious diseases and sepsis. it is also possible that the endothelium is fragile in this population [ ] . male sex was another risk factor for pure viral sepsis, consistent with data that men typically have more chronic comorbidities and a higher incidence of cap than women [ ] . we observed that viral sepsis was not a risk factor for in-hospital mortality in patients without septic shock. our data support those of previous studies in which respiratory viruses were frequently found in critically ill patients with pneumonia but mortality rates did not significantly differ between patients with bacterial infection and those with viral infection [ , , ] . this highlights the need to identify patients at higher risk of viral sepsis and the importance of a complete microbiological diagnosis in cases of cap. we could not find other studies addressing the issue of pure viral sepsis (defined according to the sepsis- criteria) in case of cap in a large inpatient adult cohort. finally, we observed that % of patients with viral cap received oseltamivir therapy, without differences between patients with and those without sepsis. compared with other previous studies [ , ] , our population received a higher proportion of antiviral therapy. however, future studies are needed to investigate why the frequency of antiviral therapy use among patients hospitalized with cap is not high, since current guidelines strongly recommend early treatment with oseltamivir in patients with influenza [ ] . some limitations must be addressed. first, although the protocol used for cap diagnosis in our hospital did not change substantially during the -year study, we cannot discount the effect of changes in microbiological diagnosis over this period. second, regarding microbiological diagnosis, morerapid pcr diagnostic tests for influenza virus and respiratory syncytial virus were used during the influenza season. third, the indications for oseltamivir therapy were only extended in , before which it was only used to treat severe cases of viral infection. in conclusion, in our cohort, pure viral sepsis affected % of patients with a diagnosis of viral cap, supporting the importance of stratifying patient risk for viral sepsis and making a complete microbiological diagnosis in cases of cap. notes viral pneumonia cdc epic study team. community-acquired pneumonia requiring hospitalization among u.s. adults microbial aetiology of community-acquired pneumonia and its relation to severity viral infection in community-acquired pneumonia: a systematic review and meta-analysis an international perspective on hospitalized patients with viral community-acquired pneumonia the third international consensus definitions for sepsis and septic shock (sepsis- ) viral sepsis in children infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults viral infection in patients with severe pneumonia requiring intensive care unit admission respiratory viral infections are underdiagnosed in patients with suspected sepsis nac calidad group. predictors of severe sepsis among patients hospitalized for community-acquired pneumonia shared features of endothelial dysfunction between sepsis and its preceding risk factors (aging and chronic disease) risk factors for community-acquired pneumonia in adults in europe: a literature review lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia oseltamivir use among children and adults hospitalized with community-acquired pneumonia we thank all medical and nursing colleagues for their assistance and cooperation in this study.financial support. this work was supported by ciber de enfermedades respiratorias (ciberes cb / / ), support to research groups of catalonia ; idibaps. dr cilloniz is the recipient of a postdoctoral grant (strategic plan for research and innovation in health-peris - to c. c.) and separ fellowship (to c. c.).potential conflicts of interest. all authors: no reported conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- - lj bfli authors: luo, honglin title: interplay between the virus and the ubiquitin–proteasome system: molecular mechanism of viral pathogenesis date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: lj bfli the ubiquitin–proteasome system (ups) plays a central role in a wide range of fundamental cellular functions by ensuring protein quality control and through maintaining a critical level of important regulatory proteins. viruses subvert or manipulate this cellular machinery to favor viral propagation and to evade host immune response. the ups serves as a double-edged sword in viral pathogenesis: on the one hand, the ups is utilized by many viruses to maintain proper function and level of viral proteins; while on the other hand, the ups constitutes a host defense mechanism to eliminate viral components. to combat this host anti-viral machinery, viruses have evolved to employ the ups to degrade or inactivate cellular proteins that limit viral growth. this review will highlight our current knowledge pertaining to the different roles for the ups in viral pathogenesis. interplay between the virus and the ubiquitinproteasome system: molecular mechanism of viral pathogenesis honglin luo , the ubiquitin-proteasome system (ups) plays a central role in a wide range of fundamental cellular functions by ensuring protein quality control and through maintaining a critical level of important regulatory proteins. viruses subvert or manipulate this cellular machinery to favor viral propagation and to evade host immune response. the ups serves as a double-edged sword in viral pathogenesis: on the one hand, the ups is utilized by many viruses to maintain proper function and level of viral proteins; while on the other hand, the ups constitutes a host defense mechanism to eliminate viral components. to combat this host anti-viral machinery, viruses have evolved to employ the ups to degrade or inactivate cellular proteins that limit viral growth. this review will highlight our current knowledge pertaining to the different roles for the ups in viral pathogenesis. viruses have evolved to exploit the host cellular machinery to establish productive infection. in eukaryotic cells, the ubiquitin-proteasome system (ups) is the major intracellular pathway for degradation and functional modification of cellular proteins. it plays a key role in the regulation of many fundamental cellular processes, including apoptosis, cell cycle regulation, signal transduction, antigen processing, and transcriptional regulation [ ] . ubiquitin is a small ( amino acids) and highly conserved protein present in almost all eukaryotic cells. for ups-mediated proteolysis, protein substrates are first labeled with ubiquitins (a process called ubiquitination), and then recognized and degraded by the s proteasome [ ] . ubiquitination occurs via a sequential reaction mediated by three enzymes, i.e. the ubiquitin-activating enzyme (e ), the ubiquitin-conjugating enzyme (e ), and the ubiquitin ligase (e ). the substrate specificity to the ubiquitin conjugation system is determined by the e ligases [ ] . after multiple rounds of ubiquitination, a poly-ubiquitin chain is formed and serves as a signal for substrate recognition and degradation by the s proteasome. ubiquitin is then released through the activities of deubiquitinating enzymes (dubs) [ , ] . in addition to ubiquitin-dependent degradation, some cellular proteins can also be destroyed by the proteasome in a ubiquitin-independent manner. this process requires the function of proteasome activator (pa , also known at reg) [ ] . besides the role of poly-ubiquitination (most commonly linked with lysine ) in proteasomal degradation, ubiquitination is also involved in regulating protein function without targeting for degradation. mono-ubiquitination or lysine -linked poly-ubiquitination has been shown to play key roles in a wide range of cellular functions, including protein subcellular localization, transcription, dna repair, and signal transduction [ , ] . apart from ubiquitination, target proteins can also be post-translationally modified by several ubiquitin-like proteins, such as the small ubiquitin-like modifiers (sumo, four isoforms are identified in humans, sumo , sumo , su-mo , and sumo ) [ , ] and the interferon-stimulated gene (isg ) [ , ] . protein modification mediated by sumo and isg (termed sumoylation and isgylation, respectively) occurs in a matter similar to ubiquitination that requires an enzymatic cascade of e (aos /uba heterodimer for sumo and ube l for isg ), e (ubc for sumo; ubch and ubch for isg ), and e [ ] [ ] [ ] [ ] . there are currently three identified sumo e s, that is, ranbp , pias and the polycomb protein pc [ , ] and three known isg e s, that is, herc , hhari, and trim [ , ] . these processes can be reversed via the action of de-sumoylating or de-isgylating enzymes. the sumoylation and isgylation conjugation systems have been implicated in the regulation of many cellular functions, including transcriptional regulation, anti-viral immune response, signaling pathways, and vesicular trafficking [ ] [ ] [ ] [ ] . become increasingly evident that viruses interact with the ups at multiple levels. they can either directly encode proteins with e -like or dub-like activities or modify specific aspects of the host ups function for their own advantages [ ] [ ] [ ] . the ups plays a dual role in viral pathogenesis: it has both pro-viral and anti-viral effects [ ] [ ] [ ] . the ups can enhance the function of viral proteins via post-translational modification mediated by ubiquitin or ubiquitin-like proteins. it can also facilitate viral infection through controlling the stability of both viral and cellular proteins. however, on the other hand, ups-mediated viral protein degradation may also constitute a host defense process against viral infections. viruses have developed sophisticated mechanisms to counteract this anti-viral immune response. this review will focus on how viruses evolve to interact with the host ups to favor their propagation, to escape host immune response, and contribute to viral pathogenesis. some cellular proteins can function as restriction factors to limit viral infection by directly inhibiting viral replication or through controlling the state of their infected cells, in particular cell survival/apoptosis and cell cycle progression. for example, the tumor suppressor protein p has been shown to block viral replication by suppressing viral gene activation and/or through promoting host cell apoptosis [ , ] . viruses can recruit the cellular e ligases to target anti-viral proteins for degradation. several proteins encoded by dna tumor viruses, such as the human papillomavirus (hpv) e and e proteins [ , ] and the adenovirus e b k/e orf proteins [ ] , have been shown to induce the assembly of an e ligase complex that contains both viral protein and cellular e to catalyze the ubiquitination of p and subsequent degradation by the proteasome. for instance, the hpv e protein binds to the cellular e ligase e -associated protein (e ap) to form an e complex to mediate p degradation [ ] . in addition to p , the hpv e protein also targets another tumor suppressor protein, retinoblastoma protein (prb), for proteasomal degradation, which is mediated through a cullin e complex [ ] . beside ubiquitin conjugation, the expression of p can also be regulated at the levels of deubiquitinating and proteasome activity. it was shown that the epstein-barr nuclear antigen interacts with the ubiquitin-specific protease (usp , a cellular dub) to enhance p degradation, presumably through inhibiting the activity of usp and thereby preventing p deubiquitination [ ] . in the case of coxsackievirus infection, it was found that proteasome activator pa g is relocated from the nucleus to the cytoplasm where it facilitates the turnover of p through the proteasome [ ] . another example of viral manipulation of the ups for cellular protein degradation is provided by lentivirus destruction of the samhd (sterile alpha motif domain-and histidine-aspartate domain-containing protein ) protein. samhd is a cellular deoxynucleoside (dntp) triphosphohydrolase that inhibits human immunodeficiency virus (hiv) infection by suppressing the activity of reverse transcriptase through depleting cellular dntps [ ] . it has been recently demonstrated that the viral protein x encoded by hiv- and some simian immunodeficiency virus redirects a cullin-ring e ligase to samhd to target it for proteasomal degradation in the nucleus [ ] . a common strategy for viral evasion of host immune surveillance is to target host immune adaptor and signaling molecules (e.g. molecules involved in type i interferon (ifn) response and mhc class i antigen presentation) for proteasomal degradation or to prevent the destruction of immune-related transcription factor inhibitors (e.g. ikba, an inhibitor of the nuclear factor kappa b (nfkb) by sequestering it in the cytoplasm). early studies have revealed that human cytomegalovirus (hcmv)-encoded proteins, us and us , induce the dislocation of mhc class i from the endoplasmic reticulum to the cytosol, where ubiquitination and proteasomal degradation of mhc molecules take place [ , ] . recent work has identified additional cellular targets (integrin a-chains, cd , interleukin- , ptprj (protein tyrosine phosphatase, receptor type, j), and thrombomodulin) for us protein, which are ubiquitinated and degraded through the recruitment of the cellular e ligase (trc ) [ ] . together, these studies suggest that us acts as a degradation hub modulating multiple host immune responses to hcmv infection. in addition to mhc class i, the janus kinase-signal transducers and activators of transcription (jak/stat) and nfkb pathways also play critical roles in host anti-viral defense. viruses have developed distinct mechanisms to utilize the ups to dampen these host innate immune responses. one such example is the v protein of paramyxoviruses, including mumps virus, simian virus , and parainfluenza virus type , which promotes ups-dependent stat degradation through co-opting a host cellular e ligase [ ] [ ] [ ] . similarly, the dengue virus ns protein was also found to stimulate proteasomal degradation of stat , thus blocking type i ifn signaling [ ] . moreover, it was recently reported that the orf protein encoded by simian varicella virus and varicella zoster virus prevents ubiquitination and degradation of ikba, most likely through interacting with b-transducin repeat containing protein, a subunit of the skp -cul -f-box (scf) e complex, thereby suppressing nfkb-mediated immune responses [ ] . rna silencing is an important host defense mechanism against viral infection in plants [ ] . to combat this anti-viral immunity, several plant viruses encode proteins to target key components of rna silencing, such as the argonaute protein [ , ], for proteasomal degradation. together, hijacking the ups is a common viral strategy to evade host immune response. in addition to its pro-viral function usurped by viruses as discussed above, the ups-mediated cellular protein degradation may also represent a host defense mechanism against viral infection. for example, the rar protein, a critical downstream product of the n gene of tobacco, was shown to interact with sgt , a highly conserved component of scf e complex, and cop signalosome, a multiprotein complex involved in ups-mediated protein degradation [ ] . inhibition of sgt and cop abolishes the n gene-mediated resistance to tobacco mosaic virus, suggesting a key role for the ups in the regulation of plant innate immune response [ , ] . further study revealed that this anti-viral effect can be counteracted by geminivirus-encoded c protein, which inhibits scf activity and interferes with the function of cop signalosome [ ]. the ups serves as either a pro-viral or anti-viral mechanism in the context of controlling the levels of viral proteins. proper ratio of structural over non-structural viral proteins is critical for productive viral infection [ , ] . viruses have employed the ups to keep some viral proteins, mostly non-structural proteins, such as rdrp that has been demonstrated to interfere with viral packaging and even become anti-viral at high amounts [ , ], at a relatively low level. multiple studies have shown that the abundance of rdrp encoded by turnip yellow mosaic virus [ ] , sindbis virus [ ] , hepatitis c virus (hcv) [ ] , and hepatitis a virus (hav) [ ] is tightly controlled via the ups. similarly, previous reports on picornavirus have revealed that the c protease of encephalomyocarditis virus and hav is rapidly degraded via the ups and present in low concentrations in infected cells [ ] [ ] [ ] . furthermore, hcv-encoded proteases ns / , were found to be degraded following viral infection in a phosphorylationdependent manner mediated by casein kinase [ ] . another example of viral non-structural protein degradation is provided by the hpv e , which is ubiquitinated and degraded through two independent pathways [ , ] . one involves the ifn-g-inducible suppressor of cytokine signaling- (socs ), a member of the stat signaling pathway, and takes place in the cytoplasm [ ] . the other requires the scf e complex, which induces e ubiquitination with the assistance of e enzyme ubch and subsequent degradation in the nucleus [ ] . in addition to being a viral strategy for its effective infection, it is also conceivable that maintaining a low level of viral proteins represents a viral mechanism to evade recognition by the host immune system. alternatively, degradation of viral proteins constitutes a host defense mechanism. in the latter case, some viral structural proteins are demonstrated to be the targets of the ups. for example, west nile virus capsid protein is ubiquitinated by the cellular e ligase, makorin ring finger protein , followed by proteasomal degradation [ ] . it was also shown that the core protein of hcv is degraded via proteasome in both ubiquitin-dependent through recruiting e ligase e ap and -independent manner mediated by proteasome activator pa g [ ] . the movement proteins of several plant viruses are also degraded through the ups [ , ] . moreover, it was found that the host protein rsp p, a member of the nedd (neuronal precursor cell-expressed developmentally downregulated ) family of e ligases, binds to the p replication protein of tomato bushy stunt virus and promotes its degradation and consequent inhibition of viral replication [ ] . thus, degradation of some viral proteins can also be a host anti-viral defense mechanism. post-translational modification of protein with ubiquitin and ubiquitin-like proteins constitutes an important mechanism to regulate viral protein function. studies from different groups have shown that ubiquitination of the gag protein of retroviruses is required for its function in viral budding and release [ ] [ ] [ ] . the late budding domain in the gag protein carries conserved motifs, such as ppxy and ptap, that recruit host ww domain-containing hect e ligase, nedd to catalyze the ubiquitination of gag [ , ] . other than nedd , the tumor susceptibility gene (tsg ), also binds to the consensus motif in gag and such interaction is required for viral budding and release [ , ] . tsg is a protein of the endosomal sorting complex required for transport (escrt) complex and is involved in vacuolar protein sorting and biogenesis of multivesicular body. similarly, it was found that the p replication protein of tomato bushy stunt virus is ubiquitinated and this modification does not affect its stability, but instead enhances its interaction with escrt proteins, contributing to effective viral replication [ , ] . moreover, ubiquitination of the hiv- tat protein and the human t-cell leukemia virus type tax protein was demonstrated to enhance their transactivation activities without targeting them for degradation [ , ] . the rna-dependent rna polymerase (rdrp) encoded by coxsackievirus provides another example of viral protein ubiquitination and activation during infection [ ] . finally, certain viral structural proteins, such as the envelope protein of severe acute respiratory syndrome coronavirus (sars-cov) [ ] and structural proteins of several plant viruses [ ] , were also found to be ubiquitinated although the functional consequence of such modification remains elusive. some viral functions also require sumo modification. for example, the immediate-early (ie) ie and ie proteins of hcmv are covalently modified by sumo following infection [ , ] . although the exact role of sumoylation in viral pathogenesis is still unclear, sumoylation-resistant mutant of hcmv ie was found to exhibit attenuated viral growth, indicating a role for sumo modification in regulating viral protein function and replication [ ] . the adenoviral e b k protein is another example of viral protein modification by sumo and this modification appears to be required for its function in modulating cell cycle progression and apoptosis as a sumoylation-deficient mutant of e b k fails to interact with p and inhibit p -mediated transactivation [ , ] . furthermore, it was reported that the papillomavirus e protein interacts with sumo e enzyme ubc and e enzyme pias to support viral replication and disruption of their association leads to reduced viral virulence [ , ] . besides animal viruses, viral protein modification by su-mo was also observed in plant viruses. it was found that the repac /rep protein, encoded by tomato golden mosaic virus and tomato yellow leaf curl sardinia virus, binds to sce (sumo-conjugating enzyme), a plant homology to ubc , and sumoylation plays an important role in viral replication [ ] . it was also demonstrated that the rdrp of turnip mosaic virus undergoes sumo modification via its interaction with sce and such modification is required for viral infection [ ] . isg and the isgylation conjugation system represent an important host defense mechanism against infection of a broad spectrum of viruses, including sindbis virus, viral interaction with the host ubiquitin-proteasome system (ups): pro-viral and anti-viral function of the ups in viral pathogenesis. the ups, including modification of key signaling molecules involved in innate immunity by ubiquitin or ubiquitin-like modifiers (e.g. sumo and isg ), represents an important host anti-viral defense mechanism. many viruses have evolved to subvert or manipulate this cellular machinery to favor their growth and to escape host immune response. in some cases, viruses encode proteins with e activity or even becoming part of cellular e complex to degrade cellular proteins (e.g. p , mhc-i, samhd , argonaute ) that limit viral growth (blue arrow). in others they generate proteins with de-ubiquitinating or de-ubiquitinating-like activity to interfere with the host ubiquitin or ubiquitin-like (e.g. sumo and isg ) conjugation system to combat the host anti-viral signaling pathways (red lines). viral protein modification by ubiquitin or sumo is often associated with increased viral release and replication, whereas modification by isg results in reduced viral protein function. viral protein degradation is either a reflection of viral clearance by host immune response or a viral strategy to maintain an optimal level of viral protein to ensure efficient viral production. abbreviations: e , ubiquitin/sumo/isg -activating enzyme; e , ubiquitin/sumo/isg -conjugating enzyme; e , ubiquitin/sumo/ isg ligase; isg , interferon-stimulated gene ; mhc-i, major histocompatibility; samhd , sterile alpha motif domain-containing protein and histidine-aspartate domain-containing protein ; infa/b, interferon-a/b. viral interaction with the host ubiquitin-proteasome system luo table interplay between the viruses and the ubiquitin-proteasome system [ ] . expression of isg is highly inducible by type i ifn upon viral infection [ ] . animal studies have shown that mice with isg À/À or ube l À/À (which lack the isg conjugating enzyme) are more susceptible to various viral infections and develop more severe tissue damage [ ] [ ] [ ] . the mechanism of the anti-viral property of isg remains largely unclear. recent studies suggest that it involves the protective function of unconjugated isg against viral infection and posttranslational modification of both host and viral proteins [ ] . it has been shown that expression of isg blocks the activity of nedd , thereby inhibiting ubiquitination of hiv- gag and tsg proteins and ebola viral matrix proteins (vp ), which are necessary for viral budding/ release [ , ] . moreover, viral protein isgylation has been revealed to also contribute to the type i ifn-mediated anti-viral response. although the precise mechanisms remain to be established, available evidence supports a loss-of-function mechanism of isg modified viral proteins. it was reported that isgylation of the influenza a virus ns protein via the function of the e ligase uerc results in impaired viral replication [ ] . similarly, modification of the hpv l capsid protein by isg is linked to decreased viral production [ ] . in coxsackievirus infection, it was found that isg modification of a protease attenuates its activity in cleaving host eukaryotic translation initiation factor g, thereby counteracting host translation shutoff during viral infection [ ] . in addition to their role in regulating viral protein function, modification by ubiquitination and sumoylation has emerged as a central host anti-viral mechanism through modulating the function of key signaling molecules involved in innate immunity, such the pattern recognition receptor signaling pathway and the nfkb pathway (see reviews [ , , ] for the details). there is increasing evidence that viruses have evolved strategies to block these processes to evade host innate immune responses. for example, herpes simplex virus encodes the largest tegument protein, ul , which acts as a dub to remove ubiquitin chains from tnf receptor associated factor (traf ) and consequently inhibits ifn-b signaling [ ] . similarly, the orf protein encoded by kaposi's sarcoma-associated herpesvirus hydrolyzes ubiquitin chains from retinoic acid inducible gene (rig-i), resulting in decreased production of ifn [ ] . besides viral proteins, a number of anti-viral host proteins, such as interferon regulatory factor [ ] , rig-i [ ] , and protein kinase r [ ] , have been shown to undergo isgylation, resulting in a gain-of-function of these proteins and consequent anti-viral immune response. to overcome host innate defense, viruses have evolved to interfere with the isg conjugation system to antagonize its anti-viral activity. for instance, it was reported that the influenza b virus ns [ ] and vaccinia e protein [ ] interact with isg to prevent it from binding to ube l, thus inhibiting the formation of isg conjugates. furthermore, some viruses can encode their own deisgylating enzyme to disrupt isg conjugation. for example, the papain-like protease of sars-cov [ ] and the ovarian tumor domain-containing protease of nairoviruses and arteriviruses [ , ] possess both deisgylating and deubiquitinating activity that counteracts the post-translational modification of signaling molecules involved in the innate immune response. growing evidence has indicated a dual role for the ups in viral pathogenesis (summarized in figure and table ). the ups may represent a host defense mechanism against viral infection, or they may be hijacked by the virus to enhance its infectivity. viral manipulation of the ups has emerged as a central immune evasion mechanism. viruses evolve to inhibit many facets of the host anti-viral immune response. in some cases, viruses produce proteins to mimic the function of the ups or to redirect the cellular e to allow for targeted degradation of cellular factors or viral proteins that might hinder viral replication. in others cases, they encode proteins to interfere with the host ubiquitin or ubiquitin-like (e.g. sumo and isg ) conjugation system to inactivate the host anti-viral signaling pathways. in many cases, these mechanisms work together to dynamically modulate the function of the ups to gain maximal viral infection. a better understanding of the complicated interaction between the virus and the host ups with further identification of the new targets for viral e s or dubs and careful characterization of the functional consequence of the ubiquitin modification or degradation events will provide novel insights into the mechanism of viral pathogenesis, facilitate the discovery of new immune modulators, and promote the development of efficient antiviral interventions. nothing declared. themes and variations on ubiquitylation ubiquitin branches out sumoylation: a regulatory protein modification in health and disease concepts in sumoylation: a decade on emerging role of isg in antiviral immunity interferon-stimulated gene and the protein isgylation system ubiquitin and plant viruses, let's play together the ubiquitin-proteasome pathway in viral infections ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection the tumor suppressor protein p strongly alters human immunodeficiency virus type replication downregulation of p by double-stranded rna modulates the antiviral response the hpv- e and e -ap complex functions as a ubiquitin-protein ligase in the ubiquitination of p the e oncoprotein encoded by human papillomavirus types and promotes the degradation of p degradation of p by adenovirus e orf and e b k proteins occurs via a novel mechanism involving a cullin-containing complex transgenic expression of the d polymerase inhibits theiler's virus infection and demyelination the ubiquitinproteasome system regulates the accumulation of turnip yellow mosaic virus rna-dependent rna polymerase during viral infection sindbis virus rna polymerase is degraded by the n-end rule pathway interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis c virus rnadependent rna polymerase signals in hepatitis a virus p region proteins recognized by the ubiquitin-mediated proteolytic system evaluation of the susceptibility of the c proteases of hepatitis a virus and poliovirus to degradation by the ubiquitin-mediated proteolytic system identification and characterization of a protein destruction signal in the encephalomyocarditis virus c protease kinetic analysis of the conjugation of ubiquitin to picornavirus c proteases catalyzed by the mammalian ubiquitin-protein ligase e alpha hepatitis c: virus ns protein is phosphorylated by the protein kinase ck and targeted for degradation to the proteasome socs [corrected] inhibits hpv-e -mediated transformation by inducing degradation of e protein the papillomavirus e oncoprotein is ubiquitinated by ubch and cullin -and skp -containing e ligase mkrn induces degradation of west nile virus capsid protein by functioning as an e ligase proteasomal turnover of hepatitis c virus core protein is regulated by two distinct mechanisms: a ubiquitin-dependent mechanism and a ubiquitin-independent but pa gamma-dependent mechanism stability in vitro of the k movement protein of turnip yellow mosaic virus is regulated by the ubiquitin-mediated proteasome pathway degradation of tobacco mosaic virus movement protein by the s proteasome the nedd -type rsp p ubiquitin ligase inhibits tombusvirus replication by regulating degradation of the p replication protein and decreasing the activity of the tombusvirus replicase ubiquitin is part of the retrovirus budding machinery proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv- and hiv- a role for ubiquitin ligase recruitment in retrovirus release sudol m: ww domains and retrovirus budding proteins related to the nedd family of ubiquitin protein ligases interact with the l domain of rous sarcoma virus and are required for gag budding from cells overexpression of the n-terminal domain of tsg inhibits hiv- budding by blocking late domain function tsg and the vacuolar protein sorting pathway are essential for hiv- budding ubiquitination of tombusvirus p replication protein plays a role in virus replication and binding to the host vps p escrt protein cdc p ubiquitin-conjugating enzyme is a component of the tombusvirus replicase complex and ubiquitinates p replication protein a non-proteolytic role for ubiquitin in tat-mediated transactivation of the hiv- promoter ubiquitination of human t-cell leukemia virus type tax modulates its activity ubiquitination is required for effective replication of coxsackievirus b the envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein and is ubiquitinated ubiquitinated conjugates are found in preparations of several plant viruses evaluation of interactions of human cytomegalovirus immediate-early ie regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme ubc sumo- modification of human cytomegalovirus ie /ie sumoylation of the human cytomegalovirus -kilodalton ie protein facilitates expression of the -kilodalton ie protein and promotes viral replication sumo- modification required for transformation by adenovirus type early region b -kda oncoprotein cross-talk between phosphorylation and sumoylation regulates transforming activities of an adenoviral oncoprotein bovine papillomavirus e protein is sumoylated by the host cell ubc protein proteins of the pias family enhance the sumoylation of the papillomavirus e protein interaction between a geminivirus replication protein and the plant sumoylation system sce , the sumo-conjugating enzyme in plants that interacts with nib, the rna-dependent rna polymerase of turnip mosaic virus, is required for viral infection this paper demonstrated that post-translational modification of the rnadependent rna polymerase of turnip mosaic virus by sumo enhances viral infectivity the antiviral activities of isg this review summarized the functional role of the isg in regulating immune response against viral infection and discussed the current understanding of how viruses have evolved to interfere with the isg pathway and the mechanisms involved virgin hw: isg arg and the isg -conjugating enzyme ube l are important for innate immune control of sindbis virus mice lacking the isg e enzyme ube l demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza b virus infection ifnstimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg isg inhibits ebola vp vlp budding in an l-domain-dependent manner by blocking nedd ligase activity isg conjugation system targets the viral ns protein in influenza a virus-infected cells the isg conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg ubiquitinlike protein isg (interferon-stimulated gene of kda) in host defense against heart failure in a mouse model of virus-induced cardiomyopathy this study demonstrated that isg is induced in patients with viral cardiomyopathy and mice with isg knockout develop severe myocarditis and have a significant increase in viral titers and mortality interplay between viruses and host sumoylation pathways viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways this review discussed the role of post-translational modification by ubiquitin in regulating the stability, activity, and subcellular localization of key signaling molecules involved in the host anti-viral immunity and how viruses have evolved various mechanisms to counteract these innate anti-viral immune responses herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf this article showed that the ul protein of herpes simplex virus acts as a deubiquitinating enzyme that removes polyubiquitin chains on traf and consequently inhibits ifn-b production inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf positive regulation of interferon regulatory factor activation by herc via isg modification negative feedback regulation of rig-i-mediated antiviral signaling by interferoninduced isg conjugation human isg conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways influenza b virus ns protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg protein vaccinia virus e protein prevents the antiviral action of isg structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease this study identified the key sites required for the deubiquitinating and deisgylating activities of the papain-like protease of sars-cov ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling this work was supported by the canadian institutes of health research (cihr, mop- , and cci- ). key: cord- - q k k authors: gu, xiaoqiong; tay, qi xiang martin; te, shu harn; saeidi, nazanin; goh, shin giek; kushmaro, ariel; thompson, janelle r.; gin, karina yew-hoong title: geospatial distribution of viromes in tropical freshwater ecosystems date: - - journal: water res doi: . /j.watres. . . sha: doc_id: cord_uid: q k k this study seeks to understand the general distribution of virome abundance and diversity in tropical freshwater ecosystems in singapore and the geospatial distribution of the virome under different landuse patterns. correlations between diversity, environmental parameters and land use patterns were analyzed and significant correlations were highlighted. overall, the majority ( . %) of the annotated virome belonged to bacteriophages. the percentage of caudovirales was higher in reservoirs whereas the percentages of dicistroviridae, microviridae and circoviridae were higher in tributaries. reservoirs showed a higher shannon-index virome diversity compared to upstream tributaries. land use (urbanized, agriculture and parkland areas) influenced the characteristics of the virome distribution pattern. dicistroviridae and microviridae were enriched in urbanized tributaries while mimiviridae, phycodnaviridae, siphoviridae and podoviridae were enriched in parkland reservoirs. several sequences closely related to the emerging zoonotic virus, cyclovirus, and the human-related virus (human picobirnavirus), were also detected. in addition, the relative abundance of pmmov (pepper mild mottle virus) sequences was significantly correlated with rt-qpcr measurements ( . < r < . , p < . ). this study shows that spatial factors (e.g., reservoirs/tributaries, land use) are the main drivers of the viral community structure in tropical freshwater ecosystems. viruses are the most abundant living entities on earth and all living entities are associated with at least one virus which can control microbial communities (ackermann, ) . viral metagenomics have been reported widely in water cycles, especially marine waters, to evaluate the role of viruses in microbial diversity and biogeochemical cycling (angly et al., ; breitbart et al., ; cassman et al., ; culley et al., ) . determining the factors in constructing the viral community is important for both understanding and manipulating ecosystems (dinsdale et al., ) . factors shaping the viral community in freshwater ecosystems can include temporal factors, geospatial factors, natural disturbances (e.g., typhoon) and human activities (djikeng et al., ; emerson et al., ; fancello et al., ; ge et al., ; hwang et al., ; l opez-bueno et al., ; skvortsov et al., ; tseng et al., ) . among all the factors, land use activities is a major factor in shaping waterborne viromes. land use changes are the primary drivers of the viral community and a range of associated infectious waterborne disease outbreaks (patz et al. (patz et al. , . in the water cycle, agriculture brings excess nutrients and agricultural chemicals to surface waters, causing oxygen depletion and increasing algal blooms (foley et al., ) . urbanization degrades water quality through surface runoff, and human pathogenic viruses have been detected more frequently in watersheds with dominant urban and agricultural land cover (corsi et al., ; lenaker et al., ) . furthermore, human expansion into wildlife habitats or construction of zoos and animal parks provide opportunities for humanwildlife interactions, thus, increasing the risk for the possible transmission of zoonotic viruses to human populations (patz et al., ) . these different anthropogenic activities harbor diverse and distinct viral hosts, including bacteria, plants, wild animals and humans. it is hypothesized that the environment surrounding these reservoirs may favor distinct viral predators and further change the viral community characterization. however, up till now, studies of land use impacts on the virome community in freshwater ecosystems are still limited as they mainly rely on traditional methodology (culture-based method or qpcr/rt-qpcr), which focuses on limited human virus targets without considering the whole picture of the viral community in the water environment (corsi et al., ; lenaker et al., ) . to date, there is no systematic report focusing on the geospatial distribution and diversity of viromes in natural surface waters and how they may be impacted by human activities and the effect of different land use. in addition, emerging viral pathogens of zoonotic origin could also be discovered through viral metagenomics, for which information is very limited. singapore is a highly urbanized island located in the tropics with an area of . km , and a population of . million in (department of statistics singapore, ). water is a scarce resource in this country and a total of reservoirs are used to collect rainwater and surface waters for potable water supplies. increasingly, selected reservoirs are being used as focal points for sporting events (kayaking and dragon boating) and recreational activities so that the public can enjoy and appreciate water resources. as such, good water quality is needed to protect recreational users of these water bodies. previous studies have detected waterborne viral pathogens (e.g., norovirus gi/gii, adenovirus, rotavirus and astrovirus) in singapore water bodies using qpcr/rt-qpcr (aw and gin, ; aw et al., ; rezaeinejad et al., ) . thus, continued surveillance of these viral targets as well as a broader range of viral targets expanded to the virome community coupled with land use information, could provide important data and new dimensions for managing the safety of water resources. for these reasons, understanding the geospatial distribution of viromes is needed. in this study, seven tropical reservoirs with diverse upstream land use functions (i.e., urbanized, agricultural and parkland areas) were examined. the viral community structure and virome populations specific to each of these environments were systematically investigated together with the characteristics of the watershed. by conducting a complete virome analysis of these freshwater ecosystems (especially viral pathogens and fecal viral indicators), a comprehensive picture of the links between the virome community structure and specific land use activities could be elucidated and thus, used to conduct risk assessment of associated waterborne disease. this information would be important for environmental management at a macroscopic level to protect public health. thus, the objectives of this study were to: ) investigate the overall virome distribution and diversity in diverse freshwater ecosystems (reservoirs/tributaries) in a tropical environment, ) compare the virome community based on the different land use patterns, ) assess the extent of human-related pathogenic viruses in surface waters, especially emerging zoonotic and human-related viruses, which may have been undetected before. a total of seven reservoirs and three catchments were sampled in singapore during january (northeast monsoon) and april (inter-monsoon period), (table s ). in total, sampling points were surveyed, comprising of locations in the reservoirs and locations in the tributaries. only reservoirs , and had corresponding tributary sampling points. the study sites were divided into categories based on their geospatial characteristics: urbanized, agricultural and parkland areas (table ) . apart from storing water and preventing flood control, some of the reservoirs (i.e., r - , e ) also catered for recreational activities such as kayaking, dragon boating and water skiing. all the upstream tributary sites were designated as non-recreational areas. physical-chemical parameters, including temperature, ph, turbidity, conductivity, salinity, total dissolved solids (tds) and dissolved oxygen (do) were measured on site using a hanna meter probe (hi multiparameter meter; hanna instruments). -hour rainfall data were obtained from the singapore historical daily records ( http://www.weather.gov.sg/climate-historical-daily/ ). . . . primary concentration, secondary concentration and viral nucleic acids extraction -l water samples were collected from each sampling location in three -l carboys. the raw-water sample was immediately transported to the lab and concentrated through a hollow fiber ultrafiltration unit with blocking and elution buffer (hemoflow fresenius hf s, germany) to a final volume of ml. the hollow fiber ultrafilter was purged with nanopure water for min and pre-treated with ml of blocking solution ( . g of napp in l of nanopure water) for min. after that, the water sample was recirculated until a final volume of approximately e ml was reached. an elution step was carried out by recirculating around ml of elution buffer ( . g of napp, ml of tween , and ml of antiform in l of nanopure water) for min. both retentate and eluent were combined to a final volume of ml during primary concentration. ml of primary concentrate was further processed to enrich viral particles in a secondary concentration step through polyethylene glycol (peg) precipitation ( % peg (w/v) and . m nacl) after ph adjustment (ph ¼ . ) (jaykus et al., ) . after incubation of the mixture at c for h followed by , g for min, the pellet was dissolved in ml of phosphate buffer saline (pbs, ph . ) with an equal volume of chloroform. after centrifugation at g for min, the supernatant was filtered through a . mm sterile syringe and further concentrated to a final volume of ml using a kda ultracentrifugal filter device (merck millipore, ireland). after primary and secondary concentration, ml of viral nucleic acids (dna and rna) was extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) and then stored at À c (saeidi et al., ) . in order to quantify the human pathogens in reservoirs and their tributaries using qpcr/rt-qpcr, viral targets were performed to include: ( ) four genotypes of male-specific coliphages (frna gi-frna giv); ( ) ten human viral pathogens and ( ) one plant viral pathogen/microbial indicator (i.e., pmmov) ( table s ). the majority of targets belonged to group iv ssrna (þ) except for adenovirus (group i dsdna) and rotavirus (group iii dsrna). the details of qpcr/rt-qpcr primers and probes are listed in table s . the extracted viral nucleic acids were reverse transcribed using improm-ii reverse transcription system for detecting rna viruses following manufacturer's instructions (promega, usa). rt products were stored at À c for later analysis. qpcr/rt-qpcr was carried out in a steponeplus real-time pcr system (applied biosystems, usa) using faststart universal probe master (rox) (roche, germany) following miqe guidelines (bustin et al., ). the extracted nucleic acids were reverse transcribed and amplified to obtain a sufficient quantity of dna and cdna (wang et al., ) . to ensure there was no microbial contamination, the negative control was run in % agarose gel and ascertained that no dna gel band was present in the negative control lane. after purification of viral dna and cdna, samples were sent to scelse (singapore centre on environmental life sciences engineering). in total, libraries and one negative control library using illumina truseq nano dna library kit were constructed. the sequencing libraries with a corresponding insert size and adapters were prepared accordingly as previously described (ng et al., ) . the illumina's phix control library was used as standard. the libraries were then pooled and sequenced in one lane with equimolar concentrations on an illumina hiseq sequencer in rapid mode at a final concentration of pm and a read-length of bp pairedend (v sequencing reagents). the overview of the virome datasets is detailed in table s . the effect of the negative control is ignored, as explained in the supplementary information. meanwhile, s rrna gene amplicon sequencing was applied to the same batch of samples at sites e to characterize the bacteria community. a total volume of ml water samples from primary concentration was centrifuged at , g for min. after removing the supernatant, the pellet was transferred to a power-soil ® bead tube (mo bio laboratories, inc., carlsbad, ca) for dna extraction with a final volume of ml. genomic dna (gdna) was amplified for the marker gene targeting the s rrna variable regions v to v using the universal pyrotaq primer set ( wf and r) (rinke et al., ) . samples were sequenced in the university of illinois, research resources center -dna services facility and sequence preprocessing as described previously (te et al., ) . the biological data (subsampled otus) were square-root transformed to reduce the dominance of major otus before putting into primerv for pcoa and correlation analyses. the sequencing data were trimmed to remove adaptors, low quality reads, "primer b" sequences which were used for random amplification and phix reads following the bbtools instruction (version . ). reads were then de novo assembled into contigs by clc genomics (version . . ) through cross-assembly strategy. the assembly setting in clc genomics is: minimum contig length of bp and similarity fraction . . contigs were then uploaded into the metavir and virsorter pipeline. briefly, metavir pipeline will first extract open reading frames (orfs) from contigs through the metageneannotator. all predicted orfs were then compared to the national centre for biotechnology information (ncbi) refseqvirus protein database ( . . ) using a reference-dependent taxonomic analysis, with the threshold of bit score and an e-value cut-off of À . after getting the best blast hit affiliation of each predicted gene, annotation of each contig was made based on the lowest common ancestor (roux et al., ) . in addition, metavir pipeline will compute the genes affiliated to the pfam database for function analysis. virsorter identified category viral signal (pretty sure) and category viral signal (quite sure) from the assembled sequence and the viral signal included bacterial and archaeal viruses (roux et al., ) . in particular, human-related viruses were extracted from met-avir results and further examined to search against ncbi nonredundant nucleotide database (downloaded in june ) through blastn search for nucleotide similarity and query coverage (e-value < e- ). in order to quantify the relative abundance of each contig within different libraries, reads were remapped to contigs using novoalign (hercus, ) . this absolute matrix was used for quantifying the relative abundance of taxonomy assignment. since every contig has its weight in quantifying virus concentration, to standardize and quantify every contig's relative abundance between each library, rpkm (reads per kilobase of "transcript" per million mapped reads) was used after remapping trimmed reads to each contig across different libraries (mortazavi et al., ; reyes et al., ) . rpkm was calculated as followed: rpkm ¼ trimmed reads mapped to each contig per library the total trimmed reads mapped to this library ðmillionÞ*this contig length ðkbpÞ this matrix was equivalent to an otu table for pcoa analysis. the non-normalized matrix of reads mapping to each contig per sample (absolute reads matrix) was used and rarefied to , reads per sample (the smallest sample size in absolute reads matrix of taxonomy affiliated viruses was , ) to calculate a-diversity. "observed species" and "shannon diversity index" were calculated for each sample using macqiime (version . . ) with the iteration set at . multivariate analysis of viral community composition under different treatments (e.g., landuse, reservoir/tributaries, monsoon/ inter-monsoon season) was conducted using anosim in primerv and permanovaþ. the similarity of each pair of samples in terms of biotic characteristics was calculated using bray-curtis similarity. rpkm of the total contigs and of the virsorter category and phages were log plus one transformed before inputting into pri-merv for pcoa and correlation analyses. all the reference genomes of qpcr/rt-qpcr viral targets were downloaded from ncbi and accession numbers are detailed in table s . mapping was performed using the clc genomics workbench . . based on the default setting (mismatch gap ¼ , with the linear gap cost, length fraction: . , similarity fraction is based on the specific targets, at medium ( %) medium-high ( %) and high ( %, % or %) three levels) (table s ). the threshold of three levels could represent the nucleotide variation within groups of these viral targets (de graaf et al., ; doceul et al., ) . the proportion of mapping reads equals the number of mapping reads in each library divided by the total reads in the library. all geospatial analyses were performed using arcgis v. (esri, aylesbury, uk) and the percentage of different land use in different reservoirs and their tributaries are detailed in table . land-use shape files were obtained from the singapore land authority and pub (singapore's national water agency). four different layers of map were used for analysis: (i) catchment land-use shape files, (ii) river shape files, (iii) sub-catchment shape files and (iv) drain line maps. wgs_ _web_mercator_auxiliary_sphere was used as the projection coordinate system while gcs_wgs_ /svy singapore was used as the geographic coordinate system. population data was downloaded from the singapore statistics website (http://www.singstat.gov.sg/statistics/browse-by-theme/ geographic-distribution, basic demographic characteristic ) and the population density was calculated based on the total population within the planning area divided by the planning area which covered the drainage area for that sampling point. the shape file of master plan of planning boundary no sea was used as the reference map for the planning area classification (https://data. gov.sg/dataset/master-plan- -planning-area-boundary-no-sea, department of statistics singapore). all the statistical tests including one-way anova and spearman rank correlations were performed using ibm spss (ibm, portsmouth, uk). environmental parameters were log plus one transformed before correlating with a-diversity index as the raw data were right skewed. graphs were plotted using microsoft excel ( ) and originpro (originlab, northampton, ma). virome datasets were deposited in ncbi sequence read archive (sra) under accession no. srr - . s rrna sequences were deposited in ncbi sra under accession no. srr - . environmental parameters of the study sites (i.e., reservoirs and tributaries points) are illustrated in table s . the temperature remained relatively stable for all the sampling points, ranging from to . c with a mean value . c. ph ranged from . to . with a mean value . . other environmental parameters varied more, including conductivity ( . e . ms/cm, mean: . ms/cm), tds ( . e ppm, mean: ppm), do ( . e . ppm, mean: . ppm) and turbidity ( . e . fnu, mean: . fnu). the -hour rainfall and -day rainfall ranged from to . mm (mean: . mm) and e . mm (mean: . mm), respectively. the -hour rainfall and -day rainfall had higher mean values (i.e., . mm and . mm, respectively) during the northeast monsoon (jan) and lower mean values (i.e., . mm and . mm, respectively) during the inter-monsoon (apr). table summarizes the land use percentage and the population density for the reservoir catchments. to better evaluate land use impacts on the viral community, the sampling sites were classified into three categories: urbanized areas, agriculture areas and parkland areas. sites , and included approximately e % residential and urban land use, and as such can be regarded as urbanized areas. site had less than % of residential and urban areas, with about e % green and e % agricultural areas. site was the only sampling location with a reasonable percentage of agriculture area; therefore, site was considered as agricultural. site and site had similar land use percentage with more than % of the land covered with green areas; thus, it was considered as parkland. site included % of residential and urban categories. however, unlike sites e , there was no drain directly connected to the reservoir at this site and hence, the chance for possible viral contamination was relatively small. therefore, site was classified as parkland areas as well as site and site . the land use category clustering of the seven different sites illustrated the reasonability of our land use category classification (fig. s ). the population density was obtained from the singapore department of statistics. among the sample locations, sites , and had the highest population densities (> people/km ) while sites , and had the lowest (< people/km ). a total of . hundred million high-quality hiseq sequencing reads of samples were obtained from different reservoirs and their tributaries in singapore during the monsoon and inter-monsoon periods (table s ). these were assembled across different libraries and , contigs were generated with an average length of bp (n ¼ bp). on average, . % ± . % reads mapped back to the assembled contigs per library (min: . %, max . %). of the , contigs, , contigs were annotated using metavir pipeline. based on the absolute reads matrix (the number of reads mapped to each family in each sample) and the metavir annotation result, on average, . % was annotated and a total of families were identified (fig. ) . the majority ( . %) of these reads belonged to the caudovirales (myoviridae, siphoviridae and podoviridae). this was followed by reads for phycodnaviridae and mimiviridae (whose hosts belong to algae and amoeba) which were retrieved at percentages of . % and . %, respectively. apart from these major viral families, other families such as dicistrovidae, microviridae, circoviridae, iridoviridae and poxviridae were also present in our study. . % were unclassified viral sequences and more than % were unassigned (na) sequences. function analysis of the , viral contigs identified , genes and , ( . %) of the genes encoded hypothetical proteins or conserved hypothetical proteins. the most retrieved pfam annotations showed that phage terminase enzyme, phage portal and phage integrase were responsible for the phage function (table s ) . fig. a shows the taxonomic percentage of virome in reservoirs and their tributaries (sites , and ). fluctuation could be observed within tributaries at the same sampling site. in contrast, virome composition in reservoirs was similar across several sampling points in singapore and shows a more stable pattern than the tributaries (fig. b) . on average, caudovirales accounts for . % among all the annotated viruses in all the tributaries (sites , and ) with a proportion of . % and . % in jan and apr, respectively. for the reservoirs, caudovirales comprises an average of . % among all the annotated viruses with a proportion of . % and . % in jan and apr, respectively. note that the percentage of caudovirales was higher in all the reservoirs than in all the tributaries. in contrast, the percentage of dicistroviridae was higher in tributaries ( . %) than in reservoirs ( . %) in jan (tables s and s ). the percentage of microviridae and circoviridae in tributaries was approximately . % (reservoirs: . %) and . % ( . %), respectively. the shift in composition was observed as the percentage of caudovirales decreased while the percentage of dicistroviridae, microviridae and circoviridae subsequently increased, as locations changed from reservoirs to tributaries. viral communities in different reservoirs in terms of land use impact were compared and the majority of viral communities were largely conserved and stable at the family level with myoviridae, siphoviridae and podoviridae as the main family level, and small differences observed in dicistroviridae and other families (fig. b) . both the richness and diversity (observed species and shannon diversity) was calculated on the taxonomically affiliated viruses from the subsampled absolute reads matrix. the species richness of viral samples from reservoirs with tributaries comprising mostly agriculture and urbanized areas had a similar amount of species while those from parkland areas showed lower viral richness, which was reasonable as parkland areas would generally receive less viral contamination. it was also noted that the viral species richness from tributaries was also lower than their corresponding reservoirs. the average shannon diversity of reservoirs and tributaries was computed as . and . respectively and a one-way anova test showed that the shannon-diversity of tributaries was significantly smaller than those from reservoirs (p < . ). this indicated that tributaries were more likely to be dominated by selected species compared to the reservoirs (fig. s ) and this may be related to the viral host's presence (e.g., bacteria, archaea, algae, insects, plants, animals). future studies could explore this further. a spearman rank correlation was carried out between richness and diversity (observed species and shannon index calculated by macqiime), environmental parameters and the land use pattern ( table ). the correlation coefficient showed that the richness index of observed species was significantly positively correlated with rainfall and agriculture while the shannon index was negatively correlated with ph. when looking at the b-diversity of the microbial community (< . mm) structure, the distribution of rpkm (< . mm microbial fig. a and b ). this pattern was consistent with the bacterial community geospatial distribution ( fig. e and f) . mimiviridae, phycodnaviridae, podoviridae and siphoviridae were enriched in parkland areas (spearman r ¼ . e . to pco ) whereas dicistroviridae and microviridae were enriched in urbanized areas (spearman r ¼ À . to pco ) (fig. a) . acidobacteria, actinobacteria and verrucomicrobia were enriched in parkland areas (spearman r ¼ À . to . to pco ), cyanobacteria were enriched in agricultural areas (r ¼ À . to pco ) whereas proteobacteria were enriched in urbanized and agricultural areas (spearman r ¼ À . to pco ) (fig. e ), comparable to a recent study conducted in singapore showing that proteobacteria were enriched in horticultural and residential samples (nshimyimana et al., ) . the microbial community (< . mm) and virsorter category and phage community in the seven reservoirs both showed distinctly different distributions within reservoirs/tributaries and within different sites ( fig. c and d) . different sampling points of reservoirs at sites and did not show any difference. for sites , and , the microbial community shared a similar contig profile even though they did not share the same land use pattern (i.e., sites and : urbanized area; site : agriculture area). noticeably, urbanized area site showed a slightly different pattern when compared with the other three reservoirs, perhaps due to potential different viral host communities (site is near the construction sites). the microbial community in the tributaries for sites , and were much more variable spatially and temporally, compared to the reservoir microbial community. in our study, contigs were affiliated to human picobirnaviruses and contigs were affiliated to cycloviruses. they had a higher relative abundance in the urbanized and agricultural reservoirs and all the tributaries (fig. ) . possible viral contamination could be from urban drains including those from housing estates, sewer leakage and construction sites. although previous studies have detected enteric viruses in urban catchments in singapore (aw and gin, ; aw et al., ; liang et al., ; rezaeinejad et al., ) , our metagenomics sequencing was unable to find contigs assigned to the enteric viruses commonly detected in singapore surface waters (e.g., norovirus gi, norovirus gii, adenovirus, rotavirus). however, several fecal indicators including pmmov, frnagi (ms ) and frnagiii (qbeta), were detected in our samples. interestingly, in our study, the pmmov-affiliated contigs had a higher nucleotide similarity ( . %e . %) with the average query coverage of . % (table ), suggesting that pmmov is a highly conserved virus with a lower evolution rate in the surface water environment. this result is similar with a recent study, where pmmov detected in % of surface water samples shared e % nucleotide identity with pmmov reference genome (rosiles-gonz alez et al., ). . . correlation of metagenomics data with molecular assays for selected viral targets qpcr/rt-qpcr was conducted to detect and quantify the concentration of the common enteric and zoonotic viruses in the surveyed communities. viral targets ( viral pathogens, fþ malespecific coliphages and plant virus) were measured and the detection frequency was between and . % across all the samples (table s ). the viral pathogens detected in this study using qpcr/rt-qpcr included enteric viruses (adenovirus, astrovirus, rotavirus, norovirus gi, norovirus gii, enterovirus and aichi virus) and other zoonotic viruses (e.g. hepatitis e virus, saporo virus). reads mapping to human-infective viruses were rare and were not observed to assemble into contigs. high-quality reads from different libraries were aligned to the viral pathogen database (vipr/ird) and mapped to viral reference genomes in ncbi (table s ). few reads ( e reads per library) were assigned to the human-infective viruses norovirus and saporo virus using both vip pipeline and standalone pipeline (results not shown) (li et al., ) , and e reads per library were mapped to human viral pathogens reference genome at the e % nucleotide similarity level. no significant correlations were observed between qpcr measurements and read mapping for these viruses. these results suggest that metagenomics is less sensitive at detecting lowabundance viral pathogens against a background of a large proportion of bacteriophages. a total of targets were found in both the metagenomics (contigs) and qpcr data. of these, pmmov showed a good spearman rank correlation (p < . ) ( table ). spearman rank correlation was carried out between the pmmov molecular results and the corresponding metagenomics data in terms of contigs level and reads level (table ). in terms of contigs level, all pmmovaffiliated contigs showed a significant good correlation between molecular results and metagenomics data ( . < r < . , p < . ). in terms of reads level, out of the libraries, libraries ( . %) had reads mapping to the pmmov reference genome (nc_ . ) with % identity to the reference genome. based on all the samples, the spearman rank correlation coefficient was . (p < . ) ( table ) . this study is the first to correlate land use impact with freshwater water viromes in an urban tropical environment using illumina hiseq sequencing. it is noteworthy that metagenomics of dna viruses and rna viruses have been often considered separately and that studies of dna viruses are far more than rna virome analysis. this disparity is largely a result of technical challenges presented by rna metagenomics as rna is fragile and needs to be reverse transcribed into dna. thus up till now, the majority of the virome metagenomics papers studied either dna or rna (but not both). moreover, the amplification protocol reported in these earlier studies may have influenced the final outcome (l opez- bueno et al., ; rodriguez-brito et al., ) . for dna viruses, the genomiphi kit (ge healthcare) has been used for amplification through dna polymerase phi but it was discovered that it has a bias towards single-stranded circular dna viruses. for rna virome analysis, random priming mediated sequence independent single primer amplification (rp-sispa) was used in lake needwood, maryland (djikeng et al., ) . wang et al. ( ) developed a protocol capable of amplifying dna and rna viruses simultaneously. this was subsequently tested in reclaimed water, plasma samples, wastewater and ballast water (bibby and peccia, ; kim et al., ; rosario et al., ; wylie et al., ) . rna viruses account for more than % of the viral pathogens due to their adaptive abilities (mutations, recombinations or reassortments) to infect novel hosts easily (temmam et al., ) . in our study, . % of viruses are dna viruses whereas . % are rna viruses among annotated viral families. among all the viral pathogens detected by metagenomics, contigs affiliated to human picobirnaviruses are dna viruses whereas contigs affiliated to cyclovirus are rna viruses. the simultaneous application of combining both dna and rna metagenomics enabled the identification of a much larger numbers of viral sequences, especially for human-related viruses. this was the approach taken in our study here. in this geospatial metagenomics study, we identified virome abundance and diversity in different reservoirs and tributaries. due to insufficient viral genome sequencing information in the ncbi, . % of the virome genome had no significant hits and bacteriophages were the most abundant organisms in the matrix, which is in agreement with previous studies of surface water viromes (mohiuddin and schellhorn, ; skvortsov et al., ; tseng et al., ) . phages, considered to be the most abundant and diverse biological entities on earth, play an important role in a ms belongs to the group of frna gi (animal-specific indicator) and qbeta belongs to the group of frna giii (human-specific indicator). spearman rank correlation between qpcr (gc/ ml) and metagenomics data (rpkm) on pmmov (n ¼ ). shaping biological and geochemical processes at the global scale (díaz-muñoz and koskella, ) . they may also be responsible for human health in spite of the fact that they do not infect humans. this is because some phages can convey new properties of coding for toxin production to the host bacteria, thus converting harmless bacteria into pathogens (grabow, ) , for example, the shiga toxin-converting phages have been known to change the pathogenicity of e. coli o :h (muniesa and jofre, ) . tributaries were found to have a higher proportion of microviridae and dicistroviridae. the hosts of microviridae belonged to enterobacteriaceae and obligate parasitic bacteria (roux et al., ) . most of the enterobacteriaceae taxa strains are pathogenic, for example, virulent strains of e. coli and klebsiella pneumonia. enterobacteriaceae occurred more in tributaries, which may contribute to the distribution and abundance of microviridae in tributaries. the hosts of dicistroviridae are soil-inhabiting invertebrates (e.g., aphids and ants, which are common in tropical singapore). thus, the fact that tributaries harbor the viral hosts, indicate that the viruses may be released from catchments and be washed into the tributary periodically. the smaller spatial variation pattern of larger reservoirs compared with tributaries was not surprising, as reservoirs tend to be more resilient to urban and storm water runoff than their tributaries. in previous studies, freshwater microbial communities have been found to be resilient to natural disturbances (tseng et al., ) . activities on a-diversity and community structure when correlating a-diversity with environmental parameters and land use factors, it was found that ph had a significant correlation among all the environmental parameters (r ¼ À . , p < . ). ph is a key factor in determining virus infectivity where low ph (ph < ) has been found to significantly reduce phage survivability ( e %) (jurczak-kurek et al., ) , even though the ph range in this study is relatively small ( . e . ), it is likely that these differences could make a difference in viral a-diversity in our study. a moderate correlation with rainfall (r ¼ . , p < . ) was observed which could be due to heavy rainfall flushing terrestrial bacteria, viruses and nutrients to the reservoir (tseng et al., ) . precipitation was found to be the major factor affecting the microbial community in a subtropical reservoir in taiwan (tseng et al., ) . singapore is characterized by two main monsoon seasons: the northeast monsoon (decemberemarch) and southwest monsoon season (juneeseptember). -hour rainfall during the sampling times during the monsoon and inter-monsoon season showed a significant difference between january ( . mm) and april ( . mm) (one-way anova, f ¼ . , pvalue ¼ . ), which could have contributed to the characterization of the virome composition. based on land use category, observed species were also found to be significantly correlated with agriculture land use (r ¼ . , p < . ). this could be expected as intensive agriculture has been reported to bring nutrients and agricultural chemicals to the water cycle (foley et al., ) , which in turn, disturbs the microbial community indirectly (tseng et al., ) . agricultural intensification has been associated with pathogen emergence transmission between wildlife and domestic animal populations and human populations, which in turn, could increase the species of zoonotic viruses (pulliam et al., ) . jones et al. ( ) concluded that agricultural practices are one of the socioeconomic drivers in the spatial distribution of emerging infectious diseases. however, the non-significant correlation between diversity and other land use types does not necessarily suggest weak connections between the viral community and land use factor, since a-diversity indices (shannon diversity and observed species) are just one aspect of characterizing the viral community and does not necessarily represent a complete view. b-diversity analysis indicated that mimiviridae, phycodnaviridae, siphoviridae and podoviridae were enriched in reservoirs of parkland areas while dicistroviridae and microviridae were enriched in tributaries of urban areas (fig. a) . siphoviridae, podoviridae and microviridae belong to bacteriophage, which may be associated with parkland and urban enriched bacteria (i.e., actinobacteria, verrucomicrobia, chloroflexi, acidobacteria and proteobacteria) in our study (fig. e) . overall, viral diversity in the surveyed reservoirs ( < h < ) was higher than that reported in a subtropical reservoir in taiwan ( . < h < . ) (tseng et al., ) . these differences could be due to the geographical (latitudinal gradient) differences in microbial diversity, as low-latitude tropical ecosystems tend to lead to higher biological diversity (chown and convey, ; fuhrman et al., ) . kim et al. ( ) reported that viruses had higher richness near the equator and lower richness at higher latitude, similar to human pathogen species (guernier et al., ) . however, these comparisons between the diversity in freshwater virome studies need further confirmation due to different indices used (chao , simpson index, shannon index, and observed species) and diverse calculation methods applied (e.g., phaccs, qiime, catchall). by using phaccs based on the contig spectra generated by circonspect, tseng et al. ( ) derived a shannon diversity (h) range from . to and from . k to . k viral genotypes in one subtropical freshwater reservoir, which was lower than the ocean's virome shannon diversity in british columbia, the gulf of mexico and the sargasso sea (h of . , . and . , respectively) (angly et al., ) . the difference in methods used in published papers makes it difficult to draw comparable conclusions. a standard pipeline in deriving the viral diversity in ecology is required in order to make comparisons of viral diversity across diverse aquatic ecosystems from different studies. the graphs of pcoa analysis in both reservoirs and tributaries indicated that the land use pattern around the surveyed areas had an important impact on characterizing the virome community. even though the viral communities at the family level were conserved across different reservoirs, the pcoa plots suggested a dynamic shift in differences between contig levels of the viral community in terms of land use. the geospatial distribution patterns in the surface water environment could have resulted from both direct and indirect factors. on the one hand, the different land use patterns could also introduce foreign viral contamination into water bodies directly through urban/agriculture runoff, precipitation, leaking sewers, etc., while the viral community itself can be indirectly changed and characterized by the relationship to their hosts in specific environments. the runoff from the surrounding areas can also bring specific bacteria, or other vectors and nutrients into the reservoirs and the tributaries (tseng et al., ) . even though previous studies have shown the correlation between land use and water-borne viral pathogens (corsi et al., ; lenaker et al., ) , ours is the first study correlating land use cover with the whole viral community, thus overcoming the limitations of investigating specific viruses and providing comprehensive information on the community structure with the relationship of land use cover. viral pathogens could be introduced into reservoirs and tributaries through urban and storm water runoff. although enteric viruses (e.g., adenovirus, norovirus, rotavirus, enterovirus, etc.) could not be detected with viral metagenomics in our study, past studies have shown that they are prevalent in tributaries using qpcr and thus, need to be carefully monitored for quantifying risk assessment and providing guidelines for water recreational activities (aw and gin, ; aw et al., ) . vergara et al. ( ) quantified illness risks of norovirus in an urban catchment in singapore by using quantitative microbial risk assessment. the finding reported mean probability of illness associated with norovirus were . and . in the scenario of primary contact recreation of adults and children, which is below the usepa guideline value of . (usepa, ) . in addition, other potential emerging viral pathogens (e.g., hepatitis e virus, coronavirus, cyclovirus, bird flu virus), which may originate from wildlife and indigenous animals in tropical forests or animal parks can also spread to neighboring tributaries, potentially causing disease to humans. according to jones et al. ( ) , the emerging infectious diseases are dominated by zoonoses ( . %) and the majority of these are from wildlife ( . %), with an increasing trend and more hotspots concentrated in lower-latitude developing countries. in this study, we observed sequences related to the humaninfective virus, human picobirnavirus and the emerging zoonotic virus, cyclovirus. human picobirnavirus, a bi-segmented doublestranded rna virus, has been detected in both healthy and unhealthy human beings. it has also been found to be prevalent ( % detection frequency) in raw sewage samples collected in the united states (symonds et al., ) . the pathogenicity of human picobirnaviruses has not been established and it has been suggested as an opportunistic pathogen which might cause diarrhea (giordano et al., ; grohmann et al., ) . as metagenomics can only provide relative abundance, further studies are needed using qpcr in order to determine absolute concentrations and to better evaluate health risks. viruses belonging to the circoviridae (approximately . % of the virome) may be involved in disease in vertebrate animals and plants. a large proportion was found to belong to swan circoviruses ( %) and circoviridae ldmd- ( %). two contigs found in the present study were assigned to the suggested cyclovirus-vn which originated from human samples in vietnam (garigliany et al., ) . indeed, cyclovirus vn was initially reported to be restricted to central and southern vietnam but was subsequently detected in both farm animals and human clinical samples from africa, indicating their geographic transmission capacity (garigliany et al., ; van doorn et al., ) . in singapore, human cyclovirus vs (cycv-vs ) was previously found in singapore harbor water by using a metagenomics approach (kim et al., ) . the contig shared a . % nucleotide similarity with human cyclovirus vn (kf ) with a query coverage of . %. the discovery of cyclovirus vn in freshwater tributaries in the densely populated area of site in our study indicates a possible transmission of the emerging human cyclovirus in the singapore urban water cycle. further risk assessment of host populations should be conducted for these water environments (van doorn et al., ) . samples from sites , and had lower occurrence rate (both rpkm and absolute reads matrix) and concentration of human picobirnavirus and cyclovirus affiliated viruses, consistent with lower human population density (< people/km ) except for reservoir . the exception of reservoir (explained in results . ) suggested that drainage points could also be significant drivers in shaping the viral communities as well as land use cover and population density. higher population density could result in high occurrence of human-related viruses (lenaker et al., ) . here, the population density to some extent could reflect the hotspots of human-related viruses. limitation of using population density to quantify human activities and human-related viral hosts in our study exists as the population density referred to is the resident population. however, the mobile population such as those associated with modern transportation, tourism, business travel and immigration could also contribute to dissemination of these highimpact pathogens (arguin et al., ). in addition, public holidays could introduce variation in population numbers to commercial and recreational areas based on lifestyle choices. thereafter, more detailed data will be needed in order to track and investigate the human-related virus transmission patterns through virus-host interaction. in our sequencing data, the absence of the majority of enteric viruses in surface waters is reasonable. a potentially relatively low abundance of human-related viruses in our surface water system and insufficient sequencing coverage could have resulted in rare sequences not being assembled into contigs for further downstream analysis. in contrast with our freshwater ecosystem, a previous study of sewage sludge samples revealed a large number of human viral pathogens, unveiling types of human viruses (bibby and peccia, ) . this result is expected as the sludge matrix harbors large amounts of human-related viruses and the concentrations of viral pathogens are much higher. in our study, a good spearman rank correlation ( . < r < . , p < . ) for the indicator virus pmmov was discovered as the qpcr/rt-qpcr concentrations of this target are relatively higher (pmmov geomean: . gc/l). this suggests that viral metagenomics, to some extent, is a conservative estimate of the true viral abundance, based on validation of rt-qpcr data with rpkm in relative abundance of contigs, especially for contigs with a higher qpcr concentration. similar results were also obtained in a previous study where a significant correlation (p < . ) between qpcr and rpkm across viral taxa in clinical samples was obtained (graf et al., ) . overall, viral metagenomics has its advantage in the simultaneous discovery of the entire set of targets in the community in spite of the intrinsic limitation of downstream bioinformatics (i.e., assembly efficiency and database bias). it can pave the way in finding emerging zoonotic viruses and alternative plausible fecal indicators in predicting viral pathogens in the future. after zooming into the specific and desired targets using high throughput sequencing, gene-specific pcr or qpcr (e.g., cyclovirus-vn) could be further investigated to confirm the presence of these potential viral pathogens and zoonotic viruses to reveal the epidemiology or transmission patterns of these viral pathogens (kim et al., ) . this study has shed light on the diversity of the viral communities in tropical reservoirs and their tributaries with different land use. correlations between the diversity index, physical-chemical parameters and land use patterns showed that environmental parameters (i.e., ph and precipitation) and spatial factors (e.g., reservoirs/tributaries, land use) are the main drivers 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freshwater reservoir subject to climatic disturbances identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections risk assessment of noroviruses and human adenoviruses in recreational surface waters microarray-based detection and genotyping of viral pathogens sequence analysis of the human virome in febrile and afebrile children this research grant is supported by the singapore national research foundation under its environment and water research programme and administered by pub, singapore's national water agency (ref: -iris- [idd / / ]). we would like to thank national university of singapore and center for environmental sensing and modeling (censam) for supporting this research. supplementary data related to this article can be found at https://doi.org/ . /j.watres. . . . key: cord- -qeojdpez authors: remolina, yuly andrea; ulloa, maría mercedes; vargas, hernán; díaz, liliana; gómez, sandra liliana; saavedra, alfredo; sánchez, edgar; cortés, jorge alberto title: viral infection in adults with severe acute respiratory infection in colombia date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: qeojdpez objectives: to identify the viral aetiology in adult patients with severe acute respiratory infection (sari) admitted to sentinel surveillance institutions in bogotá in . design: a cross-sectional study was conducted in which microarray molecular techniques for viral identification were used on nasopharyngeal samples of adult patients submitted to the surveillance system, and further descriptions of clinical features and relevant clinical outcomes, such as mortality, need for critical care, use of mechanical ventilation and hospital stay, were obtained. setting: respiratory infections requiring hospital admission in surveillance centres in bogotá, colombia. participants: ninety-one adult patients with acute respiratory infection ( % were female). measurements: viral identification, intensive care unit admission, hospital stay, and mortality. results: viral identification was achieved for patients ( . %). comorbidity was frequently identified and mainly involved chronic pulmonary disease or pregnancy. influenza, bocavirus and adenovirus were identified in . %, . % and . % of the cases, respectively. admission to the intensive care unit occurred in . % of the cases, while mechanical ventilation was required for . %. the average hospital stay was . days, and mortality was . %. antibiotics were empirically used in . % of patients. conclusions: the prevalence of viral aetiology of sari in this study was high, with adverse clinical outcomes, intensive care requirements and high mortality. acute respiratory infection is one of the main causes of hospitalization and death worldwide, although identification of the aetiological agent is not achieved in a majority of cases. instead, the infections are treated empirically and often successfully with antimicrobial therapy. nonetheless, the roles of viruses in the aetiology of these infections are becoming clear, especially after the pandemic of the new influenza a subtype h n [ ] . the presence of a virus does not imply either a more benign clinical course or that systemic inflammatory responses or complications will be absent [ ] . due to its implications for public health, the efforts in reinforcing and improving the epidemiological surveillance of respiratory infections have increased. under this initiative, countries have developed surveillance systems by following cases of influenza-like illness and severe acute respiratory infections (saris), which are clinically diagnosed among patients with fever, coughing or sore throat, difficulty breathing and the need for hospitalization [ ] . the main aims of surveillance have been to provide information on circulating viruses and the susceptibility of influenza to available antivirals and also to promote and define vaccination needs in different populations. the true impact of viral infections in the aetiology of acute respiratory disease requiring hospitalization is unknown [ ] . the aims of this study were to identify viral aetiologies in hospitalized adult patients with sari in bogotá in and to describe the characteristics and clinical outcomes among these patients. this study was performed in colombia's capital city of bogotá, which is located in the andes in south america, near the equator and , metres above sea level. a total of tertiary care hospitals performing sentinel surveillance of sari during participated in the study. such hospitals forwarded all respiratory samples from patients with sari to the district health department. sari was defined as any respiratory infection with a possible viral and/or bacterial origin requiring inpatient management and a clinical presentation of fever of less than days after onset and higher than °c, shortness of breath, cough, hypoxia and systemic compromise (systemic inflammatory response syndrome or organ failure), depending on symptom severity [ ] . the samples were taken via nasopharyngeal aspiration or swab. the samples were sent, together with the required basic epidemiological data collection form, through the epidemiological surveillance system (sistema de vigilancia epidemiológica nacional-sivigila). samples were sent in a viral transport medium to the public health laboratory of the district health department, where they were stored between and °c in refrigerators intended for this purpose. need for intensive care, the use of mechanical ventilation and the length of hospital stay. inclusion criteria consisted of patients over years of age who provided a respiratory sample in at one of the hospitals performing sentinel surveillance. patients with incomplete medical records from the sampling institution were excluded. the following research and ethics committees of each participating institution approved the study: , with the exception of one institution that requested written informed consent to access medical records (fundación cardioinfantil, instituto de cardiología). written consent was obtained from participants or their proxies (in case of death) from that institution ( patients were not found, did not respond or refused to provide written consent). written informed consent, in the cases in which was requested and obtained, was kept at universidad nacional de colombia. a total of nasopharyngeal aspiration or swab samples were taken during from adult patients of the hospitals. the sample was randomly selected, with the only inclusion criteria being patients older than years of age with sari reported to the surveillance system during that year. a sample size calculation found that respiratory samples were needed for a prevalence of %, with the poorest accepted result of %, % power and a % confidence level. however, because molecular tests could be processed, the sample was increased to patients. subsequently, the medical records were reviewed, and after the approval and authorisation of the research and ethics committee of each institution, the information was gathered from each hospital. a microarray diagnostic assay using clart pneumovir equipment by genomica (madrid, spain) was used to identify the viruses involved [ ] . this assay detects and characterises viruses that most frequently cause respiratory symptoms in humans. the following viruses were analysed: respiratory syncytial virus (rsv) a and b, influenza a (h n , h n , a/h n pdm), b and c virus, parainfluenza virus subtypes , , , a and b, metapneumovirus a and b, adenovirus, enterovirus, rhinovirus, coronavirus subtype e and bocavirus. this kit amplifies specific fragments of the viral genome via a reverse transcription polymerase chain reaction (pcr) or via a pcr with hybridisation detection using specific capture probes [ ] . the following variables were collected from the patients' medical histories according to international definitions: age, gender and comorbidities such as chronic obstructive pulmonary disease (copd), diabetes mellitus and heart failure. the inclusion criteria took into account the systemic inflammatory response syndrome (sirs), which was defined as a heart rate over beats per minute, a breathing rate of over breaths per minute, leukocytosis of over , cells per millilitre or leukopenia of fewer than , cells per millilitre and fever; pneumonia among sari patients was diagnosed from radiological findings of consolidation or alveolar infiltrates. the confusion, urea, respiratory rate and blood pressure (curb)- scores for pneumonia were applied using the criteria of age greater than or equal to years, impaired consciousness, blood urea nitrogen (bun) over mg/dl, breathing rate above breaths per minute and a systolic arterial pressure under mmhg or a diastolic pressure under mmhg [ ] . additionally, severe pneumonia was considered among patients complying with the following major and minor criteria of the american thoracic society/infectious disease society of america (ats/idsa): shock, need for mechanical ventilation, thrombocytopenia (platelets under , ), an arterial oxygen partial pressure to fractional inspired oxygen ratio (pao /fio ) ratio, multilobar compromise, impaired consciousness, leukopenia (under , ) and bun over mg/dl [ ] . shock was also considered when the systolic arterial pressure was below mmhg or when the diastolic arterial pressure was under mmhg. moderate oxygenation impairment was defined by a pao /fio below mmhg and over mmhg, and severe oxygenation impairment was defined by an index less than or equal to mmhg. institutions that identified viral antigens following institutional protocols performed and interpreted these tests in their hospitals. colonisation of the airway was defined as the presence of microorganisms in a gram test or in a culture of the airway. gram tests and sputum cultures or other respiratory samples were performed based on clinical decisions made by the doctor from the institution. a viral co-infection diagnosis was made when or more different viruses were identified in the same sample. the chi-square test and fisher's exact test were used to compare categorical variables, and the comparison of continuous variables was performed with either student's t test or the mann-whitney u test on a case-by-case basis. the variable analysis was performed using stata (ver. . ), and p values less than . were considered significant. odds ratios (or) with corresponding % confidence intervals (ci) were used to analyse outcomes. in in bogotá, samples from respiratory secretions were collected in the sentinel surveillance system from patients over years of age. through randomised sampling, cases of sari were selected from those respiratory samples (fig ) . the medical histories of patients with sari reported through the surveillance system were examined. these histories were obtained from bogota hospitals in according to the following distribution: ( . %) were from the san rafael university hospital clinic, ( %) were from fundación cardioinfantil, ( %) were from suba hospital, ( . %) were from el tunal hospital, ( %) were from santa clara hospital, ( %) were from san ignacio university hospital, and ( %) were from the occidente de kennedy hospital. of the patients, showed disease progression beyond days and were dismissed from the final analysis (fig ) . of the patients included in the analysis, ( %) were female; the patients' average age was . years (range, to years). table shows the most frequent comorbidities identified. the average length of disease progression was . days, with a minimum duration of hours and a maximum duration of days (table ) . radiographical abnormalities were observed in patients. the following radiological findings of chest images were described in the medical histories, in order of frequency: patients had interstitial infiltrates ( . %), had alveolar infiltrates ( . %), had consolidation ( . %), had multilobar compromise ( . %), and had pleural effusion at admission ( . %). pleural effusion was found in . % of the patients with alveolar infiltrates and . % of the patients with interstitial infiltrates. we among the sari patients, ( . %) were treated with antibiotics; patients received between and antibiotics, with an average of . antibiotics per patient. antibiotics were started a median of day after admission (range, to days, % ci: - days). the beta-lactam group was the most frequently used. oseltamivir was used in . % of cases ( patients), with a minimum duration of day, a maximum duration of days and an average duration of . days. oseltamivir was started a median of day after admission (range, to days, % ci: - days). at least one virus was identified in patients (table ) . influenza virus was the most common and was isolated in patients ( . %), of whom ( %) had influenza a and ( %) had influenza b (table ) . of the parainfluenza virus subtypes identified, five cases were piv , and one case was piv . in the group with more than one viral infection ( influenza and bocavirus were identified throughout the year, though cases of influenza were commonly found in may, august and september, and cases of bocavirus were commonly found in june and november. influenza b was identified only in the second semester, while the two cases of influenza a/h n were seen in may. adenovirus was seen only during the rainy season (april to may and august to november); most cases of metapneumovirus, parainfluenza and rsv were seen only in the first semester, specifically in april and may; the two cases of coronavirus were identified in may (fig ) . patients with and without viral detection did not differ in terms of comorbidity, with the exception of the frequency of diabetes mellitus. diabetes was detected in . % of patients with viral detection and . % of patients without viral detection (fisher's exact test, p = . ). of the patients with pneumonia, a virus was identified in patients ( . %). severe pneumonia was diagnosed in . % of patients without viral detection and . % of patients with viral detection. no significant differences were observed between the groups with and without viral detection with regards to sirs, neutropenia during hospitalization or shock. of the patients with sari and a history of copd, viruses were identified in ( . %), of whom had viral co-infections. the viruses were distributed as follows: cases of bocavirus, of adenovirus, of influenza a, of parainfluenza, of rsv, of metapneumovirus, of coronavirus and of rhinovirus. of the patients, were active smokers and ( . %) had a viral infection, with bocavirus being the most frequently isolated ( . %) virus. with regards to complications, pleural effusion developed in . % of patients without viral identification and % of patients with viral identification; closed thoracotomy was required in . % of patients without viral identification and . % of patients with viral identification; inpatient infection was present in . % and . % of patients without and with viral identification, respectively; and underlying disease complications were found in % of patients without viral identification and . % of patients with viral identification. these differences were not statistically significant. patients with chronic pneumopathy showed more complications in their underlying pathologies ( . % vs. . %, or: . ; ci %: . - . ). a total of ( . %) patients died. however, two cases included either an inadequate sample for pcr or no pcr amplification; thus, the outcome analysis was performed for patients. mortality was reported for cases ( . %) in the group without viral identification and cases ( . %) in the group with viral identification (p = . , or: . ; % ci: . - . ) ( table ) . mechanical ventilation was required for patients ( . %); this intervention was invasive in ( . %) patients, non-invasive in ( . %) patients and invasive and non-invasive in ( . %) patients. this outcome occurred among patients ( . %) without viral identification and patients ( . %) with viral identification (p = . , or: . ; % ci: . - . ). the average length of mechanical ventilation was days, with a minimum of day and a maximum of days. thirty-nine ( . %) patients were admitted to the intensive care unit (icu); had unsuitable samples or non-amplified pcr results, ( . %) were from the group without an isolated virus, and ( . %) were from the group with an isolated virus (p = . , or . ; % ci: . - . ). one case with viral identification was readmitted to the unit with an identification of influenza subtype ah n . the average length of icu stay was . days, with a range between and days. the group with an isolated virus had an average icu stay of . days, with a range between and days, and the group without an isolated virus had an average stay of . days, with a range between and days (p > . ). the average hospital stay in the sari group was . days, with a range between and days, with average stays of . and . days for patients with and without a virus, respectively (p = . ). among patients without a virus, the hospital stay varied between and days, while patients with a virus had hospital stays that varied between and days. of the patients from the viral infection group who died, bocavirus was isolated in , influenza was isolated in (all cases of influenza a), metapneumovirus was isolated in , and rsv was isolated in . of the patients with bocavirus isolation, . % died. death occurred in . % of those in whom influenza was identified, % of those in whom metapneumovirus was identified and % of those in whom rsv was identified. among patients with viral identification who died, ( . %) had viral infections of or more viruses (p = . , or: . ; % ci: . - . ), and fewer had only one virus detected. no statistically significant differences were observed in the outcomes of the remaining patients with mixed viral infection, which does not confirm higher morbidity in patients with more viruses isolated. the diagnosis of acute respiratory infections is common and apparently easy to perform; however, the determination of the infection's causal agent is more complex, as current diagnostic tools are limited and rarely available in primary health care centres or even in hospitals in much of the world [ ] . the role of viruses and their prevalence is a matter of debate and discussion, and findings vary worldwide. the results from studies in new zealand, spain and more recently in the united kingdom report prevalence rates of % [ ] , % [ ] ,and % [ ] , respectively. in our study, viruses were identified as the most frequent causal agents of sari requiring hospitalization in , with most cases showing a high rate of viral co-infection, a high degree of morbidity, prolonged hospital stays and frequent needs for icu management and mechanical ventilation. the definition of sari used in this study is of great utility from an epidemiological surveillance perspective, which is known as syndrome surveillance or surveillance of syndromes [ ] . nonetheless, the results reported here demonstrate its clinical relevance and potential utility in this field, as it appears to identify potentially severe patients and those with high complication rates; thus, the definition's routine use should be considered during the performance of clinical duties. furthermore, current challenges in the epidemiological surveillance of viral respiratory tract infections include the early and fast identification of aetiological agents, especially at the beginnings of outbreaks, and the optimal and timely management of a large number of samples [ ] . our study suggests that molecular technology makes it possible to closely follow circulating viruses in these groups of patients. in contrast to the epidemiological definition used, the curb- index of pneumonia was not useful, as only ( %) scored for general hospitalization and none scored for icus using these criteria. this finding contrasts with the high number of our patients who required icu care, which agrees with findings in the literature that applied this scale in cases of viral pneumonia during the influenza a subtype h n pandemic of ; these results demonstrate its low value for detecting either severity or the need for icu admission in patients with viral pneumonia [ ] . these findings represent a marked difference in severity stratification between influenza-related pneumonia and pneumonia caused by other aetiological agents, indicating the importance of clinical judgement in this scenario [ ] . the application of these scales in non-influenza viral pneumonia has yet to be assessed. moreover, a recent multicentre study of adults with radiographically confirmed pneumonia has shown that viruses are now the most commonly identified pathogens. human rhinovirus and influenza virus are more frequently found than streptococcus pneumoniae [ ] . together, viruses represent a quarter of patients and more than half of the pathogens identified. the mortality rate was relatively high for patients both with and without communityacquired pneumonia. a study performed in the united states reported a low mortality rate in patients hospitalized for viral infection; nonetheless, bacterial co-infection increased both the morbidity and the mortality [ ] . this variable should be considered for our patients. countries with marked seasons report a correlation of influenza with high mortality due to respiratory infections, which is usually related to community-acquired pneumonia [ ] . this seasonal pattern of acute respiratory infections, especially viral infections due to influenza, is dependent on temperature, humidity and host factors, such as serum vitamin d levels [ ] . colombia is located in the tropics and thus lacks seasons; however, this study confirms that these infections, especially those caused by influenza and rsv infections, increase during the rainy season in countries at these latitudes [ ] . this pattern shows the importance of epidemiological surveillance, especially during the seasons when viruses circulate, because it may increase control and prevention strategies such as timely vaccinations. techniques based on identifying nucleic acids, such as those used in this study, can obtain more rapid and precise results for diagnosing viral infections in order to provide appropriate and managed medical care [ ] . there is limited information on the diagnostic utility of these new tests. a study by sultankulova et al. compared viral isolation of influenza a with dna [ ] . microarray technology showed a higher sensitivity ( . %) and similar specificity ( . %) to real-time pcr. however, another study with the same microarray kit used in this study showed a high specificity ( %) and low sensitivity ( %) in the clinical scenario of atypical pneumonia [ ] . a study in japan using near patient automated microarray technology showed not only a higher sensitivity and specificity compared to immunochromatographic antigen detection (the gold standard used was virus isolation) but also quicker results for children infected with influenza and rsv [ ] . a recent multicentre study in the united states using real-time pcr technology was able to precisely and reproducibly detect all adenovirus infections in a group of children and adults with respiratory infections, which increased the possibility of establishing a clear diagnosis [ ] . taken together, use of molecular technology for the diagnosis of viral infections can improve detection and identify the cases in which antibiotic use might be inadequate. the use of antibiotics in acute respiratory infections is indiscriminate and excessive, and according to worldwide literature, is employed in more than half of all respiratory infections [ ] . this study determined that % of the cases are treated with antibiotics, although most of the infections were viral in origin. additional strategies, such as the measurement of procalcitonin, can identify patients who would not benefit from antibiotic therapy [ ] . this study has several limitations. although it was multicentre in design, the information was gathered from only one city in colombia. one important limitation is that it is a retrospective study; thus, it was not possible to control viral and bacterial sampling, and there was limited access to relevant data, such as previous influenza or pneumococcal vaccination history or co-morbidity measurement. the sample size was smaller than expected, although the prevalence of viral infection was higher than expected. together, all of these limitations lead to difficulties in establishing significant comparisons among the groups and in appropriately assessing the impact of co-morbidity and bacterial co-infection in the outcomes, especially in relation to the role of pneumococcus [ ] . other limitations include the difficulty of defining the actual roles of certain viruses, such as bocavirus 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complications of respiratory tract viral illness: a comprehensive evaluation estimates of mortality attributable to influenza and rsv in the united states during - by influenza type or subtype, age, cause of death, and risk status roles of humidity and temperature in shaping influenza seasonality epidemiology and seasonality of respiratory tract virus infections in the tropics utilization of nucleic acid amplification assays for the detection of respiratory viruses comparative evaluation of effectiveness of iavchip dna microarray in influenza a diagnosis microorganisms in respiratory tract of patients diagnosed with atypical pneumonia: results of a research based on the use of reverse transcription polymerase chain reaction (rt-pcr) dna microarray method and enzyme-linked immunosorbent assay the clinical utility of a near patient care rapid microarray-based diagnostic test for influenza and respiratory syncytial virus infections in the pediatric setting multi-center evaluation of the adenovirus r-gene us assay for the detection of adenovirus in respiratory samples antibiotic prescribing in ambulatory care settings for adults with colds, upper respiratory tract infections, and bronchitis procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections a role for streptococcus pneumoniae in virus-associated pneumonia we acknowledge the following institutions for their participation in this study: suba hospital, hospital de occidente kennedy, santa clara hospital (gerson arias), el tunal hospital (diana marcela bejarano), san ignacio university hospital (sandra valderrama), fundación cardioinfantil (alvaro arango), and hospital san rafael (carlos restrepo). key: cord- -b wlr t authors: engstrom-melnyk, julia; rodriguez, pedro l.; peraud, olivier; hein, raymond c. title: chapter clinical applications of quantitative real-time pcr in virology date: - - journal: methods in microbiology doi: . /bs.mim. . . sha: doc_id: cord_uid: b wlr t abstract since the invention of the polymerase chain reaction (pcr) and discovery of taq polymerase, pcr has become a staple in both research and clinical molecular laboratories. as clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has pcr performance. through optimisation, the present-day uses of real-time pcr and quantitative real-time pcr are enumerable. the technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology. complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time pcr provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. all of these serve vital roles in the continuum of care to enhance patient management. beyond this, quantitative real-time pcr facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. processes, small sample volumes and can be utilised in a wide variety of applications, making it the method of choice in today's molecular laboratories. through the aid of fluorescent signalling probes to measure amplification of dna at each pcr cycle, at the point of exponential dna accumulation, real-time pcr is able to provide broader linear dynamic ranges and increased assay performance as determined by sensitivity, specificity, precision, and reproducibility. due to the consistency in signal intensity changes during the exponential growth phase of pcr, it is also easily adaptable for quantitative reporting. however, there are three properties that are uniquely associated with quantitative real-time pcr: quantification, standardisation, and lower limit resulting. the accumulation of fluorescence signal is measured at each pcr cycle of the reaction and the cycle at which this signal exceeds a predetermined background fluorescence threshold during the logarithmic phase of amplification is referred to as the cycle threshold (c t ). the c t value is inversely proportional to the viral copy number in the specimen, and through comparisons of this value to an external calibration curve or an internal quantitation standard, the initial nucleic acid target concentration can be calculated (heid, stevens, livak, & williams, ; livak & schmittgen, ) . however, accurate quantitation within each sample is hindered when relying solely on an external standard as amplification efficiencies for each individual sample may be variable and inconsistent. by utilising a standard internal reference template, with the rationale that any variable influencing amplification efficiency should example of amplification and detection of target nucleic acid by real-time pcr. affect both template and target similarly, inhibition and amplification effects are compensated for which allows for more accurate quantitation ( figure ). this control can be further enhanced when incorporating an internal reference that utilises the same primer sequence as the target since any potential additional effects on pcr efficiency for each of the two targets is eliminated. thus, the competitive real-time pcr strategy is the most reliable approach for nucleic acid quantitation (diviacco et al., ; gilliland, perrin, & bunn, ; stieger, demolliere, ahlborn-laake, & mous, ; wang, doyle, & mark, ; zentilin & giacca, ) and is the basis for the majority of present-day virology assays. it is equally important to utilise appropriate quantitation standards, when available, to ensure accurate quantitative results, inter-laboratory correlation, and overall standardisation. standardisation of reported viral loads ensures not only interlaboratory consistency but also high clinical utility of viral load monitoring, sets the foundation for establishing clinical correlations and critical thresholds leading to better management of infections and treatments, and are critical for the development of clinical guidelines (miller et al., ) . with the wide availability of assay methods, viral targets, specimen type, and lack of standard reference material (hayden et al., ) , viral load variability across laboratories can range significantly, as high as . log copies/ml . specifically, results from proficiency testing/external quality assessment programmes as well as interlaboratory specimen exchange studies have demonstrated that there is significant variability in quantitative results for assays that lack appropriate standards quantitation of viral target using competitive quantitation standard (qs). the qs compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation viral target in each specimen. the competitive qs contains sequences with identical primer binding sites as the viral target to ensure equivalent amplification efficiency and a unique probe binding region that distinguishes the two amplicons. the competitive qs is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation, reverse transcription (when applicable), simultaneous pcr amplification, and detection. viral target concentration in the test specimens is calculated by comparing the viral target signal (solid line) to the qs signal (dashed line) for each specimen and control (a, b) . in the presence of inhibitors, both qs and viral target are equally suppressed and yield accurate viral load calculations (c). introduction (hayden et al., ; pang et al., ; preiksaitis et al., ; wolff, heaney, neuwald, stelrecht, & press, ). findings such as these reinforce the fact that with this high degree of variability and discrepancy, clinicians are unable to compare test results between two different laboratories and, further, clinically relevant cut-offs set by one test would not apply to results of another . without standardisation, the quality of patient care is dramatically impacted, preventing meaningful inter-laboratory comparison of patient results and influencing disease prevention and management programmes (kraft, armstrong, & caliendo, ) . this is especially critical for transplant patients, who may be initially monitored at one institution and then transferred to another for longer-term follow-up receiving results that no longer correlate. therefore, whenever possible, viral load monitoring tests must report results in iu/ml and be fully traceable to the higherorder first who international standard. they must generate highly accurate and reliable results based on a robust calibration methodology and have excellent reproducibility across the dynamic range of the test with demonstrated co-linearity to the who standard. lastly, there exist two distinct end-points with quantitative real-time pcr, which should be of consideration for result interpretation and reporting: the lower limit of detection (llod/lod) and the lower limit of quantitation (lloq/loq). these two limits are assessed differently and are not equivalent in either definition or, in some cases, their assigned values. the lod (also referred to as analytical sensitivity) represents the lowest viral load level at which ! % of tested samples are detected (clsi ep -a, ); theoretically, viral levels at or below the lod are not detected ! % of the time. it differentiates between 'detectable' and 'undetectable' results. the lloq, on the other hand, is the lowest viral level that is within the linear and analytically acceptable range of the assay (clsi ep -a, ) . in other words, the lloq is the lowest point at which an accurate viral load can be assigned and determines which 'detectable' sample will have a reported viral load. a common misconception is that the lod of the assay is the minimum viral level for a 'detected' result but 'undetectable' and 'detectable' viral levels are never differentiated by a single theoretical viral threshold as viral levels less than the lod may still have a high probability of being detected. this probability spans a broad range in which the lower the viral titre, the more likely the 'undetectable' result. ultimately, the statistical probability will favour the 'undetectable' result ( figure ). and because the lloq can be equal or greater than the lod on some viral load assays, it is not unusual for 'detectable but below the loq' (detectable/bloq) result reporting (cobb et al., ) . further, the 'detectable/bloq' results should not be inferred that the actual viral concentration of the sample is between the lod and loq. the clinical demand has driven and shaped the evolution of pcr and continues to do so as we gain a greater understanding of the infections we monitor and treat. through the study of the natural history and disease progression attributed to specific viral infections, the need for sensitive, accurate, precise, reproducible, and reliable quantitative measurements of viral levels has become a necessity. with the deeper understanding of the natural history of human immunodeficiency virus (hiv) infections, it is now well understood that progressive immunosuppression and the onset and development of clinical disease are strictly associated with increasing viral burden (furtado, kingsley, & wolinsky, ; ho, moudgil, & alam, ; mathez et al., ; nicholson et al., ; schnittman et al., ) . thus, quantitative real-time pcr is critical for monitoring patients infected with hiv (hufert et al., ; mellors et al., ) and those undergoing antiretroviral therapy (art) to ensure viral replication is sufficiently and effectively suppressed and to monitor potential for viral resistance to the medication (dhhs hiv, ) . this monitoring and maintained viral suppression is absolutely necessary not only to maintain progression-free survival of hiv-infected patients but also to reduce subsequent hiv transmission (cohen et al., ; diffenbach, ) . due to the significance of viral load monitoring and maintaining viral suppression, the demand for likelihoods of different test results given different viral concentration. when the viral concentration tends to , the proportion of 'target not detected' increases to (dotted line), increasing the likelihood of 'not detected' results. as the concentration tends to lloq (dashed line), the likelihood of 'detected but <lloq' results peaks. when the concentration tends to infinity, the proportion of quantitative results tends to (solid line), resulting in a continual increase in the likelihood of 'detected: quantitative' results. at any concentration, the sum of the three types of reported results is always % and throughout the concentration continuum, variations in result reporting exist. as the concentration of viral levels approaches the lloq, near equal likelihood of 'detected: quantitative' and 'detected but <lloq' results are possible, as are 'not detected' results. decreasing concentrations will further shift the likelihoods and increase the chance of 'detected but <lloq' or 'not detected' results. diagram assumes lloq ¼ llod. increasingly more sensitive assays for hiv has driven the innovation of diagnostic tests and continues to push the limits of the lod and loq ever lower (glaubitz et al., ; sizmann et al., ) . additionally, the amount of viral dna in samples from either hepatitis b virus (hbv) or cytomegalovirus (cmv) chronic carriers is indicative of active viral replication in liver cells and is correlated with liver disease progression (chen, lin, et al., ; chen, yang, et al., ; emery et al., ; humar et al., ; humar, kumar, boivin, & caliendo, ; iloeje et al., ; wursthorn, manns, & wedemeyer, ) , highlighting the need for quantitative viral levels in chronic disease monitoring. similarly, hepatitis c virus (hcv) levels have been linked to prognosis and treatment outcomes (trepo, ) , treatment response (pearlman & ehleben, ; zeuzem et al., ) , as well as assessing sustained virologic response (svr), or 'cure', following treatment (pearlman & traub, ) . more recently, as new antiviral therapies for hcv treatment rely on targeting specific biological steps of the viral replication cycle, reliable quantitative monitoring of the viral burden at specific time-points to ensure treatment compliance and resistance emergence during treatment is recommended (au & pockros, ; aasld/idsa/ias-usa, ). alongside the changing clinical needs, several instrument and manufacturing innovations have been introduced to meet these newer requirements. the first pcrbased diagnostic test and the first automated system were both introduced in the early s, allowing for improved standardised technique, increased efficiency, and reduction in error and contamination. in , to meet the clinical needs of monitoring patients infected with hiv, the first 'personalised healthcare' diagnostic test was fda approved, that monitored whether art was working and whether a patient was on optimal treatment. entire systems were introduced in the early s that automated the up-front sample preparation step, leading to further reduction of hands-on time, increased reliability and reproducibility. with the introduction of real-time pcr techniques to further enhance assay performance, the first pcr tests that allowed for simultaneous amplification and detection were introduced in . throughout the decade, improvements in assays and instrumentation began pushing the boundary for hiv- detection, achieving greater and greater sensitivity, a feat that has proven to be incredibly relevant in understanding risks of viral load rebound and virologic failure (doyle et al., ; estevez et al., ; pascual-pareja et al., ) . more recently, shortly after the release of two new higher-order standards for cmv (first who international standard and nist cmv standard), fully automated real-time quantitative pcr assays were fda approved to meet laboratory and clinical demands for standardisation. not only does the instrumentation reduce assay design variability and inconsistency across laboratories, but also the assays are standardised to the who and report in international units, providing accuracy across the entire dynamic range that is only achieved by calibration and traceability to these higher-order standards. but it is not until these fda-approved tests gain widespread use will inter-laboratory agreement for cmv viral load results truly improve (kraft et al., ) . the objective of test innovation is to evolve and adapt to clinical and laboratory needs. as patient outcome gaps are identified, and as we gain greater understanding and insight into the clinical progression of disease and disease management, technological achievements facilitate advancements in quality of diagnostics and patient outcomes. life-threatening illnesses convert to chronic/manageable disease with the aid of viral load monitoring. and pressure exists to develop more precise, accurate, sensitive assays, which, in turn, drives the development of more efficacious drugs. this synergy demonstrates the value of diagnostics: it is an integral part of the patient care continuum. applications of quantitative real-time pcr for virology are extensive. it is especially necessary when antibody seroconversion is delayed after an acute infection and early diagnosis are essential (dibiasi & tyler, ; thomson et al., ) , in immunocompromised patients that may not have an optimal antibody response (kadmon et al., ) , and for the diagnosis of congenital or perinatally acquired viral infections (park, streicher, & rothberg, ; young, nelson, & good, ) . additionally, it is critical in maintaining a high level of patient care at each stage of the disease and infection (figure ). screening tests are designed with one key parameter in mind: exceptional sensitivity (herman, gill, eng, & fajardo, ) . they must ensure that disease is not missed in a population that is generally free from risk to allow for appropriate early intervention, thereby effectively reducing mortality and morbidity. although traditional applications of real-time pcr in the continuum of care and patient management. population-based screening programmes and recommendations do not often utilise nucleic acid testing (nat) for virology targets, relying more on immunoassays and antigen testing, nats and real-time pcr assays are still integral components. donor-eligibility determination ensures that a donor is eligible to donate cells or tissues to be used as human cells, tissues, and cellular and tissue-based products (hct/ps), which can include haematopoietic stem/progenitor cell, organ, semen, and other types of donations. in part, living and cadaveric hct/p donor eligibility is granted if screening shows that the donor is free from risk factors for, and clinical evidence of, infection due to relevant communicable disease agents and diseases such as hiv type and , hbv, hcv, and west nile virus (wnv) (fda testing, ) . the fda testing recommendations stipulate that hct/ps tests for hiv and hcv may include fda-licensed nat blood donor screening and that, specifically, an fda-licensed nat should be used to assess infection with wnv. despite the fact that these nat tests provide qualitative as opposed to quantitative results, the molecular technology utilised is often real-time pcr due to its enhanced accurate and reliable resulting (fda assays, ) . additionally, pre-emptive virology screening post-transplant may reduce subsequent complications and provide a more cost-effective management strategy (evers, ) . pre-emptive therapy utilises routine viral screening to initiate therapy at the first indications of viraemia, prior to clinical manifestation of disease. this strategy reduces the overall morbidity and mortality post-transplant compared to strategies in which treatment is initiated at the onset of clinical disease (sch€ onberger et al., ). the pre-emptive treatment model utilising routine screening by quantitative realtime pcr of asymptomatic post-transplant patients has been shown to be especially effective for paediatric patients undergoing haematopoietic stem cell transplant, who are uniquely at a high risk of cmv and epstein-barr virus (ebv) infections (evers, ) . this strategy and prospective screening utilising a quantitative real-time pcr assay for bk polyoma virus (bkv) is recommended as part of routine posttransplant follow-up of all kidney transplants since early identification and management of bkv infection may prevent future incidence of polyoma virus-associated nephropathy (hirsch et al., ; kdigo, ) . nats and real-time pcr are also utilised as vital parts of certain screening algorithms. the center for disease control (cdc) currently recommends that hcv rna testing be utilised for anyone who may have been exposed to hcv within the preceding months. in addition, nats would identify active hcv infection among persons who have tested anti-hcv positive or those with an indeterminate antibody test indicating need for referral for further medical evaluation and care (cdc hcv, ). viral diagnostic tests are used to determine presence or absence of current or previous infection. because many viral infections present with similar symptoms, accurate diagnosis is critical as each requires unique and vastly different interventions and/or management strategies. historically, diagnosis of viral infections has relied on viral growth in cell culture, immunoassays, antigen assays (elisa), haemagglutination testing, and electron microscopy (krishna & cunnion, ) . the introduction of molecular methods including nats and real-time pcr assays has vastly improved viral diagnosis with their superior sensitivity, specificity, and rapid result reporting (emmadi et al., ) . nat-based infectious disease testing, providing rapid results, aids in outbreak detection (ebola), genotype identification (hcv), and identification of possible drug resistance (hiv- ), which can lead to rapid clinical therapeutic decisions and early infection control to prevent spread of disease (espy et al., ) . viral culture was traditionally considered to be the 'gold standard' for viral diagnosis because of increased sensitivity compared to rapid antigen-testing methods. however, a significant limitation of viral culture was a long time to result, reaching days for cmv (gleaves, smith, shuster, & pearson, ) . improvements in culture included the introduction of shell viral culture, which reduced the result turnaround-time (tat) for cmv to h. although considered to be a vast improvement, certain viruses require immediate treatment intervention to prevent life-threatening infection and therefore, a -h tat is simply much too long. further, reliance on viral culture for diagnostics testing introduces other pronounced drawbacks, the most noteworthy being that not all routine viruses grow in culture and that virus viability, and thus its culture ability, could be impacted by sample collection, transport conditions, or prior patient treatment. with these limitations in mind, nat testing has tremendous advantages and has replaced culture for most viruses due to its greatly reduced tat (typically less than h) while still retaining high sensitivity. the applications of nat testing, and more specifically, real-time quantitative pcr technology, in viral diagnostics and confirmatory testing continue to expand. the cdc-updated recommendations for hiv testing state that specimens that are reactive on the initial antigen/antibody combination immunoassay and non-reactive or indeterminate on the hiv- /hiv- antibody differentiation immunoassay should be tested with an fda-approved hiv- nat test (branson, ; branson et al., ) . additionally, nats have demonstrated utility in high-risk populations, in which antibody testing alone might miss a considerable percentage of hiv infections that are otherwise detectable by nat virologic tests (priddy et al. ; stekler et al., ) . particularly, immunoassays for hiv diagnosis are limited by the marked delay between infection and seroconversion, a time when hiv viral levels are at their peak ( figure ). for this reason, nats are the recommended method for diagnosis of hiv during the acute phase of infection ( - days post-infection). hiv viral rna is the first marker to manifest itself approximately days post-infection at initiation of the acute phase of infection (lindback et al., ) . not until - days after initial detection of hiv rna do the hivp antigen levels rise to detectable levels using fourth-generation immunoassays. this marked delay and the need for rapid diagnosis by the identification of hiv rna is especially critical when an early diagnosis is medically warranted. during the acute phase of the hiv infection, patients typically present with flu-like symptoms, which can often lead to a misdiagnosis. misdiagnosing those at the highest risk of infection such as prison inmates, iv drug users, and men who have sex with men (msm), also increases the risk of further disease spreading (chu & selwyn, ) . therefore, the use of a real-time pcr testing method that is able to identify the presence of the hiv virus at this initial stage is critical. additionally, nats are critical for early diagnosis of congenital or perinatally acquired viral infections, especially infants born to hiv-infected mothers (tang & ou, ) . the maternal antibodies against hiv can persist in exposed infants for up to months of age, which, in turn, prevents the use of antibody-based assays for early diagnosis of infection. there is a high level of morbidity and mortality during the first years of life for infected infants; therefore, it is of high importance to determine infection status quickly in the exposed infant to implement the appropriate art therapy early (van rossum, fraaij, & de groot, ) . in addition to hiv, additional viruses have been demonstrated to exhibit perinatal transmission including respiratory virus, cmv, herpes simplex virus (hsv), varicella zoster virus (vzv), hbv, enterovirus, rotavirus, and human papilloma virus (prober et al., ) . for all these, nat testing may serve as a valuable tool for early intervention, especially in this critical neonate population that lacks fully developed immune systems. in addition to their applications for hiv diagnosis, nat testing is also utilised as a diagnostic for hcv. although diagnostic hcv rna real-time pcr tests are predominately used as confirmation of a positive hcv antibody result (cdc hcv, ), nat testing is recommended for the confirmation of a non-reactive hcv antibody test for patients suspected of having recent hcv exposure. hcv rna can be detected as early as weeks post-exposure, whereas hcv antibodies are not detectable until - weeks post-infection (ghany, strader, thomas, & seeff, ). therefore, similar to strategies outlined for hiv, nat testing is the most suitable for detection and diagnosis during the acute phase of hcv infection. further, immunocompromised patients, those with weakened immune systems can include those that are hiv positive, on immunosuppressive drugs, or undergoing chemotherapy, may lack the ability to generate an appropriate immune response required for an anti-hcv test. in these special circumstances, the cdc recommends considering hcv rna testing for diagnosing viral infections. rapid diagnostic testing is also very important for viruses with high potential to create an epidemic or outbreak. throughout history, influenza has caused many such outbreaks, the most notable being the spanish flu thought to be responsible for an estimated million deaths (taubenberger & morens, ) . in response to the possibility of future outbreaks, the world health organization (who) has established that pcr-based influenza testing is now the first-choice diagnostic test for both humans and animals (who influenza, ) . the conversion to molecular tests was based on the fact that these assays are more sensitive and specific for detecting influenza viruses compared to other non-molecular methods. nat methods also have a lower likelihood of false positive or false negative results, and therefore, result interpretation is less impacted by community-based influenza prevalence (cdc influenza, ) . the introduction of rapid molecular testing, which can provide results in as little as min, is extremely beneficial especially in hospitals, nursing homes, and chronic care facilities where early influenza identification can prevent outbreaks. the number of molecular-based diagnostic tests has expanded even further with the introduction of tests for wnv, respiratory syncytial virus, hsv, rotavirus, and even more recently, ebola. as development and improvements continue to be made to real-time pcr technology that will reduce both cost and time to result even further, the number of nat diagnostic tests will continue to increase and the applications for real-time pcr in diagnostics will also expand. once a patient is appropriately diagnosed and linked to care, partly through the aid of quantitative real-time pcr, clinicians will often consider several baseline factors, which collectively help to guide therapeutic decision. these decisions are based on a series of questions including: are treatment options available? what treatment regimen should be prescribed? for how long will the patient need to be on therapy? factors influencing these therapeutic decisions include disease complications, patient predisposition, prior treatment experience, viral genotype, viral resistance profile, and host genetic profile, among others. and depending on the viral infection, a quantitative baseline viral load may also serve an important role in helping to guide treatment decision (table ) . over the past years, pharmaceutical development and clinical trials investigating cutting-edge antiviral treatments have relied heavily on the data generated from pcr-based-and eventually quantitative real-time pcr-based-technology to determine safe and effective drug use (cobb et al., ; mackay, arden, & nitsche, ) . practice guidelines continue to reference registrational and nonregistrational studies utilising quantitative real-time pcr to guide both clinicians and laboratories in the proper implementation of treatment and testing in order to deliver the most effective personalised care to patients (aasld/idsa/ias-usa, kotton et al., ) . because of this extensive co-utilisation of real-time pcr, it is widely accepted as the gold-standard technology for measuring a patient's quantitative viral load before, during, and after the course of treatment. once the decision to initiate treatment for chronic hcv infection is made, several baseline factors are routinely considered (aasld/idsa/ias-usa, ). among these are complications like liver fibrosis stage, co-infection with hiv, hepatocellular carcinoma, end-stage liver disease, genetic variations like hcv genotype, subtype and resistance markers, and prior treatment experience and outcome. the association of baseline viral load, measured by quantitative real-time pcr, to chronic hcv treatment outcome has been well documented with earlier therapies (pegylated-interferon plus ribavirin) but, given the lack of alternative therapeutic options, has not been recommended as a therapeutic decision factor (jensen et al., ; pawlotsky, ) . historically, practice guidelines have recommended the measurement of baseline viral load to serve only as an initial time-point required for effective monitoring of treatment without any prognostic indication (yee, currie, darling, & wright, ) . currently, tremendous advancements in the treatment of chronic hcv infection that employ direct-acting antivirals (daas) reported cure rates (as determined by svr) of > % for even the once most difficult to treat hcv genotype- patients, the most predominant in the united states. because of this high potency of these drugs across patient populations and the greater importance of numerous other factors, including hcv genotype and prior treatment experience, in determining the appropriate course of treatment, the most recent aasld/idsa practice guidelines still do not recommend a baseline quantitative viral load as a therapeutic decision factor. however, in the rapidly evolving field of hcv treatment, the recent fda approval of a fixed-dose combination drug consisting of two daas (sofosbuvir and ledipasvir) for the treatment of hcv genotype- , the manufacturer's drug label now includes a new indication for quantitative real-time pcr. it is indicated that treatment naïve and non-cirrhotic patients with a specific baseline viral load are eligible for shortened therapy, an indication with tremendous implications. according to the prescribing information, patients with a baseline viral load below million iu/ml are eligible to have shorter therapy duration of weeks, much shorter than the -or -week duration for other patient populations (harvoni, ) . this therapeutic decision practice is the first of its kind in treatment of chronic hcv infection and is likely to be a recurring theme as daa manufacturers strive to develop high efficacy regimens requiring shorter treatment durations. additionally, shorter treatment durations are more favourable to patients and payers when considering the cost of achieving svr with daas and may improve patient drug adherence and completion of therapy (hep c online, ) . as much as quantitative real-time pcr helped to develop this claim for this particular regimen, this technology will also be employed by numerous laboratories to aid in this part of therapeutic decision. in contrast to chronic infection, treatment of patients presenting in the acute phase of hcv infection, within the first months after exposure, is not recommended by aasld/idsa for patients in whom hcv infection spontaneously clears (aasld/ idsa/ias-usa, ). therefore, careful monitoring of hcv rna by a sensitive nucleic acid test is required in order to confirm spontaneous clearance, defined as hcv rna negative at two specific measurements. quantitative and qualitative real-time pcr assays are both widely used for this purpose, given their comparable sensitivity. factors influencing art decision for hiv-infected patients include determination of pregnancy, aids-defining conditions, acute opportunistic infections, low cd counts, hiv-associated nephropathy, potential drug interactions, co-infection with hcv or hbv, hiv resistance testing, and prior treatment experience (dhhs hiv, ). plasma hiv rna viral load, performed widely by quantitative real-time pcr, is also recommended as a pre-art decision factor specifically for treatment naïve patients. the department of health and human services (dhhs hiv) recommends that only art-naïve patients with a plasma hiv viral load below , cp/ml can be prescribed various regimen options, which they otherwise should be restricted from taking with higher viral load. this is primarily due to inferior virologic responses in patients with higher viral loads observed in clinical studies (sax et al., ) . these clinical trial studies employed quantitative real-time pcr in order to help determine this cut-off and many labs have utilised the same technology to help guide hiv-treating clinicians in this decision. in the case of chronic hbv infection, several studies have shown that hepatitis b 'e' antigen (hbeag) and high levels of hbv dna are independent risk factors for the subsequent development of cirrhosis and hepatocellular carcinoma (chen, lin, et al., ; chen, yang, et al., ; iloeje et al., ) . however, due to the fluctuating nature of chronic hbv infection, the prognostic utility of one high hbv dna level at a single time-point is limited. thus, hbv baseline dna viral load, along with hbeag, alanine aminotransferase (alt) levels, and fibrosis, collectively aids in the decision to treat with antiviral agents as well as which hbv antiviral regimen to choose and duration of treatment (lok & mcmahon, ). typically, patients with an hbv dna viral load > , iu/ml, signs of liver disease (i.e. high alt levels and/or significant fibrosis), and loss of hbeag are considered for immediate treatment with antivirals, whereas patients < iu/ml are closely monitored for viral load changes prior to treatment. patients who fall in between this range are monitored for persistent viraemia and signs of liver disease before deciding to treat. quantitative real-time pcr, therefore, plays a crucial role in the care of chronic hbv patients who, if not treated at the appropriate time with the appropriate regimen and duration, are at greater risk of liver complications. unlike treatment guidelines for hcv, hiv, and hbv, management of cmv after solid organ transplant is not associated with specific quantitative cmv viral load cutoffs in order to make therapeutic decisions (kotton et al., ) . this is partly due to the historical lack of an international standard and varying assay designs, which has led to poor inter-institutional correlation of quantitative nats. in addition, the widespread practice of universal prophylaxis, where cmv antiviral medication is administered to patients early in the post-transplant period and continued for a finite period of time, has diminished the clinical utility of baseline viral loads for making therapeutic decisions. however, with the recent availability of the who cmv international reference standard, the establishment of viral load cut-offs that can be applied to pre-emptive monitoring of patients prior to treatment initiation may soon become more widely accepted . until then, institutions are required to determine their own test performance characteristics and clinical cut-offs. several studies have shown that a low cmv virologic threshold (e.g. detectable viraemia) using quantitative real-time pcr should be used for starting pre-emptive therapy especially in high-risk cases where the organ donor screens positive and the receptor screens negative for cmv serology (atabani et al., ; couzi et al., ; sun, cacciarelli, wagener, & singh, ) . among a variety of baseline risk factors that may indicate longer cmv treatment duration, significant predictive value has been demonstrated with higher baseline viral loads where longer treatment duration may prevent cmv disease relapse (kotton et al., ; sia et al., ) . clinical trial studies supporting the recent fda approval of a quantitative real-time pcr cmv test calibrated to the who international standard also demonstrated clinical value for baseline testing of patients with cmv disease who are undergoing treatment with the anti-cmv drugs ganciclovir or valganciclovir (razonable et al., ) . data from this study suggested that patients with a baseline cmv viral load < , iu/ml are likely to resolve cmv disease more rapidly than those who have a higher baseline viral load. further studies are needed to determine universal thresholds for pre-emptive therapy initiation and predictive value for cmv baseline viral load in defining optimal treatment duration. there exists a clear application for quantitative real-time pcr technology in baseline determination of patients with significant viral infections, and in fact, quantitative viral load determination plays a critical role in therapeutic decision for many other viral infections. high baseline viral load has been shown to correlate with advanced disease during infection with numerous viruses such as bkv, hsv- , ebv, and adenovirus and may potentiate the need for longer duration therapies in certain scenarios (cincinnati children's hospital medical center, ; domingues, lakeman, mayo, & whitley, ; gustafson et al., ; randhawa et al., ) . after the patient's baseline assessment or pre-emptive monitoring suggests if treatment is available, which treatment regimen to choose and perhaps the duration of therapy, the patient can move on to therapeutic administration. quantitative realtime pcr has helped and continues to set the stage for decisions that potentially saves lives, reduces complications, decreases morbidity, and lessens the economic burden to both the patient and the healthcare system. serial measures of viral load serve as an individualised map of a viral infection through the estimation of the amount of virus found within an infected person. tracking viral load in the continuum of care is a vital tool used predominantly to monitor treatment response and its effectiveness, early signs of resistance emergence during therapy of chronic viral infections, and viral activation or reactivation in immunocompromised patients following bone marrow or solid organ transplantation. while the goal of treatment for chronic hcv infection is svr, patients may fail therapy due to non-response, on-treatment breakthrough, or post-treatment relapse ( figure ). the early change in quantitative viral load over time may be predictive of treatment efficacy and a shorten therapy for patients who respond rapidly to treatment (yee et al., ) . this 'response-guided therapy' (rgt) is best exemplified during treatment of chronic hcv patients. specifically, the sooner a patient becomes hcv rna undetectable during treatment, the lower the relapse rate when treatment is shortened. conversely, the longer it takes for a patient to become hcv rna undetectable, the longer they need to remain on treatment to limit relapse. however, given the poorer efficacy of earlier regimens, not all patients who received therapy achieved svr. for this reason, 'futility rules' or 'stopping rules' were also developed, which required that failure of a patient to respond (target not detected or viral load cutoff ) by a given time-point indicated the need to immediately discontinue therapy. monitoring hcv viral loads during treatment. despite advances in treatment for hcv patients, failure to achieve svr is still a reality. patients who do not achieve svr fall into four categories: ( ) null responders (black line) achieve less than -log decrease in hepatitis c viral load upon treatment; ( ) partial responders (red line; light grey in the print version) experiences at least a -log decrease in hepatitis c viral load during hcv treatment but fail to proceed to an undetectable viral load level; ( ) breakthrough patients (orange line; light grey in the print version) have an undetectable hcv viral load, but the virus rebounded during treatment; ( ) relapsers (blue line; dark grey in the print version) have had an undetectable hcv viral load, but the virus rebounded after they completed hcv treatment. although these rgt notions were originally developed from observations made during treatment with the older therapies, peg-ifn and ribavirin, rgt was also required during treatment with the much more potent first-generation daas, telaprevir and boceprevir, and stopping rules were put in place during treatment with the second-generation daa, simeprevir (aasld/idsa/ias-usa, ; ghany et al., ; yee et al., ) . newer ifn-free daa regimens targeting hcv, which are better tolerated by patients and by virtue of the targets they inhibit, have a higher barrier to resistance, yield more rapidly declining viral kinetics, and, thus, do not contain treatment indications for rgt in their prescribing information (harvoni, ; olysio, ; sovaldi, ; viekira, ) . while rgt was a major driver for regular viral load monitoring during antiviral therapy, it is not the only reason to monitor hcv viral load. in the interval between baseline measurement and assessment of svr, the aasld/idsa guidelines also include recommendations for monitoring initial response (week on treatment with a repeat at week if detectable) and end of treatment in order to provide an assessment of drug compliance/early efficacy and predict treatment outcomes, respectively (aasld/idsa/ias-usa, ). in the most recent revision to these web-based guidelines, it is recommended that an hcv viral load increase of greater than -fold on repeat testing at week (or thereafter) should prompt a discontinuation of hcv treatment. many clinicians also closely monitor and report the declining viral loads to their patients in order to demonstrate treatment efficacy, motivating patients to continue treatment and remain adherent to the drug regimen until the next follow-up appointment (fusfeld et al., ) . regardless of monitoring during hcv treatment for rgt, adherence/compliance, patient motivation, early treatment efficacy, etc., quantitative real-time pcr is widely used by laboratories due to its sensitivity, accuracy, and reproducibility of each consecutive viral load test. for patients infected with chronic viral infections, such as hiv, the lifelong regimen of highly active art aims to suppress hiv viral levels to near undetectable levels, ensuring progression-free survival (delay or all together prevention of the progression to aids) and reducing potential transmission. alongside monitoring immune function and immunologic efficacy through cd t-cell count, hiv viral levels are critical in the clinical evaluation and assessment of hiv-infected patients undergoing art. determining a patient's hiv viral load is indicated prior to entry into care, at the initiation of art, at - weeks after art initiation, and then typically every - months while on treatment: ( ) to establish a baseline level of hiv viral load; ( ) to establish viral response to the therapy to assess the virologic efficacy of art; and ( ) to monitor for abnormalities that may be associated with antiretroviral drugs (dhhs hiv, ) . the baseline hiv viral load is not only linked to treatment options (sax et al., ) but also helps to establish the magnitude of viral load decline after initiation of art and provides prognostic information about the probability of progression to aids or death (marschner et al., ; murray, elashoff, iacono-connors, cvetkovich, & struble, ; thiebaut et al., ) . once treatment is initiated, the goal is to reach and maintain suppressed hiv replication as determined by undetected viral levels utilising highly sensitive nat tests, which is generally achieved within - weeks after art initiation. the need for sensitive assays to effectively assess viral suppression hinges on the need to suppress hiv replication to the extent that viral evolution and drug resistance mutations do not emerge, which typically do not occur in patients whose hiv rna levels are maintained below the llod of current real-time quantitative pcr assays (kieffer et al., ) . due to the introduction of more sensitive real-time pcr assays, which can detect as few as viral copies/ml, natural variability in hiv viral levels over time, even in patients with effective suppression, is much more evident (lima, harrigan, & montaner, ; gatanaga et al., ; willig et al., ) . although controversy exists between the clinical significance of viral loads between llod and < copies/ ml, there are reports suggesting that this low-level viraemia is predictive of virologic rebound (doyle et al., ; eron et al., ; laprise, de pokomandy, baril, dufresne, & trottier, ) , virologic failure (estevez et al., ) , and indication of drug resistance (taiwo et al., ) , signifying the need for highly sensitive assays. viraemic blips, a single detectable hiv viral load (< copies/ml) in an otherwise seemingly suppressed patient (figure ) , however, do not indicate subsequent virologic failure or development of resistance mutations (castro et al., ; lee, kieffer, siliciano, & nettles, ; nettles et al., ) . blips are not unusual (havlir et al., ) and appear to be more common in winter, suggesting that host-related and seasonal factors are associated with the occurrence of viraemia (van sighem et al., ) . on the other hand, persistent hiv rna levels ! copies/ml are often evidence of viral evolution and accumulation of drug resistance on-treatment hiv patient monitoring. (a) hiv viral loads will fluctuate as patients are on treatment, and, in most instances, will remain 'undetectable' (at or below dotted line); viral 'blips' are not uncommon and will result in transient 'detectable' and even quantifiable results (above the dashed line). (b) virologic failure will lead to a sustained high-level viral titre that, without intervention, will increase with time. mutations (aleman, soderbarg, visco-comandini, sitbon, & sonnerborg, ; karlsson et al., ) . once treatment failure is confirmed, immediate intervention is recommended to avoid progressive accumulation of resistance mutations and effective response of new regimen (dhhs hiv, ), which is benefited by low hiv rna levels and/or higher cd cell counts (eron et al., ) , and even a brief interruption in therapy may lead to a rapid increase in hiv rna and a decrease in cd cell count and increases the risk of clinical progression (deeks et al., ; lawrence et al., ) . with the development and administration of newer drugs that target specific biological processes of hiv, routine and clinical monitoring of viral loads using a real-time quantitative pcr assay continues to be critical to predict treatment failure and early emergence of drug resistance mutations, within a timeframe that would increase subsequent treatment success. viral load monitoring is also essential when the recipient of a solid organ transplant is cmv seropositive and the decision is made to initiate treatment only once the cmv levels predictive of disease are reached. this strategy, known as pre-emptive therapy, utilises intensive monitoring for cmv activity by sensitive real-time quantitative pcr methods and short-term antiviral treatment is given only to those with significant viral counts before symptoms occur. cmv is one of the most common opportunistic pathogens that infect solid organ transplant recipients (fishman, ) and is associated with increased morbidity and mortality (sagedal et al., ; schnitzler et al., ) . following primary infection, the virus establishes a lifelong latent infection in several sites of the body and may reactivate in the presence of immunosuppression, such as in transplant recipients. once reactivated, cmv is able to modulate the immune system and is known to be a potent upregulator of alloantigens (razonable, ) , increasing the risk of chronic allograft dysfunction (reischig, ; sagedal et al., ; smith et al., ) and acute rejection (sagedal et al., ) . pre-emptive therapy reduces the incidence of cmv disease (khoury et al., ; reischig et al., ) , which has been documented as a serious problem in randomised trials upon completion of universal antiviral prophylaxis therapy (kalil, levitsky, lyden, stoner, & freifeld, ; lowance et al., ; paya et al., ) . long-term studies have demonstrated that patients receiving preemptive therapy, when compared to prophylaxis therapy, were less likely to develop moderate-to-severe kidney scaring and atrophy and significantly better survival of the transplanted organ (reischig et al., ) . however, challenges still exist around defining appropriate thresholds to initiate pre-emptive therapy (humar & snydman, ) . but with new standardised real-time pcr assays, widespread adoption, and utilisation of these tests, pre-emptive therapy relying in intensive viral load monitoring may become the standard for certain at-risk patients. test of cure, or end of treatment response, is assessed following a given therapeutic regimen for signs of treatment efficacy. in few cases, a quantitative viral load measurement serves as a way to establish a cure rate, but, in others, may only be used as a confirmation of virologic suppression as clinical cure may not yet be possible with current therapies or technical limitations by real-time pcr that limits the overall sensitivity of viral detection. regardless of the clinical utility for measuring a virologic suppression, quantitative real-time pcrs with their current limits of detection and limits of quantitation are valuable tools in measuring low-level viraemia and establishing undetectable viral loads. utilisation of quantitative real-time pcr to assess virologic cure is perhaps best exemplified by treatment of patients with chronic hcv. according to the aasld/ idsa guidelines, patients who have 'undetectable' hcv rna in the serum, when assessed by a sensitive pcr assay, or more weeks after completing treatment, are deemed to have achieved a sustained virologic response . achieving an svr is considered a virologic cure of hcv infection since, in these patients, hepatitis c-related liver injury stops and recurrence of infection is marginal, detected in < % of patients after years post-treatment (aasld/idsa/ias-usa, ; manns et al., ) . in agreement with these guidelines, the fda recommendation to pharmaceutical daa manufacturers also stipulates that viral rna clearance at svr- be measured in clinical trials using an fda-approved sensitive and specific quantitative hcv rna assay (fda hcv, ) . according to prescribing information accompanying the current daas, the threshold of svr- is defined as a quantitative threshold of hcv rna < iu/ml at weeks after the end of treatment (feld et al., ; kowdley et al., ; lawitz et al., ) . this is somewhat dissimilar to the aasld/idsa guidelines as 'undetected' viral levels are not equivalent to 'detected but below the limit of quantitation' (figure ). but, with the benefit of high sensitivity and reproducibility, quantitative real-time pcr has a clear established role in assessment of hcv virologic cure in both clinical trials and clinical practice and is able to meet the needs for assessing svr. quantitative real-time pcr may also play a critical role in the assessment of cmv disease resolution. the consensus guidelines recommend that two consecutive negative samples be obtained with a minimum treatment course of weeks before treatment is discontinued, which is thought to minimise the risk for development of resistance and disease recurrence (asberg et al., ; chou, ; sia et al., ) . still, some transplant centres may extend treatment (secondary prophylaxis) in patients with compartmentalised disease for as long as necessary to reduce the likelihood of recurrent cmv infection (kotton et al., ) . resolving cmv disease has the long-term benefits of reducing mortality, potential allograft rejection, and the risk of bacterial, fungal, or viral opportunistic infections, among many other transplantand non-transplant-specific effects (arthurs et al., ; fishman, ) . although there is currently no cure for hiv infection, highly sensitive quantitative and qualitative real-time pcr tests targeting total hiv dna and rna have been used in clinical studies for both sterilisation (elimination of hiv-infected cells) and functional (controlled hiv in the absence of art) cures (kibirige, ; lewin & rouzioux, ) . improvements in real-time pcr technology may lead to profound increases in assay sensitivity and the ability to achieve single-copy detection ( cp/ml) may lead us to a better understanding of hiv virology and what may be needed therapeutically to achieve a cure (alidjinou, bocket, & hober, ) . if therapeutic strategies are one day able to achieve an hiv cure, these highly sensitive tests will no doubt play a key role in the continuum of care for patients and, most importantly, in the confirmation of cure. clinical laboratories have undergone changes to become more efficient and flexible while delivering the same high-quality results. when choosing to implement new testing, even beyond viral targets, laboratories have to consider first and foremost the performance and medical value of the test and then factors such as tat, ease of use, and cost. real-time pcr with its wide dynamic range, high specificity, and high sensitivity is considered the gold standard for the quantification and identification of a variety of targets including bacteria, fungi, viruses, or oncological mutations (klein, ) . furthermore, the multiplexing capability of real-time pcr increases the number of targets and information gathered from the same test, further improving laboratory workflow, tat, and costs (deshpande & white, ) . while novel technologies have entered clinical laboratories including mass spectrometry and next-generation sequencing, real-time pcr remains a staple and an attractive option for clinical laboratories aiming to create molecular laboratory-developed tests (ldts). in addition, pcr can quickly be adapted to provide a robust test for the identification of emerging disease and molecular testing is now able to reach beyond the clinical laboratory and further enhance healthcare (farrar & wittwer, ; foudeh, didar, veres, & tabrizian, ) . most molecular tests used in clinical laboratories are fda-approved and commercially available. there are instances, however, when a test may not be available for a specific virus or the sample type and/or clinical indication used by the laboratory differs from those of the fda-approved assay, typically leading a laboratory to design its own pcr-based test or modify existing assays. fda defines an ldt as 'a type of in vitro diagnostic test that is designed, manufactured, and used within a single laboratory' and recognises that 'ldts are important to the continued development of personalised medicine' (fda ldt, ) . laboratory developed tests can be grouped into three categories, fda-cleared or approved test that have been modified, tests that are not subject to fda clearance or approval, and tests for which no performance specifications have been provided by the manufacturer (e.g. analytespecific reagents or asrs) (burd, ; code of federal regulations, ). with alternative sample types or applications, fda-approved tests are often modified to fit the testing needs of laboratories, including alternative collection media and sample types or expanded clinical applications. as an example, a recent gap was created in the hcv-screening algorithm for the confirmation of a positive enzyme immunoassay result following the discontinuation of the only fdaapproved confirmatory test (alter, kuhnert, & finelli, ) . in response, the cdc published recommendation for the use of fda-approved tests detecting hcv viraemia (cdc hcv, ), despite the fact that most of these assays did not have specific claims for confirmatory testing; as a result, several laboratories chose to validate these assays as ldts to meet the screening needs for hcv. additionally, ldts are the only option for the identification of the aetiologic agents of viral infections that can occur in transplant patients, such as ebv, adenovirus, vzv, and bk virus, that often present with non-specific clinical manifestations (razonable, ) and for which fda-approved assay options are lacking. ldts are an integral part of molecular laboratory testing. whether created from the ground-up or modified from fda-approved assays, ldts are answering the clinicians' needs for information as an aid for diagnosis or treatment of patients. as with any clinical tests, ldts have to meet the minimum standards set forth by clia prior to report patient results (code of federal regulations, ). in july , fda informed congress of the agency's ldt regulatory oversight framework (fda ldt, ) . fda aims to address concerns over high-risk ldts with inadequately supported claims, lack of appropriate controls, and falsification of data that may lead to inadequate treatment, possible harm to patients, and unnecessary healthcare cost. presently, there is still a high degree of uncertainty as to what the final regulation scope will be and the possible impact on molecular laboratories will have to be seen. palaeopathology confirmed the truism that humanity, since its inception, has been exposed to genetic and infectious diseases with early documentation of trachoma ( b.c.e.), tuberculosis ( b.c.e.), and pneumonia (ca. b.c.e.) (aufderheide & rodreguez-martin, ; hershkovitz et al., ; roberts & manchester, ; webb, ) . even today, emerging infectious diseases (eids) continue to appear unpredictably driven by changes in human demographic, land use, and population behaviour (lederberg, hamburg, & smolinski, ; sehgal, ; taylor, latham, & woolhouse, ) . these infections can be classified as either newly emerging/a previously unknown disease or re-emerging infectious diseases/a previously known disease, that reappears after a significant reduction in incidence or elimination (morens & fauci, ) . eids are a threat not only to human health but also to global stability and economy. efforts to monitor these eids are in place both at the global level spearheaded by the who and at the national level. in the united states, governmental agencies (department of health and human services, united states agency for international development, department of defense) are supporting activities to detect, assess, and respond to potential outbreaks. specifically, pcr and real-time pcr are easily adaptable to detect nucleic acid targets that are unique to each given pathogen, and as such, they play essential roles in the identification and detection of infectious pathogens and have been routinely used by health organisation agencies during epidemic outbreaks such as severe acute respiratory syndrome (sars), h n , h n , and ebola (shuaib et al., ; who influenza, ) . the sars epidemic appeared in november , in the chinese province of guangdong before reaching the adjacent hong kong in (who sars, . this sars eventually spread to countries and resulted in more than cases. in response, the cdc triggered its emergency operations centre and issued a draft genome in april , days after the initial who global alert (cdc sars, ) . soon after, real-time quantitative pcr assays were described and put in use for the diagnosis of sars (drosten et al., ; peiris et al., ) . a host of measures were taken in order to contain this epidemic, and the molecular identification and diagnosis of the infectious agent by pcr played a key role in providing critical information to address the situation and contributed to the care of the patients infected. additionally, the re-emerging ebola epidemic (cdc ebola, ; who ebola, ) started in guinea in march of before spreading to nearby west african countries and eventually reaching the united states and europe (who ebola, ) . at the height of the epidemic, fda issued an emergency use authorisation (eua) for the use of the first real-time rt-pcr assay (fda eua, ) and less than months later, five additional realtime pcr tests were authorised under an eua (fda eua, ) to provide an early diagnosis of the ebola viral disease (cdc ebola, ). eids remain a constant and unpredictable threat to human health. the flexibility of real-time pcr technology continues to show how promptly it can be used for the detection of infectious agents. by providing a rapid diagnostic, real-time pcr can help in starting the appropriate treatment right away and maximise the chances of a positive outcome. the goal of point-of-care testing (poct) is to quickly obtain a test result that will be used to implement the appropriate treatment for an improved clinical outcome. by definition, poct is laboratory testing that takes place at or near the site of patients (cap poc, ) . the advantages of poct are an improved tat and result availability regardless of normal core laboratory hours, access to care in remote areas, and greater patient involvement. the fight against aids largely contributed to the development of poct devices with viral load capabilities (hong, studer, hang, anderson, & quake, ; lee et al., ; marcus, anderson, & quake, ; tanriverdi, chen, & chen, ; unitaid, ; vulto et al., ) . originally developed to meet the difficult conditions associated with remote places, far from any core laboratory facility often found in the developing world, the design and convenience of a portable poct device with fast turnaround and accurate results extends the reach of healthcare. with this in mind, these poct systems could easily be used in developed nations at hospitals, within clinics a physicians' offices, pharmacies, correction facilities, or mobile health units, to target pathogens that benefit from immediate actionable results, for which not only accurate but also quick results are critical (kiechle & holland, ). ultimately, test menu available on these platforms will drive its implementation as a complement for the clinical laboratory core testing. the ideal molecular poct system that includes medical value, simplicity, fast tat, and ruggedness remains an ongoing engineering challenge. however, the latest advances in microfluidics are a great example of the potential of these devices and brings the real-time pcr lab-on-a-chip closer to mainstream diagnostic use. this is an exciting time for molecular poct and the upcoming years should bring new systems and perhaps a paradigm change in the world of healthcare. as the needs of the clinicians, laboratory, and patients continue to evolve, so do the applications of molecular diagnostics and pcr. over the past decade, quantitative real-time pcr technology has been increasingly phased into clinical practice and all of the potential present-day applications of real-time pcr-based methods are enumerable. they serve to advance experimental approaches within biological fields, pushing the boundaries of what we know and what we can learn, as well as to diminish empiric medical identification and management of viral diseases. the high sensitivity of the technology has reduced risks of the most commonly transmitted transfusion illnesses and has become an integral part of managing a variety of viral infections by providing pretreatment prognostic information, therapeutic effectiveness through monitoring, and end of treatment response assessment. quantitative real-time pcr complements serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses and are able to predict therapeutic failures sooner than traditional methods, allowing for a more timely management response. real-time pcr assays can be rapidly developed in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. the next few years are likely to see an even further increase in the expansion of the clinical applications of nucleic acid quantification, particularly following bone marrow and solid organ transplantation for which the newest standardised assays may provide an avenue for the development of consensus management guidelines for initiating pre-emptive anti-cmv treatment. further, with the drive towards hiv eradication and complete elimination of the virus from within cells of infected patients, innovations in quantitative real-time pcr assay design will continue to push the boundaries of detection and 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progression of hiv- infection according to the viral response during the first year of antiretroviral treatment delayed anti-hcv antibody response in hiv-positive men acutely infected with hcv genotype and viral load as prognostic indicators in the treatment of hepatitis c hiv/aids diagnostics technology landscape efficacy of highly active antiretroviral therapy in hiv- infected children immunologic, virologic, and clinical consequences of episodes of transient viremia during suppressive combination antiretroviral therapy viekira pak™ (ombitasvir, paritaprevir, and ritonavir tablets a microchip for automated extraction of rna from gram-positive bacteria. transducers quantitation of mrna by the polymerase chain reaction prehistoric eye disease (trachoma?) in australian aborigines world health organization. global alert and response: ebola outbreak world health organization. the use of pcr in the surveillance and diagnosis of influenza world health organization. severe acute respiratory syndrome cost ramifications of increased reporting of detectable plasma hiv- rna levels by the roche cobas ampliprep/cobas taqman hiv- version . viral load test multi-site pcr-based cmv viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology natural history: the importance of viral load, liver damage and hcc management and treatment of hepatitis c viral infection: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program office discordant human immunodeficiency virus infection in dizygotic twins detected by polymerase chain reaction. the pediatric infectious competitive pcr for precise nucleic acid quantification expert opinion on the treatment of patients with chronic hepatitis c key: cord- -fmnro kw authors: hoshino, y.; zimmer, j. f.; moise, n. s.; scott, f. w. title: detection of astroviruses in feces of a cat with diarrhea date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: fmnro kw astroviruses were detected by electron microscopy in the feces from a month old kitten with diarrhea. the mean diameter of the viral particles was . nm, and they showed characteristic five- or six-pointed star-shaped surface configurations. the clinical disease manifested by the cat and the observed morphology of the viral particles are consistent with previous reports on astroviruses of other species. astroviruses were detected by electron microscopy in the feces from a month old kitten with diarrhea. the mean diameter of the viral particles was . nm, and they showed characteristic five-or six-pointed star-shaped surface configurations. the clinical disease manifested by the cat and the observed morphology of the viral particles are consistent with previous reports on astroviruses of other species. . viral gastroenteritis is one of the leading causes of morbidity and mortality in neonatal and young animals, birds and man throughout the world ( , , , , ) . the development of methods for identification of viruses in fecal samples by direct electron microscopy (em) has led to the discovery of a number of previously unrecognized enteric viruses, including astroviruses. astroviruses are isometric, approximately nm in diameter viral particles which, when negatively stained, give the impression of a white, five-or six-pointed star superimposed on the particle. astroviruses have been detected in the feces of humans ( , , , , t , , ) , calves ( ) , lambs ( ) and turkeys ( ) with diarrhea. since attempts to propagate the mammalian and avian astroviruses in cell cultures have been unsuccessful, their size and morphology as determined by em are the primary criteria by which they are identified. viral antigen was detected by indirect immunofluorescent staining within the cytoplasm of cultured cells infected with fecal material of human ( , ) and bovine ( ) astroviruses. however, these infections were abortive in y. hos~l-¢o, j. f. zi~iet~, n. s. molse, and f. w. scott: that no specific fluorescence was seen in passaged celt cultures, t~ecently, t-ierrii~g et al. ( ) reported that ovine astrovirus has a single-stranded rna genome which resembles that of the picornaviruses and ealiciviruses but the polypeptide composition is unlike that of either of these groups. we report here the detection by direct. em of astroviruses in feces from a month old kitten with diarrhea. an intact female -month-old kitten was presented to the small animal hospital at the new york state college of veterinary medicine, cornell university, on october , . the chief complaint was that the kitten had had diarrhea, characterized as loose and watery, for approximately one week. a tentative diagnosis of parasite infestation and a diet-induced diarrhea was made, and the cat was vaccinated for feline panleukopenia, rhinotracheitis, and eaticivirus. the cat was returned five days later (october ) with the report that the diarrhea had continued and that the cat had been anorectie for three days. the diarrhea was reportedly more severe than on the initial visit. physical examination revealed slight dehydration, poor body condition, gas-distended loops of small intestine, and a green, watery diarrhea. a fecal sample was submitted for em examination for viral particles. two days later (october ), the kitten was hospitalized because of more severe dehydration and diarrhea. the results of a hemogram were normal, but the kitten was mildly acidotic and hypokalemic. during the subsequent nine days of hospitalization, the kitten's feces improved in consistency. the cat was discharged on october . re-examinations one week and months after discharge indicated that the kitten improved steadily and made an uneventful recovery. the feces had become normal, and the appetite was good. fecal sample was examined by em for ~ ral particles using the procedures reported previously ( ) . a percent (w/v) fecal suspension was prepared in eagle's minimum essential medium (mem) with antibiotics and without serum. this suspension was centrifuged at , × g for minutes (crude fecal suspension), and the supernatant was then ultra.centrifuged at , × g for minutes (clarified fecal suspension). one to drops of crude fecal suspension were mixed with drops of distilled water, one drop of . percent bovine serum albumin and three to four drops of percent phosphotungstie acid adjusted to ph . . this mixture was applied to a carbon-parlodion-coated copper grid with an all-glass nebulizer and examined in a philips electron microscope at kv. direct em examination demonstrated the presence of astrovirus-like particles, mostly in aggregates, in the fecal suspension (fig. ) . these viral particles were roughly spherical in outline, ranged in size from . nm to . nm in diameter and h ad a mean diameter of . nm. the surface structure had the appearance of characteristic five-or six-pointed stars ( fig. , arrows). this star-shaped configuration was not always apparent on all particles. bridging structures between virus particles were frequently seen (fig. t, arrowheads) . these structures have been observed on human ( ) and ovine ( ) astroviruses. it is speculated that these bridging structures may be surface fibers similar to those on adenoviruses. attempts at viral isolation with cell cultures were carried out as previously reported ( ) . confluent monolayer cultures ( × mm culture tubes) of first transfer cells of feline kidney and an established fetal rhesus monkey kidney celt line (ma ) were inoculated with . ml of clarified fecal suspension. after one hour adsorption at ° c, cell cultures were washed once with phosphate buffered saline, p h . , fed with . ml of maintenance medium (eagle's mem supplemented with . percent laetalburnin hydrolysate, . percent sodium! bicarbonate, antibiotics and no serum), and incubated at ° c. three blind passages were made at one week intervals. attempts to propagate this feline astrovirus in cell cultures have been unsuccessful. no calicivirus or other eytopathogenic virus was isolated in cell cultures. experimental transmission experiments with mammalian astroviruses indicate that. they induce mild, constitutional symptoms ( , , , ) . studies on the pathogenesis of astrovirus infection in lambs have shown the site of virus multiplication to be the mature villous epithelial cells of the small intestine ( , ) . there is a clinical impression that astrovirus-associated gastroenteritis in man and animals is generally milder than that associated with rotaviruses. asymptomatie excretion of astrovirus particles has been reported in man ( , ), calves ( ) and lambs ( ) . studies on the pathogenicity of feline astrovirns have been hampered by a shortage of material. as this is the first report of the identification of a feline astrovirus and since no serological test. is available, it is unknown how widespread and how significant this virus is. since eats frequently have close contact with humans, it will be important to study the relationship between feline and human astrovirnses. astrovirus-associated gastroenteritis in children the role of viruses in acute diarrhoeal disease viral intestinal infections of animals and man. comp. i m m u n . microbiol purification and characterization of ovine astrovirus viral gastroenteritis coronavirus-tike particles in the feces of normm cats isolation and characterization of feline rotavirus small spherical viruses in faeces from gastroenteritis patients astrovirus-associated gastroenteritis in a children's ward astrovirus infection in volunteers astroviruses detected by immunofluorescence (letter) ). : viruses in infantile gastroenteritis (letter) -n~ particles in feces in infantile gastroenteritis (letter) comparison of the features of astroviruses and caliciviruses seen in samples of feces by electron microscopy viruses in the stools detection of astroviruses in turkey faeces by direct electron microscopy the tecumseh study. x i . occurrence of acute enteric illness in the community coronavirus et "astrovirus" observ@s d ans ies selles d'enfants atteints de gastroentdrites recent advances in viral gastroenteritis detection and transmission of -nm virus particles (astroviruses) in faeces of lambs with diarrhoea pathogenesis of diarrhoea caused b y astrovirus infections ultrastructure of the small intestine in astrovirus isolation of small viruses resembling astroviruses and ealiciviruses from acute enteritis of calves key: cord- -hbpfz rt authors: glingston, r. sahaya; deb, rachayeeta; kumar, sachin; nagotu, shirisha title: organelle dynamics and viral infections: at cross roads date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: hbpfz rt viruses are obligate intracellular parasites of the host cells. a commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. organelles enable favourable intracellular environment for several viruses. however, studies reporting organelle dynamics upon viral infections are scant. in this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins. a unique feature of eukaryotic cells is the presence of distinct membrane bound structures called organelles. this subcompartmentalization of eukaryotic cells is very essential for its optimum function. organelles achieve this due to the presence of a unique set of proteins and distinctive lipid composition that determines their function. they are dynamic and interact with the surrounding organelles to regulate their biogenesis and function [ , ] . association of organelle dysfunction with human diseases/ disorders is reported and widely acknowledged [ ] . it is interesting that not only organelles are required for proper functioning of cells, but are also required for the successful infection of a virus [ , ] . it is well established that viruses develop alternate strategies to survive in the cells. the steps of the viral life cycle include entry, translation, replication, assembly and egress [ ] . moreover, viruses have developed remarkable ways to complete their life cycle by targeting specific cell organelles and processes. organelles like mitochondria, er, peroxisomes play an important role in innate immunity and host defence [ ] . recently lipid droplets have also been reported to be essential for innate response against viral infection [ ] . the viral life cycle is also a dynamic process that leads to the extensive host cellular reorganization. characterization of this spatiotemporal reorganization is a key step in order to understand the molecular mechanism of viral infection. localization of the viral proteins to an appropriate sub-cellular compartment is part of the strategy to hijack the host machinery and necessary pathways leading to establishment of an infection [ , ] . with the advent of several modern microscopy and proteomics methods, it is now clear that the localization and function of many host proteins are altered due to viral infections. not only the host proteins but the intracellular localization of the viral proteins and their interactions with the host proteins are also extensively studied using these methods [ ] . however, currently our understanding of organelle dynamics during viral infections is still at its infancy. current review briefly summarizes our knowledge of the various cell organelles/compartments following virus infection. this topic at the interjection of virology and cell biology represents an emerging research area of molecular investigations in virology. presence of nucleus that houses the genome is what distinguishes eukaryotic cells from prokaryotes (fig. ). it is a double membrane organelle and is the site for gene regulation and transcription. nuclear envelope comprising of nuclear pores is the barrier between the nuclear content and the rest of the cell. nucleolus is the region where ribosomal subunit assembly takes place in the nucleus. sub nuclear structures comprising of several proteins which regulate many cellular processes like apoptosis, dna damage, etc are called pml-nbs. many dna and rna viruses depend on the host nuclear proteins for their replication [ ] . several strategies are used by viruses in order to deliver its genome to the host cells [ ] . usually, when cells undergo mitosis, there is a temporary disassembly of the nuclear envelope (ne) which allows some viruses to enter into the nucleus [ ] . entry and integration of murine leukemia virus (mlv) into host nucleus depends on the ne breakdown during mitosis [ ] . various human immunodeficiency virus (hiv) preintegration complex proteins, such as the matrix [ ] , vpr [ ] , integrase [ , ] were found to contain nuclear localization signal (nls) in their genome sequence. this nls interacts with the nuclear transport receptors, which transports the viral proteins through the nuclear pore complex (npc). similarly, the influenza virus nucleoprotein (np) present in the viral ribonucleoprotein complex (vrnp) contains nls , nls and nls , which helps in its binding to cellular importins resulting in transportation of the vrnp to the nucleus [ e ] . in another strategy, the capsid of certain viruses gets attached to the cytoplasmic side of the host npc either directly or with the help of importins. this interaction acts as a signal for capsid disassembly followed by entry of the viral genome along with their proteins through the npc into the nucleus [ ] . studies on the herpes simplex virus- (hsv- ) infection on vero, bhk- and ptk cells reported transportation of viral tegument-capsid by dynein to the cytoplasmic side of npc [ , ] . the hsv- capsid binds to npc with the help of importin b and subsequently releases dna into the nucleus through the npc [ ] . similarly, the capsid protein of adenovirus (ad) on binding with the nuclear transport protein nup attaches to the cytoplasmic side of npc and releases the genetic material into the nucleus [ ] . some small viruses such as hepatitis b virus (hbv), baculovirus, etc were found to cross the npc and release their genome at the nuclear side of npc or within the nucleus [ ] . the capsid protein of hbv was reported to interact with npc via the nuclear receptor nup in xenopus laevis oocytes [ ] . furthermore, this binding depends on importin a, b and phosphorylated core protein present on its capsid [ ] . upon infection, hbv capsid enters the nucleus through the npc and subsequently releases its genome into the nucleoplasm [ ] . similarly, capsid proteins of simian virus (sv ) were found to enter the nucleus through npc [ ] . it has been reported that the disruption of the ne and nuclear lamina temporarily aid the nuclear entry of viruses such as parvoviruses [ ] . disruption of ne in xenopus oocytes and both ne and nuclear lamina in mouse fibroblast cells upon parvovirus infection and minute virus of mice (mvm) was reported using electron microscopy analysis [ , ] . presumably, viruses evolved different mechanisms to enter nucleus in order to facilitate their replication cycle and survivability inside the host cells. after the successful entry into the nucleus, both dna and rna viruses exploit various nuclear components such as nucleolus, promyelocytic leukaemia nuclear body (pln) and/or nuclear proteins in order to facilitate their replication (fig. ) [ ] . nucleolin, fibrillarin and b (nucleophosmin) are examples of nucleolar proteins reported essential for various functions like post transcriptional process, ribosome assembly, etc [ ] . these multifunctional host nucleolar proteins were reported to be incorporated into the replication and translation complex of many viruses [ ] . many dna viruses like hsv and ad were reported to be involved in relocalization or disruption of the host nucleolar proteins [ ] . for instance, the transfection of the recombinant ul protein of hsv- , tagged with an n-terminal hemagglutinin in vero cells resulted in the redistribution of nucleolin and b [ , ] . another nucleolar protein fibrillarin was also found to be redistributed in spots throughout the nucleus but in an ul independent manner [ ] . the core protein of ad was found to be associated with the nucleolus in hela cells [ ] . further studies showed that this association results in the relocation of the nucleolin to the cytoplasm of the infected cells [ ] . the ad-induced relocation of nucleolin was proposed to be a strategy used by virus to suppress its activity [ ] . additionally, infection of hela cells with ad resulted in inhibition of synthesis and maturation of rrna [ ] . nucleolar localization of the viral capsid and rna binding proteins of many rna viruses has been reported [ ] . for example, the encephalomyocarditis viral (emcv) proteins a and bcd and human rhinovirus hrv c protease were found to localize to the nucleoli resulting in inhibition of cellular rna transcription [ , ] . it has been reported that the expression of tat and rev proteins of hiv- in cos cells resulted in their accumulation in dense fibrillar component in the nucleolus [ ] . moreover, rev protein was reported to be involved in deforming the nucleolar architecture [ ] . similarly, the viral n protein was found to be localized to the nucleolus in addition to the cytoplasm upon infection of coronavirus resulting in delayed cell cycle to facilitate viral assembly [ ] . poliovirus (pv) or rhinoviruses interact with nucleolin and blocks the nuclear import leading to accumulation of nucleolin in the cytoplasm of the infected cells [ ] . the accumulated nucleolin interacts with the internal ribosome entry site (ires) element present in the upstream end of the viral genome to stimulate its translation [ ] . on the other hand, these changes result in the shutdown of cellular transcription leading to the downregulation of the host defence mechanism [ ] . infection of human respiratory syncytial virus (hrsv) in a cells resulted in depletion of nucleolin [ ] . although, both dna and rna viruses behave differently towards acquiring the nuclear niche however the goal being to establish control over cellular transcription and favour its genome over the host. pml nb is another subnucleolar component, which contains proteins such as pml and sp . these proteins are induced by interferons and hence pml nb can act as a target for viruses to escape the antiviral signalling response [ ] . redistribution of the pml nb has been reported upon infection of hsv- to bhk- cells [ ] . similarly, ebna lp protein of epstein-barr virus (ebv) was found to displace sp from pml nbs in burkitt's lymphoma and hep cells [ ] . the viral e -orf protein induced reorganization of pml nbs into elongated track like structures upon infection of cv cells with human ad [ ] . later, it was found that this reorganization was responsible for the downregulation of the host antiviral response [ ] . another example is the infection of human foreskin fibroblasts (hffs) with human cytomegalovirus (hcmv) resulting in the accumulation of pp at pml nbs in the infected cells [ ] . further studies showed that pp was responsible for the proteasomal degradation of pml-nb protein daxx, which is important for inducing intrinsic immune response against hcmv infection [ ] . some rna viruses also result in the redistribution and degradation of host pml nbs in order to neutralize the antiviral response. the ring finger protein z of lymphocytic choriomeningitis virus interacts with pml protein and leads to the redistribution of pml-nbs from the nucleolus to the cytoplasm [ ] . the expression of c protease of emcv in cho cells was reported to target pml-nbs and promote its degradation in proteasome and by sumo-dependent mechanism [ ] . interestingly, cho cells infected with rabies virus were reported to contain enlarged pml nbs [ ] . disruption of the nucleocytoplasmic trafficking in the host cells is another major alteration caused due to viral infections ( fig. ) [ ] . two conserved proteins pul and pul of hcmv were reported to remodel the nuclear lamina of helfs (primary human lung fibroblasts) for the budding of virions [ ] . interaction between human papillomavirus (hpv) and host mitotic chromosomes has been documented [ ] . infection of porcine bone marrow cells with african swine fever virus (afsv) resulted in fragmentation of the nucleus [ ] . the organelle found in continuation with the nucleus in a cell is the er (fig. ) . protein synthesis and folding (rough er), lipid synthesis, calcium regulation (smooth er), etc are some of the important functions of the er. multiple structural domains of the er are reported which enable it to serve as a site for various important functions in a cell. several morphological alterations of er such as vesicle formation, invagination of the membrane, zippered appearance, etc have been reported upon viral infection in different cell lines (fig. ) . the large malleable surface of the er aids viruses to form protective compartments to set up their replication machinery. these compartments known as viroplasm not only concentrate viral and host proteins required for viral genome replication but also protect the viral genome from cellular nucleases [ ] . in order to construct these compartments, viruses alter host's fatty acid metabolism, induce rearrangement of the membrane constituents and also recruit cellular machinery to produce proteins essential for its replication [ , ] . infection of vaccinia virus (vacv) in bs-c- , hela and rk- cells leads to the formation of vacv membranes derived from the er membrane [ ] . in case of dengue virus (denv- ), the genomic rna was observed to localize over the rough er in c / aedes albopictus cells [ ] . denv infection in human hepatoma (huh ) cells leads to invagination of er membrane resulting in the formation of spherules or vesicles containing double stranded rna [ ] . hela cells and cos- cells on infection with pv cause formation of single membrane tubules which later mature into double membrane vesicles (dmvs) [ , ] . similarly, severe acute respiratory syndrome corona virus (sars-cov) induced formation of er derived dmvs upon infection of vero cells [ , ] . zippered appearance of er was observed upon infection of infectious bronchitis virus (ibv) on mammalian, avian and tracheal epithelial cells [ ] . studies with the plant viruses like the potato virus x (pvx) reported tgbp protein induced reorganization of the er and appearance of vesicular structures in tobacco plants [ ] . effect of the viral protein expression on sterol biosynthesis apart from the alterations in er morphology have also been reported [ , ] . several viral proteins need to be glycosylated at their n-terminal to ensure proper folding and for the incorporation into virions [ ] . hence, a common phenomenon observed upon viral infection is interference with the host post translational machinery and competition with the cellular proteins to undergo these modifications [ ] . for example, protein porf of hepatitis e virus (hev) gets glycosylated in the er upon its expression in cos- cells and huh mammalian cells [ , ] . this increased biosynthetic load is proposed to increase in accumulation of malformed proteins resulting in er stress (fig. ) . er gets relieved from this stress by inducing a pathway called unfolded protein response (upr) pathway [ ] . upr serves to enhance the cell's degradation ability in order to establish er homeostasis [ ] . transport of misfolded, misassembled proteins from the er to the cytosol and clearance by the ubiquitin proteasome system takes place via the er associated degradation (erad) pathway [ ] . however, some viruses use erad pathway for their advantage by co-opting erad to disassemble and gain access to the cytosol of the host cell [ ] . upon sv infection of cv- , hela and t cells, induction of erad factors in turn induce er membrane reorganization into distinct subdomains called foci and the accumulation of sv in these foci was observed [ ] . later, sv particles travel across the er membrane to reach the cytosol [ ] . many enveloped and non-enveloped viruses that exploit er functions like upr, erad to facilitate their replication have been discussed elsewhere [ ] . in addition to the above listed mechanisms, er mediated nlinked glycosylation plays a very important role in the survival of the viruses. it has been very elegantly demonstrated that the biogenesis of influenza could modulate the host immune response [ ] . similarly, the role of n-glycans in the host immunity against hiv has been documented [ ] . these studies corroborate to a common point where the level of glycosylation in the viral surface glycoproteins could alter their antigenicity. mitochondria are double membrane bound organelles that comprise their own genome ( fig. ). they are involved in various functions like fatty acid metabolism, apoptosis, calcium homeostasis, etc. considered evolutionarily the oldest organelle of a eukaryotic cell, they are indispensable as power house of the cell. mitochondrial morphology is maintained by a series of interlinked events namely mitochondrial fusion, fission and mitophagy [ ] . mitochondrial fusion helps in exchanging matrix metabolites and intact mitochondrial dna while mitochondrial fission helps in sorting impaired mitochondria from the healthy population which are further eliminated by a process called mitophagy [ ] . all the above dynamic events are altered upon viral infection in order to facilitate its replication (fig. ) [ ] . for example, it was reported that hbv infection in the cell induces mitochondrial fission followed by mitophagy in order to attenuate apoptosis [ ] . on the other hand, expression of orf- b protein of sars-cov virus promoted mitochondrial fusion in hek cells [ ] . upregulation of mitophagy and degradation of the mitochondrial antiviral signalling protein (mavs) in order to attenuate the antiviral immune response in non-small cell lung cancer (nsclc) cells was reported upon measles virus infection [ ] . the expression of matrix protein (m) of human parainfluenza virus type (hpiv ) in hek t and hela cells was reported to induce mitophagy resulting in the suppression of type interferon response [ ] . hbv induces mitophagosome formation which upon fusion with lysosomes leads to mitophagy and prevents apoptosis, thus facilitating persistent infection in the huh cells [ ] . similarly, newcastle disease virus (ndv) was reported to induce mitophagy, which promotes ndv replication by preventing caspase dependent apoptosis in human non-small cell lung cancer a cells [ ] . viruses can alter the intracellular distribution of mitochondria either to prevent the release of mediators of apoptosis or to meet their energy requirement during replication by concentrating them near their viral factories [ ] . hbv x protein induces the perinuclear clustering of mitochondria in huh cells [ ] and afsv leads to the transport of mitochondria to viral assembly sites in vero cells [ ] . mitochondrial dna codes for proteins essential for respiratory functions of a cell. certain viruses evade the mitochondria associated antiviral response by damaging the host cell mitochondrial dna, which is essential for synthesizing enzymes for optimum mitochondrial function [ ] . for example, mitochondrial dna degradation is induced in mammalian cells upon expression of ul . , an amino-terminally truncated ul isoform of hsv- [ ] . raji cell lines infected with hepatitis c virus (hcv) also exhibit mitochondrial dna depletion [ ] . maintenance of mitochondrial/cellular ca þ homeostasis is vital for various cellular functions. many viruses are involved in altering the mitochondrial calcium homeostasis in order to meet their needs during replication [ ] . hcmv upon infection causes calcium influx into mitochondria from er [ ] . on the other hand, expression of b protein of coxsackievirus resulted in reduced signalling of ca þ between the er-golgi and the mitochondria in hela cells resulting in suppression of apoptosis [ ] . rotavirus was also reported to alter the calcium homeostasis in the host cell throughout its life cycle [ ] . mitochondria are the major source of ros in a cell and a balance between ros production and scavenging is crucial for optimum functioning of the cells. upon viral infection mitochondria undergo oxidative stress and result in an increased production of ros which in turn reduces virus replication [ ] . interestingly, ros is also involved in activating many host cellular pathways favourable for viral replication and pathogenesis. several viruses like hiv, hcv, adv, ebv, etc result in increased oxidative stress upon infection [ ] . on the other hand, both increase and decrease of oxidative stress is employed as a survival strategy by hbv [ , ] . many viral proteins reach mitochondria through the mitochondria-associated membrane [ ] a sub-compartment of the er or are targeted directly from the cytosol and result in an altered mitochondrial permeability transition pore (mptp) [ ] . mptp is also responsible for the maintenance of mmp and provides energy for atp synthesis. altering mptp leads to passive swelling, outer membrane rupture, osmotic water flux, and release of proapoptotic factors leading to cell death [ ] . in general, increased mmp induces apoptosis, while decreased mmp prevents apoptosis [ ] . viruses are proposed to decrease mmp to prevent cell death in order to promote their replication. however, in later stages, they may trigger an increase in mmp to release the progeny virions by apoptosis [ ] . the m l protein of myxoma pox virus prevents the loss of mmp in cos- monkey kidney cells, hela cells, thp- human monocytes and jurkat t lymphocytes [ ] . on the other side, the r protein of hiv- induces the loss of mmp in cem-c and jurkat cells and results in apoptosis [ ] . viruses alter the host mitochondrial metabolic pathways in order to maintain cellular energy homeostasis essential to ensure efficient replication and to avoid mitochondrial antiviral response particularly in case of slow replicating virus [ ] . some viruses modulate the normal cells to increase aerobic glycolysis and use glucose biosynthetically, which helps especially enveloped viruses to increase their available pool of fatty acids, lipids and nucleotides during their replication [ , ] the feline leukemia virus infection on lung fibroblasts (flf- ) resulted in a e % increase in glucose uptake and lactic acid production [ ] . an increase in the lactic acid production upon infection of the chick embryo cells with rous sarcoma virus was reported [ ] . hcmv upon infection of hffs, human retinal pigment epithelial cells (arpe ), human embryonic lung fibroblasts (mrc ) and vero cells enhances glycolytic flux and directs the supply of carbon from glucose to tca cycle. this helps to facilitate fatty acid biosynthesis while hsv- upon infection of same cell lines directs the central carbon metabolism in order to induce the production of pyrimidine nucleotide components [ ] . hcv core protein suppresses mitochondrial complex activity and impaired function of electron transport cycle leading to ros accumulation in hepatoma cells of transgenic mice [ ] . mitochondrial involvement in virus survival is quite relevant to the fact that viruses require a source of energy to favour the active processes involved in their life cycle. peroxisomes are single membrane bound dynamic organelles required for b-oxidation of fatty acids and ros metabolism (fig. ) . they have developed diverse functions which are organism and environment dependent. they are unique with respect to proliferation, as they can increase in number by both growth and division of pre-existing organelles and formation of new organelles from the er. many viruses or viral proteins are reported to localize to peroxisomes and/or exploit their functions to facilitate their replication in the host cells [ ] . presence of a peroxisome targeting signal (pts) in the protein sequence is reported to be essential for targeting proteins into the peroxisomes [ ] . however, it was also proposed that viral proteins without pts may get associated with host peroxisomal proteins in the cytosol which then ferry the viral protein into the peroxisomes in a piggyback fashion [ ] . for instance, hcmv encodes a protein called vmia reported to be localized to peroxisomes in hffs and hepg cells [ ] . the vmia interaction with the host pex protein aids the viral protein localization to the peroxisomes. fragmentation of peroxisomes upon vmia expression in hepg was also reported [ ] . in addition, peroxisomal localization of two cymbidium ring spot viral proteins p and p was reported in yeast [ ] . a role for peroxisomes in the replication of tbsv has also been reported. the viral replication protein p interacts with the host peroxisomal protein pex for its targeting to the peroxisomal membrane and subsequent replication [ , ] . the host hsp protein was reported to promote the localization of the viral replication proteins to the peroxisomes in yeast cells [ ] . n-terminal protease n pro of pestivirus is another example of a viral protein that is associated with peroxisomes and facilitates its survival and replication [ ] . some members of the family tombusviridae are involved in remodelling the peroxisomal membrane resulting in the formation of vesicular structures called "profoundly modified peroxisomes" or "peroxisome derived multivesicular bodies" [ ] . for example, the infection of cymbidium ring spot virus resulted in the formation of small vesicles at the periphery of the peroxisomes in plant leaf tissues [ ] . at the later stage of infection, the entire peroxisomal matrix is occupied by these vesicles [ ] . similarly, the cucumber necrosis virus (cnv) induces the formation of peroxisomal vesicles in which the viral rna replication occurs in yeast cells [ ] . studies revealed upregulation of peroxisomal biogenesis in endothelial cells upon latent infection of kaposi's sarcoma associated herpes virus [ ] . it was also reported that proteins involved in peroxisomal lipid metabolism were essential for the survival of latently infected human dermal microvascular endothelial cells and lymphatic endothelial cells [ ] . interestingly, hiv infection reduces the number of peroxisomes in infected cells due to upregulation in the levels of micrornas that inhibit production of peroxisome biogenesis factors [ ] . it has been reported that the west nile virus (wnv) and denv infection on a and hek t cells result in the degradation of peroxisomes [ ] . the capsid proteins of both denv and wnv were reported to interact with the peroxisomal protein pex required for peroxisome biogenesis. degradation and redistribution of pex from perinuclear region to juxtanuclear region was reported in the infected cells. in addition, reduced level of the antioxidant enzyme catalase was observed in the infected cells. these alterations resulted in an impairment of early antiviral signalling of the peroxisomes. expression of the pts containing vp protein of the rotavirus, in ma cell lines resulted in peroxisomal localization [ ] . the role for such a localization was speculated to utilize the peroxisomal lipid metabolism for the supply of cholesterol for lipid raft synthesis, required for the viral replication. peroxisomal b-oxidation metabolism leads to the production of myristoyl-coa by shortening the fatty acids chain which could be exported to the cytosol for nmyristoylation of the viral proteins vp and vp of rotavirus [ ] . studies on hiv suggested an interaction between viral nef protein and the peroxisomal enzyme thio-esterase using yeast two hybrid system [ ] . further studies reported an increase in the enzymatic activity of human acyl-coa thio-esterase on binding with the nef protein [ ] . the increased activity of the peroxisomal enzyme was speculated to contribute in alteration of the subcellular morphology and in downregulation of the host antiviral response [ ] . extensive modifications of proteins for proper functioning and targeting in a cell takes place at the golgi apparatus. other functions of golgi inevitable for the cell are carbohydrate synthesis and lipid transport. the unique stacked structural organization of the golgi is essential for these functions (fig. ) . the golgi apparatus is composed of three regions namely cis golgi network (cgn), medial and trans golgi network (tgn). various viruses and viral proteins have been identified to localize to these golgi regions during their life cycle. for example, hela cells upon infection with adeno-associated virus type (aav- ) led to an accumulation of the viral particles in the tgn and in the golginetwork associated coated vesicles [ ] . in sars-cov, the transmembrane domain of the orf b protein contains the retention signal required for accumulation of the protein in the cgn and tgn [ ] . reports suggest bunyaviruses assemble in the golgi as a result of its retention signal in the glycoprotein (gn) of the virus [ , ] . fragmentation of golgi body is a common phenomenon that occurs as a result of various viral infections (fig. ) . orf virus (a parapox virus) causes disruption and fragmentation of golgi in vero cells. this structural modification affects the late vesicular export machinery and results in downregulation of the host immune response [ ] . similar phenomenon was also reported upon expression of c protease of foot-and-mouth disease virus in vero cells [ ] . infection of hrv a on wi- , t, and h -hela cells was reported to induce fragmentation of golgi body, rearrangement of the golgi membranes into vesicles which are utilized as sites of rna replication [ ] . another incidence of the virus induced golgi body fragmentation was observed upon infection of sars-cov that resulted in death of vero cells [ ] . many rna viruses were also found to alter the integrity of golgi complex of the host cells. for example, the expression of n-terminal non-structural protein of norwalk virus in crandell-rees feline kidney (crfk) and hela cells co-localizes to golgi complex and induces its disassembly into discrete aggregates [ ] . another example is the pv infection on vero cells which results in complete disruption of the cgn into fragments scattered throughout the cytoplasm. the expression of pv protein b in vero, normal rat kidney (nrk) and cos- cells also resulted in golgi bodies disassembly [ ] . similarly, expression of protein a of avian encephalomyelitis virus (aev) in chick embryo brain (ceb) cells and cos- cells resulted in depletion of golgi stacks and in severe disassembly of golgi bodies [ ] . an interesting alteration in the morphology of golgi apparatus was observed upon infection of turnip mosaic virus (tumv) on n. benthamiana plant. tumv infection was observed to induce amalgamation of golgi apparatus, er and chloroplast [ ] . mccoy mouse fibroblast cells led to accumulation of golgin- a tgn resident protein in the viral factories [ ] . similarly, a protein of some picornaviruses were also found to interact with a golgi apparatus resident protein namely golgi adaptor protein acyl coenzyme a (acyl-coa) binding domain protein (acbd /gpc ), acts as an adaptor to recruit phosphatidylinositol -kinase class iii beta (pi kiii) in infected cells [ ] . evidence shows that the tomato spotted wilt virus (tswv) glycoprotein gn localizes to golgi membranes and induces deformation of the membranes into pseudo-circular and pleomorphic structures in tobacco plant cells [ ] . upon infection of hela cells, rabbit kidney cells and mouse monocytes-macrophages cell lines with vacv, viral progeny becomes enwrapped in the membrane derived from tgn cisterna to form the enveloped virus [ ] . several viral proteins localize to the golgi body and mature by undergoing post translational modifications such as glycosylation, phosphorylation, etc. rubella virus was reported to undergo a golgi dependent maturation upon infection of bhk- and vero cells [ ] . the inner tegument protein pul of the dna virus hsv was identified to be responsible in directing the viral capsids to the tgn in order to undergo secondary envelopment in different cell lines [ ] . the glycoproteins of bunyamwera virus undergo primary maturation by modifying their sugar composition in the tgn upon infection of the bhk- and vero cells [ ] . lipid droplets are single membrane bound organelles with a lipid core that primarily consists of neutral lipids like triacylglycerols (fig. ) . they are essential for lipid storage and metabolism in a cell. these stored lipids can be used to generate and maintain energy homeostasis and hence they are central to the cellular function. lipid droplets are dynamic intracellular organelles which are required for storing lipids in a cell. they play a major role in energy homeostasis and membrane trafficking [ ] . many rna viruses exploit this energy storing capacity of lipid droplets to facilitate their replication [ ] . upon expression, various viral proteins are reported to localize to the lipid droplets [ ] . for example, upon hcv infection, the hcv ns a protein localizes to the surface of the lipid droplets with the help of a host factor diacylglycerol acyltransferase (dgat ) in huh and hek t cells [ ] . another study reported that hcv core a protein is involved in downregulating the expression of phosphoinositide -kinase (pi k)/phosphatase and tensin (pten) which in turn induces the accumulation of enlarged lipid droplets in human huh and hepg cells [ ] . in another example, denv c protein was reported to bind and interact with perilipin a protein present on the surface of the lipid droplets in hepg cell lines [ ] . a similar localization of denv c protein was also observed upon infection of denv in bhk- , hepg , and c / ht mosquito cells of a. albopictus [ ] . additionally, the denv c protein localization on lipid droplets was also found to be essential for the denv replication [ ] . denv induces autophagy and results in a reduction in lipid droplet area in huh . cells, a subline derived from huh cells [ , ] . further analysis of the infected cells showed reduced levels of triglycerides in the lipid droplets and suggests that atp generation by b-oxidation of fatty acids is essential for robust replication of viral rna [ , ] . another study reports that rotavirus recruits lipid droplets into their viroplasm compartments upon infection of ma , caco- , bsc- , and cos- cell lines [ ] . confocal microscopy studies reported that two ld-associated proteins namely perilipin a and adrp colocalize with rotaviral proteins present in the viroplasm [ ] . overexpression of the hbv x (hbx) protein was found to induce lipid accumulation in hepatic cells [ , ] . na and colleagues found that hbx induces a pathway that involves the expression of liver x receptor and its associated genes result in accumulation of lipid droplets in hepg cell lines [ ] . lysosomes are single membrane-bound organelles which house enzymes involved in the degradation of various extracellular and intracellular macromolecules such as proteins, pathogens, etc through phagocytic or autophagic pathway. plant and fungal vacuoles have similar degradative and storage functions like the lysosomes (fig. ) . the specialized acidic lumen is the unique feature of these organelles which is needed to keep the enzymes active. viruses utilize lysosomal enzymes in order to facilitate their replication and release within the host cell [ ] . it has been suggested that lysosomal enzymes are involved at different stages of vacv and mouse hepatitis virus replication [ ] . they proposed a possibility for viruses to recruit lysosomal enzymes inside phagosomes in order to uncoat and release their genome in the host cell. a possible role for the lysosomal enzymes in enhancement of glycolysis in viral infected cells was also proposed. recent studies reported the accumulation of hav progeny in the lysosome of the host hepg cells. the maturation of these viral particles was reported to be catalysed by the lysosomal protease [ ] . sv infection in bsc- and t cell lines resulted in swelling up of lysosomes followed by the release of lysosomal enzyme into the cytoplasm [ ] . since lysosomes play a major role in the host's antiviral response, they are likely to become a target for certain viruses. the x protein of the hbv leads to inhibition of lysosomal acidification leading to loss of functioning [ ] . however, the lysosome's ability to fuse with autophagosomes was found to remain unaffected. this virus induced accumulation of immature lysosomes resulting in the suppression of autophagic degradation was followed by development of hbv-associated hepatocellular carcinoma [ ] . deglycosylation of the lysosome-associated membrane proteins by neuraminidase (na) of h n influenza virus resulted in destruction of lysosomes. this was followed by cell death due to the release of hydrolytic lysosomal enzymes to the cytoplasm [ ] . shubin and colleagues studied the expression of c protease of hav in a and human lung epidermoid carcinoma (calu- ) cell lines and found that hav c protease induces the development of non-acidic cytoplasmic vacuoles which originate from several types of lysosomal/endosomal organelles [ ] . similarly several viruses like hiv, adenovirus, pv have been reported to cause lysosomal rupture [ ] . tobacco plant when infected with cucumber mosaic virus (cmv), the viral replicase complex that constitutes the replicaseassociated protein cmv a and rna dependent rna polymerase protein cmv a was reported to localize on the vacuolar membrane [ ] . singapore grouper iridovirus (sgiv) infection on grouper embryonic cells (gecs) from the brown-spotted grouper epinephelus tauvina results in the formation of a large intracellular vacuole for viral accumulation. later, the virus recruits the host cytosolic membrane-bending proteins in order to induce tubulation of vacuolar membrane [ ] . the individual vacuoles fuse together to form a large vacuole which in turn fuses with the cell membrane. these events aid in the release of virions [ ] . semliki forest virus (sfv) targets the vacuolar atpase in order to cause acidification of vacuoles resulting in low intra luminal ph [ ] . upon infection with the sfv the endocytic vacuoles with low ph trigger membrane fusion essential for the viral pathogenesis in bhk- cells [ ] . vacuolar proton atpase activity leading to low intra endosomal ph was reported to be required for the entry of reovirus into the host cells [ ] . acidification of vacuoles by cellular v-atpase in human dermal fibroblast cells upon infection of hcmv was reported to be required for the formation of the specialized compartment for virion assembly [ ] . apart from the above discussed well defined organelles of a cell, viruses also modify and hijack various vesicular structures and protein complexes of the host cell to ensure efficient infection. we discuss these essential compartments/structures in this section. endosomes are required for internalization of extracellular material into the cells and lead them to lysosomes for degradation or recycle back to the plasma membrane. multi vesicular bodies are vesicular compartment of the endocytic pathway and contain intraluminal vesicles. autophagosomes are double membrane vesicular structures that sequester the cytoplasm containing proteins, organelles, etc and direct them to degradation (fig. ) . efficient cellular entry of most viruses is via endocytic pathway that comprises of various endosomes. several viruses like influenza, sfv, vsv, sv , ebola, etc use different endocytic pathways like clathrin, caveoli or micropinocytosis to gain entry into the cells [ , e ] . viruses modify or induce the formation of vesicles like endosomes in order to facilitate their multiplication inside the host cells [ ] . sfv modifies the endosomal and lysosomal membrane of the infected bhk- cells for the construction of its replication site [ ] . further the endosomes and lysosomes were reported to fuse together resulting in the formation of cytoplasmic vacuoles [ ] . sv was reported to trigger the formation of endocytic vacuoles that migrate and fuse with the nuclear membrane upon infection of cv- cells leading to the migration of virions into the infected cell nucleus [ , ] . endosomal sorting complexes required for transport (escrt) catalyse the process of invagination of endosomal membrane and result in the formation of multiple vesicular bodies (mvb) in eukaryotic cells [ ] . mvb act as an intermediate for transporting ubiquitinated or misfolded protein to lysosomes [ ] . mvb are also reported to play an active role in endolysosomal transport and budding of the virus. several rna and dna viruses hijack the escrt machinery in order to facilitate their release from the infected cells [ ] . the matrix protein vp recruits tsg protein and escrt- complex constituting of vps , vps b and vps in order to direct the ebola virus into multivesicular bodies that assists in their budding [ ] . hoffmann and colleagues reported that upon infection of hepatoma derived cell lines by the dna virus hbv, cellular a-taxilin acts as an adaptor for the binding of large hbv surface antigen with escrt components. this aids in recruiting the escrt machinery for the release of hbv-dna containing particles [ ] . various rna viruses alter autophagosomes and induce or suppress autophagy in order to complete their life cycle or to escape from the host antiviral response [ ] . it has been reported that the coxsackie b infection triggers the formation of autophagosomes in hela and hek cells [ ] . prevention of lysosomal fusion with autophagosomes also enhanced coxsackie virus replication in the host cells. wild type mouse embryonic fibroblast (mef) and huh cells also exhibit enhanced autophagosome formation upon denv infection [ ] . the viruses modulating the phenomenon of cellular autophagy for their advantage have been reviewed elsewhere [ ] . proteasome is a complex of proteases that selectively degrades intracellular proteins. this complex is tightly regulated and identifies polyubiquitinated proteins for degradation process. recent studies identified that viruses hijack ups in several ways to enhance their infectivity [ ] . this is achieved by degradation of host proteins of ups, enhancing the function of viral proteins by modifications, hampering the modifications of signalling molecules of innate immunity, etc [ ] . virus induced ubiquitination and subsequent proteasomal degradation of p as a strategy is employed by dna viruses such as hpv, adv, etc [ ] . viral protein x of hiv- was reported to be responsible for the ubiquitination and proteasomal degradation of the host protein samhd that inhibits hiv infection [ ] . an interesting strategy by denv was reported where the viral protein ns stimulates proteasomal degradation of stat and blocks type ifn signalling and thus evades the host immune mechanisms [ ] . similarly selective degradation of ns (non-structural protein) of the zika virus via proteasome mediated pathway was recently reported [ ] . this proteasome dependant degradation of the viral protein was proposed as a strategy for the host antiviral mechanism. a role for ups has also been reported in plant viral infections. components of rna silencing such as argonaute are targeted to proteasomal degradation by viruses like potato virus x, enamovirus, etc for efficient infection [ , ] . this review summarizes the modifications that organelles encounter upon viral infection in a cell. understanding organelle dynamics under various conditions is a fundamental question that has attracted researchers for a long time. the importance of organelle dynamics and function is highlighted by many examples of diseases/disorders where it is affected. alterations in organelles such as shape, content, dynamics and eventually the function as a result of viral infections is observed. not only viruses but pathogenic bacteria have also been reported to alter organelles for their survival and infection. as emphasized in this paper, recent studies have shown that many viruses encode proteins that are targeted to various cellular organelles and control their functions. certainly there exists a close relationship between organelle dynamics and viral infections but thorough characterization will highlight their relevance to pathogenesis. advanced methods in microscopy and proteomics have enabled such characterization and the molecular details of virus-host interactions and viral replication in host cells is now understood in detail for few viruses. it is important to determine how various organelle proteins are temporally and spatially regulated upon viral infections leading to altered functions. organelles not as independent entities but a role for inter-organelle communication/inter-organelle cross talk in a cell for optimum functioning is now unequivocally accepted. this is another very interesting aspect to be explored to enhance our understanding of the virus-host interaction mechanisms to enable design of new antiviral strategies. our understanding on the organelle-virus interaction has been rapidly increasing with the advent of new molecular biology tools and advance imaging techniques. although we know the basic modus operandi of the viruses, there might be a novel virus that behaves different than the existing dogma with respect to host cellular architecture. the authors declare to no conflict of interest. ground control to major tom: mitochondria-nucleus communication the upsides and downsides of organelle interconnectivity an organelle-specific protein landscape identifies novel diseases and molecular mechanisms hiv- and the host cell: an intimate association virulence and pathogenesis virus entry by endocytosis signaling organelles of the innate immune system lipid droplet density alters the early innate immune response to viral infection virus factories: associations of cell organelles for viral replication and morphogenesis exploring and exploiting proteome organization during viral infection nuclear remodelling during viral infections how viruses access the nucleus the road to chromatin -nuclear entry of retroviruses integration of murine leukemia virus dna depends on mitosis two nuclear localization signals in the hiv- matrix protein regulate nuclear import of the hiv- pre-integration complex characterization of hiv- vpr nuclear import: analysis of signals and pathways hiv- infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway integrase interacts with nucleoporin nup to mediate the nuclear import of human immunodeficiency virus type nuclear import and export of influenza virus nucleoprotein application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza a virus strain importin alpha nuclear localization signal binding sites for stat , stat , and influenza a virus nucleoprotein function of dynein and dynactin in herpes simplex virus capsid transport microtubule-mediated transport of incoming herpes simplex virus capsids to the nucleus herpes simplex virus type entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro import of adenovirus dna involves the nuclear pore complex receptor can/nup and histone h nucleoporin arrests the nuclear import of hepatitis b virus capsids in the nuclear basket phosphorylationdependent binding of hepatitis b virus core particles to the nuclear pore complex nuclear import of hepatitis b virus capsids and release of the viral genome role of nuclear pore complex in simian virus nuclear targeting pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope parvoviral nuclear import: bypassing the host nuclear-transport machinery nucleoli: composition, function, and dynamics involvement of ul in herpes-simplex-virus- -induced dispersal of nucleolin involvement of the ul protein in herpes simplex virus -induced dispersal of b and in nuclear egress adenovirus core protein v is delivered by the invading virus to the nucleus of the infected cell and later in infection is associated with nucleoli adenovirus protein v induces redistribution of nucleolin and b from nucleolus to cytoplasm effects of adenovirus infection on rrna synthesis and maturation in hela cells encephalomyocarditis virus (emcv) proteins a and bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription rhinovirus c protease precursors cd and cd' localize to the nuclei of infected cells the post-transcriptional regulator rev of hiv: implications for its interaction with the nucleolar protein b localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus nucleolin stimulates viral internal ribosome entry site-mediated translation quantitative proteomic analysis of a cells infected with human respiratory syncytial virus ifn enhance expression of sp , an autoantigen in primary biliary cirrhosis hsv- ie protein vmw causes redistribution of pml mediation of epstein-barr virus ebna-lp transcriptional coactivation by sp adenovirus replication is coupled with the dynamic properties of the pml nuclear structure adenovirus e orf protein inhibits the interferon-mediated antiviral response inactivating a cellular intrinsic immune defense mediated by daxx is the mechanism through which the human cytomegalovirus pp protein stimulates viral immediate-early gene expression two ring finger proteins, the oncoprotein pml and the arenavirus z protein, colocalize with the nuclear fraction of the ribosomal p proteins sumoylation promotes pml degradation during encephalomyocarditis virus infection rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies viral interactions with the nuclear transport machinery: discovering and disrupting pathways remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pul and pul papillomavirus interaction with cellular chromatin phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of african swine fever virus wrapping membranes around plant virus infection inhibition of sterol biosynthesis reduces tombusvirus replication in yeast and plants endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly direct formation of vaccinia virus membranes from the endoplasmic reticulum in the absence of the newly characterized l -interacting protein a . intracellular localisation of dengue- rna in mosquito cell culture using electron microscopic in situ hybridisation composition and three-dimensional architecture of the dengue virus replication and assembly sites cellular origin and ultrastructure of membranes induced during poliovirus infection remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagylike origin for virus-induced vesicles ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes the potato virus x tgbp movement protein associates with endoplasmic reticulum-derived vesicles during virus infection tombusviruses upregulate phospholipid biosynthesis via interaction between p replication protein and yeast lipid sensor proteins during virus replication in yeast opportunistic intruders: how viruses orchestrate er functions to infect cells arms race between enveloped viruses and the host erad machinery mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis e virus orf protein cytoplasmic localization of the orf protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis bap and bip are essential for dislocation of sv from the endoplasmic reticulum to the cytosol global sitespecific n-glycosylation analysis of hiv envelope glycoprotein the interplay between mitochondrial dynamics and mitophagy mitochondrial fusion, fission, and mitochondrial toxicity mitochondrial dynamics and viral infections: a close nexus hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis sarscoronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome mitophagy in viral infections the matrix protein of human parainfluenza virus type induces mitophagy that suppresses interferon responses mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells viruses as modulators of mitochondrial functions hepatitis b virus x protein induces perinuclear mitochondrial clustering in microtubule-and dyneindependent manners migration of mitochondria to viral assembly sites in african swine fever virus-infected cells herpes simplex virus eliminates host mitochondrial dna hepatitis c virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to dna damage and stat activation human cytomegalovirus pul x induces the release of endoplasmic reticulum calcium stores the coxsackievirus b protein suppresses apoptotic host cell responses by manipulating intracellular ca þ homeostasis role of ca þin the replication and pathogenesis of rotavirus and other viral infections human hepatitis b virus-x protein alters mitochondrial function and physiology in human liver cells hbx sensitizes cells to oxidative stress-induced apoptosis by accelerating the loss of mcl- protein via caspase- cascade influence of cytosolic and mitochondrial ca þ, atp, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by -nitropropionic acid viral product trafficking to mitochondria, mechanisms and roles in pathogenesis a pore way to die: the role of mitochondria in reperfusion injury and cardioprotection the myxoma poxvirus protein, m l, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore the hiv- viral protein r induces apoptosis via a direct effect on the mitochondrial permeability transition pore a renewed focus on the interplay between viruses and mitochondrial metabolism systemslevel metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy dynamics of the cellular metabolome during human cytomegalovirus infection glycolysis during early infection of feline and human cells with feline leukemia virus glycolysis in chick embryo cell cultures transformed by rous sarcoma virus divergent effects of human cytomegalovirus and herpes simplex virus- on cellular metabolism hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production viruses exploiting peroxisomes import of peroxisomal matrix and membrane proteins peroxisomes are platforms for cytomegalovirus' evasion from the cellular immune response expression of the cymbidium ringspot virus -kilodalton protein in saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal localization of the tomato bushy stunt virus replication protein p reveals a peroxisome-to-endoplasmic reticulum sorting pathway the host pex p plays a role in peroxisomal localization of tombusvirus replication proteins a key role for heat shock protein in the localization and insertion of tombusvirus replication proteins to intracellular membranes the pestivirus n terminal protease n(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of irf by n(pro.) the fine structure of cymbidium ringspot virus infections in host tissues. iii. role of peroxisomes in the genesis of multivesicular bodies the role of the p :p /p interaction domain in rna replication and intracellular localization of p and p proteins of cucumber necrosis tombusvirus integrated systems biology analysis of kshv latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism micro-rnas upregulated during hiv infection target peroxisome biogenesis factors: implications for virus biology, disease mechanisms and neuropathology flavivirus infection impairs peroxisome biogenesis and early antiviral signaling identification of a type peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle binding of hiv- nef to a novel thioesterase enzyme correlates with nef-mediated cd down-regulation a novel acyl-coa thioesterase enhances its enzymatic activity by direct binding with hiv nef endocytosis of adeno-associated virus type leads to accumulation of virus particles in the golgi compartment the transmembrane domain of the severe acute respiratory syndrome coronavirus orf b protein is necessary and sufficient for its retention in the golgi complex a signal for golgi retention in the bunyavirus g glycoprotein orf virus interferes with mhc class i surface expression by targeting vesicular transport and golgi foot-and-mouth disease virus c protease induces fragmentation of the golgi compartment and blocks intra-golgi transport fragmentation of the golgi apparatus provides replication membranes for human rhinovirus a the open reading frame a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death norwalk virus n-terminal nonstructural protein is associated with disassembly of the golgi complex in transfected cells poliovirus infection and expression of the poliovirus protein b provoke the disassembly of the golgi complex, the organelle target for the antipoliovirus drug ro- membrane-association properties of avian encephalomyelitis virus protein a impact on the endoplasmic reticulum and golgi apparatus of turnip mosaic virus infection a trans-golgi network resident protein, golgin- , accumulates in viral factories and incorporates into virions during poxvirus infection the a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd to recruit pi kiiibeta tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum-and golgi-derived pleomorphic membrane structures in plant cells assembly of vaccinia virus: the second wrapping cisterna is derived from the trans golgi network structural maturation of rubella virus in the golgi complex inner tegument protein pul of herpes simplex virus type is involved in directing capsids to the trans-golgi network for envelopment key golgi factors for structural and functional maturation of bunyamwera virus emerging role of lipid droplets in host/pathogen interactions lipid droplets and viral infections down-regulation of phosphatase and tensin homolog by hepatitis c virus core a in hepatocytes triggers the formation of large lipid droplets dengue virus capsid protein binding to hepatic lipid droplets (ld) is potassium ion dependent and is mediated by ld surface proteins dengue virus capsid protein usurps lipid droplets for viral particle formation dengue virus-induced autophagy regulates lipid metabolism dengue virus and autophagy rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication hepatitis b virus x protein induces lipogenic transcription factor srebp and fatty acid synthase through the activation of nuclear receptor lxralpha hepatitis b virus x protein induces hepatic steatosis via transcriptional activation of srebp and ppargamma liver x receptor mediates hepatitis b virus x protein-induced lipogenesis in hepatitis b virusassociated hepatocellular carcinoma activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects lysosomes serve as a platform for hepatitis a virus particle maturation and nonlytic release lysosomal changes in lytic and nonlytic infections with the simian vacuolating virus (sv ) hepatitis b virus x protein inhibits autophagic degradation by impairing lysosomal maturation neuraminidase of influenza a virus binds lysosome-associated membrane proteins directly and induces lysosome rupture protease c of hepatitis a virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death lysosomal cell death at a glance a zinc finger protein tsip controls cucumber mosaic virus infection by interacting with the replication complex on vacuolar membranes of the tobacco plant visualization of assembly intermediates and budding vacuoles of singapore grouper iridovirus in grouper embryonic cells involvement of the vacuolar h(þ)-atpase in animal virus entry membrane and protein interactions of a soluble form of the semliki forest virus fusion protein the entry of reovirus into l cells is dependent on vacuolar proton-atpase activity cellular v-atpase is required for virion assembly compartment formation in human cytomegalovirus infection endocytosis via caveolae endocytosis of simian virus into the endoplasmic reticulum cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes membrane dynamics associated with viral infection biogenesis of the semliki forest virus rna replication complex fusion of sv -induced endocytotic vacuoles with the nuclear membrane interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus entry into cv- cell nucleus escrt complexes and the biogenesis of multivesicular bodies involvement of vacuolar protein sorting pathway in ebola virus release independent of tsg interaction identification of alpha-taxilin as an essential factor for the life cycle of hepatitis b virus divergent roles of autophagy in virus infection autophagosome supports coxsackievirus b replication in host cells autophagic machinery activated by dengue virus enhances virus replication irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting identification of three functions of the adenovirus e orf protein that mediate p degradation by the e orf -e b k complex molecular determinants for recognition of divergent samhd proteins by the lentiviral accessory protein vpx ns of dengue virus mediates stat binding and degradation viperin restricts zika virus and tick-borne encephalitis virus replication by targeting ns for proteasomal degradation the silencing suppressor p of potato virus x interacts with argonaute and mediates its degradation through the proteasome pathway the enamovirus p protein is a silencing suppressor which inhibits local and systemic rna silencing through ago degradation we sincerely apologize to our colleagues for any research study or review publication not being cited in this review. this is only due to space limitation. research in the laboratory of sn is supported by grants from science and engineering research board ( key: cord- - wju authors: beldomenico, pablo m. title: do superspreaders generate new superspreaders? a hypothesis to explain the propagation pattern of covid- date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: wju abstract the current global propagation of covid- is heterogeneous, with slow transmission continuing in many countries, and exponential propagation in others, in which the time that took to begin this explosive spread varies greatly. it is proposed that this could be explained by cascading superspreading events, in which new infections caused by a superspreader are more likely to be highly infectious. the mechanism suggested for this is related to viral loads. exposure to high viral loads may result in infections of high intensity, which exposes new cases to high viral loads, and so on. this notion is supported by experimental veterinary research. j o u r n a l p r e -p r o o f --- the patterns of propagation of the severe acute respiratory syndrome (sars) outbreak of were not explained by conventional epidemic models that assumed homogeneity of infectiousness. instead, the existing datasets were best matched by models that used negative binomial distributions in which a small proportion of cases were highly infectious (lloyd-smith et al., , mcdonald et al., , shen et al., . data and modelling supported the existence of 'superspreaders' which played a crucial role in propagating the disease by being very efficient at transmitting sars-cov- , such that in the absence of superspreading events most cases infected few, if any, secondary contacts (stein, ) . almost a decade later emerged the middle east respiratory syndrome coronavirus (mers-cov), with analogous infection dynamics involving superspreading events (hui, ) . similarly, early modelling and data suggested that a small proportion of cases of covid- were responsible for most transmission, which is evidence that superspreaders also play an important role for sars-cov- (mackenzie d, , frieden and lee, ). explanations of this superspreader status included high viral shedding due to poor immunocompetence, underlying diseases or co-infection, or elevated contact rate due active social behaviour (lloyd-smith et al., , mcdonald et al., , shen et al., , wong et al. . the propagation of sars-cov- has shown to be heterogeneous at a global scale (data publicly shared by the world health organization and johns hopkins university). after the virus started to be reported outside of china, cases were infecting fewer people than expected, compared to the rate of spread in china. by the end of february, over countries outside china had confirmed the infection, but only three of these, south korea, italy and iran, presented notable spread. in south korea, during the first month of viral propagation there were only two to three reports of new infections per day. however, the rapid spread began after one case was linked to secondary cases in daegu (shim et al., ) . in italy, the rapid surge of cases began in a cluster in lombardy after an infected man was hospitalised without precautionary measures and infected other patients (mostly elder people) and health workers. apparently, there was no calm period in iran, where the first two reported cases were fatal, two weeks later there were cases, and after one month there were over reported infections. a few weeks later, several other countries underwent a similar exponential growth in the number of cases, despite many of them taking drastic measures to control the epidemic. a notable case was the usa, page of j o u r n a l p r e -p r o o f where the infection was propagating slowly since january th until early march, when the daily growth in the number of cases went suddenly from being of one digit to surpassing %, remaining above that geometrical growth rate for almost days. this explosive spread began in new york city, where the number of cases reached in just over two weeks. in contrast, in most countries the infection has been propagating at a slow to moderate pace (e.g. thailand, singapore, egypt, finland, japan, australia, among many others). in general, there have been also contrasts in the apparent case-fatality rate (deaths/reported) depending on the speed of propagation, being much lower in countries with slow spread (e.g. . % in singapore, . % in australia, . % in thailand) compared with those where the transmission was notably high (e.g. % in italy, % in spain, % in usa). this difference might be too large to be explained solely by detection bias. it appears that within a region sars-cov- spreads gradually unless a chain reaction of transmission is triggered. independent superspreading events due to individual variation cannot explain this large-scale heterogeneous pattern of transmission. the occurrence of superspreaders may not be at random and may depend on other superspreaders. it is proposed that infections caused by contact with superspreaders are more likely to result in new superspreaders than those caused by transmission from a less infectious individual. the mechanism by which this would be possible is by exposure to differential viral load. the primary mode of transmission of sars-cov- appears to be through exposure to respiratory droplets and direct contact with infected individuals and their contaminated environment (xiao et al., ; van doremalen et al., ) . droplets may contain a few or a million viral particles, and this differential load determines how much the environment is contaminated and the infective dose a susceptible person is exposed to. a case with a high intensity of infection has the potential of being a superspreader due to high viral shedding. susceptible people exposed to this hypothetical superspreader would be exposed to a high viral dose. infections resulting from exposure to high loads of virus are expected to be of high intensity, as a large quantity of viral particles initiating replication in synchrony might overwhelm the mechanisms of resistance, and the poor control of viral replication may therefore result in a new potential superspreader. this hypothesis has support from veterinary research. for example, in a recent study calves were experimentally infected by bovine viral diarrhoea virus (an j o u r n a l p r e -p r o o f outcome of infection was dose dependent with animals given a higher dose developing severe disease and more pronounced viral replication and shedding. moreover, sentinel calves housed with the lowdose-infected group did not become infected, despite viral shedding being confirmed. other experimental infections also found that viral dose correlated positively with disease severity and viral shedding in other virus-domestic animal systems, such as feline viral rhinotracheitis in cats (gaskell and povey, ) , low pathogenic avian influenza virus in chicken (zarkov and bochev, ) , and equine influenza in ponies (mumford et al., ) . under the hypothesis posited here, cases with low-to-moderate intensity of infection would mainly yield new infections of low-to-moderate severity and viral shedding in people who are not in risk groups. replication, generating a 'domino effect'. the severity of the disease caused by high viral loads is expected to be high. this would be due to extensive cell damage caused by large amounts of virus and also due to the resulting immune response. the virulence arising from an infection by sars-cov- is related to inflammatory self-damage (quin et al., ) , and it is expected that an infection initiated by a large number of viral particles would generate a stronger immune response, compared to infections caused by a low viral dose. therefore, a case resulting from an exposure to high viral loads has the potential to develop severe disease and also of being highly infectious. it was found that in mers patients the severity of the disease was positively correlated with viral load (min et al., ) , and the same was recently reported for covid- ; zou et al., ) . it could be argued that individuals with higher viral loads are more likely to be hospitalised or die, and therefore would be less likely to contribute to community transmission as superspreaders. however, it should be taken into account that the outcome of an exposure to a high viral dose will largely depend on the tolerance (ability to reduce the damage of an infection) of an individual (råberg et al., ) . given equal resistance (ability to limit the infection), exposure to high viral loads will result in severe disease in the less tolerant and high infection intensity with few manifestations in the more tolerant. the latter case is of special concern, because in these individuals the clinical signs would be mild or absent, and therefore are likely to be undetected, exposing many people to high viral loads. on the other hand, severe cases may be important sources of disease in hospitals . for example, in j o u r n a l p r e -p r o o f argentina, % of the cases reported to date are healthcare workers (infobae, ) . therefore, the presence of superspreaders in hospitals could make them nodes where cascades of superspreading events emerge, which is consistent with what was observed in lombardy. disease is traditionally studied as a binary outcome, infected or non-infected. the concepts presented here alert us to the value of studying disease as a continuous variable (i.e. infection intensity) (beldomenico and begon, ) . measuring the intensity of an infection is crucial because it may be related to the virulence as well as the infectiousness. there are many studies of different viral diseases in which the length of viral shedding is recorded, yet very few produced data on the viral shedding load. the hypothesis posited here needs to be tested by empirical and theoretical studies, but this requires that data on viral load (viraemia and shedding) are urgently collected. if superspreaders generate new superspreaders by exposing susceptible people to large viral loads, this mechanism should be immediately acknowledged and considered in the responses being undertaken. in particular, emphasis should be placed on the isolation or strict distancing of people of risk groups, as they would not only have more chances of developing a more severe disease (with the potential of overwhelming the health system), but they could also be source of high viral loads. in addition, aggressive contact tracing and testing would allow quick identification of tolerant superspreaders, who might be key elements of propagation. are sars superspreaders cloud adults? disease spread, susceptibility and infection intensity: vicious circles? identifying and interrupting superspreading events-implications for control of severe acute respiratory syndrome coronavirus . emerg infect dis the dose response of cats to experimental infection with feline viral rhinotracheitis virus super-spreading events of mers-cov infection coronavirus in argentina: deaths among healthcare workers and the number of infected is still rising viral dynamics in mild and severe cases of covid- superspreading and the effect of individual variation on disease emergence sars in healthcare facilities comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity experimental infection of ponies with equine influenza (h n ) viruses by intranasal inoculation or exposure to aerosols dysregulation of immune response in patients with coronavirus (covid- ) in wuhan, china decomposing health: tolerance and resistance to parasites in animals superspreading sars events transmission potential and severity of covid- in south korea superspreaders in infectious diseases viral dose and immunosuppression modulate the progression of acute bvdv- infection in calves: evidence of long term persistence after intra-nasal infection aerosol and surface stability of sars-cov- as compared with sars-cov- exploring the reasons for healthcare workers infected with novel coronavirus disease (covid- ) in china the role of super-spreaders in infectious disease role of fomites in sars transmission during the largest hospital outbreak in hong kong influence of inoculation dose of avian h n influenza a virus on virus shedding and humoral immune response of chickens after artificial experimental intravenous infection key: cord- -cubh jxc authors: domingo, e.; martín, v.; perales, c.; grande-pérez, a.; garcía-arriaza, j.; arias, a. title: viruses as quasispecies: biological implications date: journal: quasispecies: concept and implications for virology doi: . / - - - _ sha: doc_id: cord_uid: cubh jxc during viral infections, the complex and dynamic distributions of variants, termed viral quasispecies, play a key role in the adaptability of viruses to changing environments and the fate of the population as a whole. mutant spectra are continuously and avoidably generated during rna genome replication, and they are not just a by-product of error-prone replication, devoid of biological relevance. on the contrary, current evidence indicates that mutant spectra contribute to viral pathogenesis, can modulate the expression of phenotypic traits by subpopulations of viruses, can include memory genomes that reflect the past evolutionary history of the viral lineage, and, furthermore, can participate in viral extinction through lethal mutagenesis. also, mutant spectra are the target on which selection and random drift act to shape the long-term evolution of viruses. the biological relevance of mutant spectra is the central topic of this chapter. the historical overview of evolutionary virology by john holland in the closing chapter of this volume documents the many indications of genetic and phenotypic instability of rna viruses, and the growing suspicion over the second half of the twentieth century that something was fundamentally different between rna genetics and dna genetics. the first evidence that an rna virus consisted of distributions of mutant genomes, and that the wild type existed only as a weighted average of many different sequences, was obtained by sampling genomic sequences of biological clones of bacteriophage qβ by charles weissmann and colleagues in zurich (review in holland, this volume). weissmann presented these experimental results to manfred eigen and his colleagues at a max planck institute meeting in klosters (switzerland) in . eigen had developed the first theoretical treatment of a system that replicated with limited fidelity, so that the replication process regularly generated mutant copies of the templates (eigen ) . this theory, later to become known as quasispecies theory (eigen and schuster ) , was developed to understand self-organization and adaptability of early life forms on earth. in seeing the experimental results on phage qβ, eigen exclaimed "quasispecies in reality!" and this represented the beginning of a productive interaction between theoretical biophysics and experimental virology (further information on these early encounters is given in domingo et al. ) . quasispecies theory is covered in the chapters by biebricher and eigen and by wilke et al. in this volume, and here we review biological implications of quasispecies dynamics for rna viruses. the extended definition of quasispecies currently used by virologists is the following: "viral quasispecies are dynamic distributions of non-identical but closely related mutant and recombinant viral genomes subjected to a continuous process of genetic variation, competition and selection, and which act as a unit of selection" (domingo ) . this definition incorporates general principles of darwinian evolution, whose effects, in the case of viruses, can be observed within short time periods (usually days and weeks) both in natural hosts, and in model systems such as alternative hosts in vivo or in cell cultures. short-term evolution of viruses underlies virus adaptation to compartments within infected organisms, that may contribute to time-dependent changes of disease symptoms and to disease progression. this is particularly evident with rna viruses, and several examples are discussed in this volume, and others are regularly reported in the current literature on virology. some terms (such as "fitness", "environment", etc.) used in this and other chapters, and that have been approached mainly with viral infections in cell culture, may seem abstract and distant from the real world of viral diseases. yet they are not. fitness values express a replication capacity, and competitive growth occurs in infected hosts when variant forms of the same virus meet to penetrate a compartment of a differentiated organism, or to replicate in the same cell subset. environment may mean specific cell types or cell assemblies in tissues and organs where virus replication takes place, or a set of physiological conditions that may affect virion stability or susceptibility of cells to viral variants. the main steps involved in middle-term and long-term rna virus evolution have some resemblance with some of the steps that determine shortterm evolution in quasispecies dynamics (fig. ) , despite their occurring with very different space-time scales. the triggering event in these evolutionary episodes is reproduction with genetic variation. then positive and negative selection, together with random sampling (drift) that take place within a host, or between host individuals, shape the genetic composition of the virus. reproductive success can be quantified as relative fitness values (quiñones-mateu and arts, this volume) or with epidemiological parameters such as the basic reproductive rate (or ratio) (ro), defined as the average number of secondary cases resulting from the introduction of a single infected case into a susceptible population. ro is a general parameter (which can be applied to demography of any type of biological entity) that can predict the long-term epidemic spread of a pathogenic agent (reviews concerning application to viral infections in may and woolhouse ). both fitness and ro values capture average values of fundamentally heterogeneous entities, and such averages may vary as an infection progresses or an epidemic spreads, thus adding an additional level of complexity to the interpretation of the reproductive success of a virus. while the reproductive ratio is generally used in epidemiological investigations, fitness finds its application in the comparison of the relative replication capacity of variant viruses within a single host or in cell culture. as indicated by biebricher ( ) , evolutionary success depends on two components of the phenotype: those that determine survival and those that determine the rate of production of viable progeny, and the combination of the two is what we call fitness. virologists have adapted growth-competition experiments to measure the relative capacity of two viruses to produce infectious progeny, thus providing estimates of relative fitness values under a given set of environmental conditions (holland et al. ) . fitness can be measured in other ways (defilippis and villarreal ) , and it is virtually impossible to design a measurement that captures in full the potential evolutionary success of viruses replicating in their host organisms (biebricher ; defilippis and villarreal ) . despite limitations in the measurements and significance of fitness values for rna viruses, variations in relative fitness, based on a b fig. a ,b schematic representation of viral quasispecies in an infected host. viral genomes are represented as horizontal lines, and mutations as symbols on the lines. a upon infection with an rna virus (even with a single particle, as depicted here, enlarged about times), viral replication leads to a mutant spectrum of related genomes, termed viral quasispecies. nucleotide sequencing of the ensemble produces a consensus sequence which includes in each of its positions (nucleotide or amino acid) the residue found most frequently in the corresponding position of the distribution (mutant spectrum). b different mutant distributions are found in different infected organs or at different sites of the same organ. as further discussed in the text, in real infections multiple mutant spectra that can amount to a large number of replicating (or potentially replicating) genomes (up to or even per infected individual) provide highly dynamic mutant repertoire viral yields in cell culture, have been immensely powerful in characterizing the population dynamics of rna viruses (see references in the reviews by domingo and holland ; quiñones-mateu and arts ; novella ; and the chapters by quiñones-mateu and arts and escarmís et al., this volume) . as usually performed, fitness values incorporate differences in replication capacity (both of viruses in isolation and when co-replicating in the same cells) at all stages of the viral life cycle. restricting fitness measurements to individual events of the replication cycle (rna synthesis, viral-specific protein synthesis, virion assembly, etc.) would entail additional ambiguities (i.e. rna replication may be coupled to translation, translation to assembly, etc.). quiñones-mateu and arts in the next chapter of this volume discuss several important implications of fitness measurements based on viral yields. figure shows a schematic view of quasispecies dynamics in relation to population size and fitness variations. but there are additional implications of the links between quasispecies theory and virus population dynamics. since rna viruses replicate as complex mutant distributions (figs. and ), determination of the consensus nucleotide sequence (or the consensus amino acid sequence of encoded proteins) provides very fragmentary information of the genetic composition and of the evolutionary potential of a virus population. analyses of individual genomic sequences of mutant spectra can be achieved by two alternative procedures. one is to isolate virus either from individual plaques developed on cell monolayers or from an infection following end-point dilution. viral rna is then subjected to reverse transcriptionpolymerase chain reaction (rt-pcr) amplification and nucleotide sequencing. this procedure leads a sequence that cannot be influenced by possible misincorporations introduced during the rt-pcr amplification (that can arise due to the limited copying fidelity of the enzymes used in the amplification). in sequence screening of biological clones, a bias may occur that favours representation of genomes that are more infectious (produce early cytopathology or larger plaques) in the particular cell line or primary culture chosen. a second means to characterize a virus population is to subject total rna extracted from the biological specimen of interest to rt-pcr, followed by molecular cloning and sequencing of dna of individual molecular clones. this procedure does not depend on infectivity of the viral rna in a cell culture system. here a bias may arise from the low fidelity of the enzymes used in the rt-pcr, which may result in an overestimate of the nucleotide sequence small arrows indicate genetic bottlenecks experimentally realized as plaque-to-plaque transfers. serial bottleneck events lead to accumulation of mutations in the consensus sequence. the large arrow represents replication of the quasispecies without population size limitations. bottom: fitness (f) variation associated with passage regime. bottleneck events lead to a decreases in fitness; however, at low fitness values a fluctuating pattern with a constant average fitness is observed (escarmís et al., this volume) . large population passages result in a fitness increase that may or may not result in a modification of the consensus sequence. again, at high fitness, a fluctuating pattern of fitness values is observed, presumably reflecting a limitation exerted by the population size on the capacity for fitness increase (novella et al. (novella et al. , . (figure adapted from domingo , with permission) heterogeneity. this can be solved by using high-fidelity polymerases, correct amplification reaction conditions (adequate ph and ionic composition, unbiased concentrations of chemically intact nucleoside triphosphates, etc.), and control experiments to determine a basal error rate for the system . another bias may come from insufficient initial template rna so that several sequences that originate from the same viral template rna may be represented among the clones analyzed. this will generally result in an underestimate of the nucleotide sequence heterogeneity. some simple calculations have been applied to derive the expected number of independent sequences (those that will represent different templates in the viral population) from the last dilution of the template rna that yields positive rt-pcr amplification (airaksinen et al. ) . our rule of thumb is that when a : dilution of the template yields positive rt-pcr amplification, we proceed with the cloning and sequencing of the amplification product of the undiluted template. when the amount of template is limiting, alternative procedures (such as multiple parallel amplifications prior to cloning and sequencing) must be sought (airaksinen et al. ) . it is a common practice to align at least genomic (or subgenomic) sequences and to determine the mutation frequency and shannon entropy (table ) . in most situations, - sequences represent a tiny minority of table the characterization of mutant spectra of viral quasispecies definition: the proportion of mutant nucleotides in a genome population. mutant frequency may refer to the proportion of genomes harbouring a specific mutation. calculation: determine the total number of mutations (counting repeated mutations only once) relative to the consensus (defined by the same set of sequences) and divide by the total number of nucleotides that have been sequenced (i.e. , when the same nucleotides of individual genomes have been determined). in some cases, it may be interesting for statistical reasons to count all mutations found (counting repeated mutations as many times as they occur), yielding a maximum mutation frequency. calculation: normalized shannon entropy, sn = − [ i (p i × lnp i )] | ln n, in which p i is the proportion of each different sequence of the mutant spectrum, and n the total number of sequences compared. definitions: genetic distance is the number of mutations that distinguish any two sequences from the population. the average for all possible pairs reflects the genetic complexity. hamming distance is the number of mutated positions in a genome with respect to the best adapted sequence (the most abundant) in a genome distribution. for quasispecies analysis, a list of hamming distances (or the average for the population) can characterize the complexity of the ensemble. calculation: align sequences and divide the number of mismatched positions between any two sequences by the number of identical positions. matrices of pair-wise genetic distances are used to determine phylogenetic trees. for distantly related sequences (rarely occurring within a quasispecies), multiple sequence alignments using clustal w and adequate scoring of gaps may be needed. based on eigen ; volkenstein ; domingo ; mount ; the total number of viral genomes in a biological specimen, and therefore this procedure must be regarded as a very crude sampling of a viral population. in some cases, a selective agent may permit the screening of minority components of mutant spectra. for example, in studies on quasispecies memory (described in sect. ) analyses of mutant spectra were based on the repertoire of mutants resistant to a monoclonal antibody that neutralized the dominant fmdv population, but did not neutralize the portion of the mutant spectra of interest (ruiz-jarabo et al. , b . despite these limitations, determination of nucleotide sequence heterogeneities in virus populations using correct reagents and adequate controls has consistently documented that most rna viruses (and also some dna viruses) consist of complex mutant spectra, with an average number of - mutations per genome (sect. ). the degree of heterogeneity within a viral population will be influenced by the fidelity of the replication machinery, the distance (in rounds of replication) from a clonal origin, constraints for variation (both at the rna and protein levels), and intervening bottlenecks in the evolutionary history, among other influences. scientists should be cautious in attributing to high-fidelity rna replication what may be a standard error level together with negative selection, which maintains an invariant consensus nucleotide sequence. the literature contains several cases of such likely misinterpretation of data. it is not surprising that comparisons of different virus-host systems have led to exceedingly broad ranges of genetic diversity within mutant spectra. this should not blur the common underlying influences and the general biological relevance of population complexity even if seemingly modest in terms of mutations per individual genome. this is justified in the next section. the relevance of viruses replicating as mutant spectra is intuitively obvious since any individual mutant genome of the ensemble can potentially differ in behaviour from other individuals or from the ensemble of genomes. the importance of such a population structure is strengthened by considering five parameters that characterize a mutant distribution: average number of mutations per genome, virus population size, genome length, mutations needed for a phenotypic change, and virus fecundity (table ) . despite all cellular organisms being highly polymorphic genetically (in that distinct alleles from a gene are represented among individuals of one biological species), the level of heterogeneity of rna virus populations confers a much greater adaptability than the levels of polymorphism estimated for cells. this is a consequence of parameters and (listed in table ) : as an example, a viral genome of , nucleotides has a total of × possible single mutants (disregarding fitness effects of mutations), which is a figure well below the population size of many natural virus populations. in contrast, the total number of possible single mutants for a mammalian genome is about , well above the population size of mammalian species. that is, the capacity to explore sequence space (a concept discussed in the chapter by biebricher and eigen in this volume, which refers to the total number of possible sequences available to a genetic system, reviewed by eigen and biebricher ) is far greater for viruses than for cellular organisms. this confers adaptability to viruses (with amply recognized biological implications) and renders viruses suitable experimental systems for probing evolutionary concepts (see domingo et al. ; flint et al. , and other chapters of this volume). one of the critical parameters in viral quasispecies is the number of mutations in an rna virus that is needed for a phenotypic change in the virus (table ) . indeed, if a relevant phenotypic change (for example, a modification in host cell tropism, resistance to neutralizing antibodies or to an antiviral agent, etc.) depended on the occurrence of - mutations in a viral genome (to invent a simple example), then the quasispecies nature of rna viruses (with the characteristic parameters we measure today; table ) would table some important parameters that influence the adaptability of viral quasispecies . average number of mutations per genome in a mutant spectrum generally it amounts to an average of - mutations per genome. (see text for reasons for broad range). variable, but very high upper limits. an acutely infected organism may include - viral particles at any given time. even a single viral plaque on a cell's monolayer can yield - particles. . genome length kb- kb . mutations needed for a phenotypic change many recorded adaptive changes depend on one or a few mutations (see text). variable. average of < - particles per cell have been measured. high fecundity promotes quasispecies dynamics, as discussed in several chapters of this volume. based on domingo et al. and references therein. be largely irrelevant for short-term adaptation. this is because in the exploration of sequence space the probability of arriving at a point corresponding to a mutant with the required - mutations is exceedingly low ( - to - !). even if we relax the requirement to multiple combinations of mutations, it can be estimated from the poisson distribution that the probability of genomes with any mutations in a population of individuals is about - genomes (on a purely statistical basis, ignoring fitness effects; additional representative figures can be found in domingo ) . as evidenced by many experimental results with rna viruses of virtually all families that have been examined, one or a few amino acid replacements are sufficient to modify a biologically relevant feature of a virus, as amply recorded in the literature (review in domingo et al. ; examples of single mutations associated with epidemiologically relevant adaptations of arboviruses are given in the chapter by weaver in this volume). because phenotypically relevant mutations can be frequently represented in mutant spectra, they have the potential to become dominant (command the development of new mutant distributions) in response to environmental demands, despite the modulating effects of mutant spectra described in sect. . multiple constraints for variability have been evidenced in viral genomes (simmonds and smith ; simmonds et al. ). therefore, many variants with one or few mutations that may confer biologically relevant, adaptive traits may not be represented in a mutant spectrum (under a set of environmental conditions) due to fitness costs. it is impressive that despite such constraints (for example, genome-scale, ordered rna structures in hepaciviruses; simmonds et al. ) , the level of heterogeneity and adaptive capacity of viral populations are the ones we record with unfailing continuity with any virus we examine in some detail. a highly significant example is the occurrence of mutants with one or a few amino acid substitutions that confer decreased sensitivity to antiviral inhibitors. this is a general phenomenon -documented with many viruses since the s both in vivo and in cell culture -which complicates enormously the treatment of viral disease (a recent overview can be found in domingo ; see also previous versions published in progress in drug research for a historical account). it is not the case that "suddenly" a virus strain "appears" that is resistant to an inhibitor. resistant mutants are generated as components of mutant spectra and then may become dominant when virus replication occurs in the presence of the inhibitor. perhaps the most dramatic example has been the selection over the years of mutants of human immunodeficiency virus type (hiv- ) resistant (or with decreased sensitivity) to the antiretroviral inhibitors (targeted mainly to reverse transcriptase, protease or surface structures involved in virus fusion or cell recognition) used in clinical practice (see the chapter by mullins and jensen, this volume). both experi-mental analyses of hiv- populations (coffin ; nájera et al. ) and theoretical predictions (ribeiro et al. ; gerrish and garcia-lerma ) suggest that such mutants may preexist in hiv- populations, even when they have not been exposed to the inhibitors. in addition, resistant mutants that may occupy very low frequency levels in hiv- mutant spectra in the absence of the inhibitor may be raised to higher frequency levels in the presence of the inhibitor. the recently described phenomenon of memory in viral quasispecies, including memory subpopulations of hiv- in vivo (sect. ), supports even further a role of genome subpopulations in the response of viruses to inhibitors . the fitness costs of inhibitor-resistance mutations, as well as relative fitness values of wild-type and resistant mutants in the absence and the presence of the inhibitor will influence the kinetics and degree of dominance of inhibitor-resistant mutants (treated in the chapter by quiñones-mateu and arts, this volume). paradoxically, the presence of a mutant with a specific phenotypic trait may not be sufficient to guarantee its dominance even when a selective constraint favours that phenotypic trait. the reason for this important phenomenon again has to do with the presence of a mutant spectrum surrounding the relevant mutant, and this is discussed next. a mutant virus potentially capable of becoming dominant in an evolving viral quasispecies (either because of its high fitness or because it harbours a selectable trait) may remain as a minority in the population, depending on the nature of the mutant spectrum in which it is immersed. this concept was first documented with a numerical example of two master sequences of small size differing in fitness by %, replicating near the error threshold (see the chapters by biebricher and eigen and by wilke et al., this volume, and sect. in this chapter). interestingly, the inferior mutant outgrew the fitter one when the inferior mutant was surrounded by closely related mutants that were somewhat better adapted than the mutants that surrounded the fitter master. this and other numerical simulations (swetina and schuster ; review in eigen and biebricher ) suggest a strong influence of the mutant spectrum on the behaviour of any particular variant and supports the view that the target of selection is not a single species, but rather the distribution of the quasispecies as a whole (eigen and biebricher ; perales et al. ) . the prediction that the mutant spectrum can affect the dominance of an individual mutant has found experimental confirmations both in vivo and in cell culture (table ). the first evidence was obtained using vesicular stomatitis virus (vsv) (de la torre and holland ) . a mutant spectrum of vsv suppressed replication of a mutant of superior fitness than the ensemble, unless the mutant was present above a certain frequency in the population. in another instance of remarkable practical relevance, it was found that in anti-poliomyelitis vaccine preparations, the dominant attenuated poliovirus (pv) suppressed neurological disease associated with minority virulent pv, unless the latter was present above a minimal concentration in the vaccine (chumakov et al. ) . some strains of the arenavirus lymphocytic choriomeningitis virus (lcmv) (see the chapter by sevilla and de la torre, this volume) induce a hormone-deficiency syndrome in mice (reviewed by oldstone ) . remarkably, some co-infecting nonpathogenic strains of lcmv suppressed expression of pathogenic strains in such a way that no disease was manifested (teng et al. ) . studies with foot-and-mouth disease virus (fmdv) have provided two additional examples (recent reviews on fmdv in rowlands ; sobrino and domingo ; mahy ) . fmdv serially passaged in bhk- cells in the presence of polyclonal antibodies directed to a specific antigenic site of the virus, generated a complex mutant spectrum of antigenic variants of low relative fitness. such mutant spectra included biological clones that manifested high fitness when separated from the mutant cloud (borrego et al. ). in the transition to error catastrophe of fmdv (sect. ), pre-extinction viral rna (which is defined as viral rna extracted from the population that, in the serial passages in the presence of mutagens, precedes the one in which virus extinction occurs) interferes with infectious rna (gonzález-lópez et al. ) . interference was documented by co-electroporation of cells with two rnas and it was exerted by pre-extinction rna but was not exerted by a defective fmdv rna (containing an in-frame deletion), unrelated viral and nonviral rnas, or the same pre-extinction rna reduced in size by chemical treatment. it was proposed that due to mutations in pre-extinction viral rna, abnormal expression patterns and expression of abnormal viral proteins may jeopardize the replication capacity of coexisting infectious rna (gonzález-lópez et al. ) . in support of this proposal, fmdv ds (polymerases) harbouring deleterious mutations have been identified in mutagenized populations (sierra et al. ; arias et al. ; see also sect. for additional information on possible mechanisms of suppressive effects of mutant spectra). an alternative model, termed positive clonal interference, taken from bacterial population genetics, was proposed to explain dominance of one vsv clone over another (miralles et al. ). in this model, the basis for the interference is competition among genomes carrying advantageous mutations during replication as large populations. in addition to a technical problem derived from the likely presence of defective interfering (di) particles in the co-infections carried out to test the model (see domingo for a more detailed discussion of this problem), recent results with fmdv show that suppression does not require comparable fitness of the two competing populations (gonzález-lópez et al. ) , in agreement with a suppressive effect exerted by a low fitness mutant distribution on genomes displaying higher fitness (sect. ). the suppression of individual genomes by mutant spectra (table ) confers additional biological relevance to population bottleneck events that may intervene during virus replication and evolution. in addition to effects on virus population dynamics (see the chapter by escarmís et al., this volume) , and in the relationship between virulence and transmission mode (bergstrom et al. ) , bottlenecks may release a portion of mutant spectrum from the complete cloud that may modulate the behaviour of minority genomes (fig. ) . also, some contradictory results seen in the literature on the frequency of deleterious mutations or epistatic effects of mutations in rna viruses may in part derive from the fact that it is not possible to "freeze" a mutant to remain as the "same" individual in the course of replication: a cloud forms immediately and unavoidably. thus, what initially was "a" genome with one or two mutations of interest will soon become a "distribution" of genomes table modulating effects of mutant spectra on individual virus variants cell culture mutant spectrum of vesicular stomatitis virus (vsv) suppresses a vsv variant of superior fitness (de la torre and holland ). low fitness antibody-escape population of foot-and-mouth disease virus (fmdv) suppresses individual antigenic variants displaying high fitness (borrego et al. ). attenuated poliovirus (pv) can suppress neurological disease in monkeys, associated with virulent pv (chumakov et al. ) . nonpathogenic lcmv can suppress manifestation of the growth hormone-deficiency syndrome in mice, associated with pathogenic lcmv strains (teng et al. ) . lines represent a complex mutant spectrum in which individual mutations have not been indicated. an individual genome characterized by two specific mutations (dot and rectangle) is surrounded by a mutant spectrum, which suppresses its replication by the biochemical mechanisms discussed in the text (see also fig. ) . a population bottleneck represented by the shaded rectangle relieves the individual genome of part of the suppressing ensemble, thereby facilitating expression of the individual genome, which may increase its frequency (right). experimental evidence and references are given in the text with additional mutations that may affect the behaviour of the genomes harbouring the initial mutations. careful monitoring of progeny sequences and mutant spectrum complexities is needed. it is tempting to suggest that some of the uncertainties that appear to characterize virus evolution in vivo (for example, that an antibody-, ctl-or inhibitor-resistant variants may raise to dominance or not) may relate to suppressive effects of mutant spectra perturbed by intra-host bottleneck events of different intensities, prompted by the replicative characteristics of the virus in its permanent interaction with of the host. as further penetration into the fine population structure of viruses in vivo becomes technically feasible, it may be possible to elucidate some of these issues. in the course of fitness determinations [usually by growth competition between the virus to be tested and a reference virus in a defined biological environment (see the chapter by quiñones-mateu and arts, this volume)] suppressive effects of mutant spectra may also influence the outcome of the competition. indeed, as discussed in sect. , the mutation rates during rna genome replication and retrotranscription render extremely unlikely the sustained synthesis of genomes without mutations (drake and holland ) (although, interestingly, evolution appears to have adjusted mutation rates to permit the presence and survival of the nonmutated class when the latter are fitter than mutant progeny). thus, despite the fact that during fitness determination a competition is established that should result in dominance of the most fit mutant distributions, modulating effects of mutant spectra of the type discussed in the previous section cannot be excluded, and they have been recently evidenced in the extreme case of fitness determinations of pre-extinction fmdv populations (gonzalez-lopez et al. ) . the converse effect, that is, complementation among mutants (moreno et al. ; agol ) , can also occur at the stage of fitness determination in which the two competing ensembles co-infect the same cells, and this effect has been modeled ) (see the chapter by wilke et al., this volume). taking relative virus yield in a given environment as a measure of fitness is justified because first, no virus genome ever exists as a "single nucleotide sequence"; and second, when it exists (as in the very beginning of a formation of a plaque on a cell monolayer) it rapidly becomes a mutant spectrum in the first infected cell. thus, initial rna replication rates are fictitious for these reasons and others summarized in sect. , supporting the adequacy of fitness values given as relative viral yields (see sect. and the chapter by quiñones-mateu and arts, this volume). it is a rooted tradition, not only among virologists but also in some schools of population genetics, to view the individual as the standard unit of selection, although this is a debated subject (as a general discussion of this topic see lewontin ; williams ) . in this section, we argue that since rna viruses consist of mutant spectra, and components of the spectra may (and often do) deviate genotypically and phenotypically from other components of the same spectrum, the behaviour of an individual may (and often will) be influenced by surrounding individuals that we call "the ensemble" (extreme cases of suppression of individual variants are discussed in sect. ). we further suggest that this feature of viral quasispecies is consistent with many observations made in classical viral genetics. viral interference provides a useful introduction to our argument. interference referred initially to the inhibition of multiplication exerted by one virus on an entirely different virus entering the same cell (see reviews in white and fenner ; youngner and whitaker-dowling ; condit ) . this led to the distinction between homologous and heterologous interference, depending on the relatedness of the two viruses under analysis. interference acquired its major impetus with the discovery of defective, noninfectious genomes (generally deletion mutants) that interfered with the corresponding standard virus and mediated the establishment of persistent infections of some rna viruses (see reviews in huang and baltimore ; perrault ; holland ). the interfering genome, whether infectious or noninfectious, often has a higher affinity than the standard (interfered) virus for a viral or host protein (or other host component). here a conceptual overlap with the concept of fitness exists (garcía-arriaza et al. ) . most fitness measurements involve a phase of intracellular competition between the two viruses to be tested. this is because even in competitions started at low moi there will be a second round of cell infection that, depending on the initial yield, may involve high moi. this point has been discussed by (novella ) regarding complementation, and it applies to fitness assays as well. when intracellular competition occurs, the difference in progeny production may have the same molecular basis as interference. this has been manifested in recent studies that have shown a fitness-dependent interference by a bipartite version of the fmdv genome (garcía-arriaza et al. , . we arrive at the dilemma that either fitness values should be determined avoiding any type of intracellular competition, or it must be accepted that fitness differences (generally measured for mutants of the same virus) are influenced by intracellular competition events akin to those underlying viral interference. intracellular competition could be avoided either by each virus infecting cells on separate culture dishes or animal hosts, or by restricting replication to the initial round of progeny production following infection at low moi; both requirements pose technical problems. interference may be mediated by interferon, which evokes a general antiviral state in a cell, as well as by a number of interactions between the interfering and interfered viruses (white and fenner ; youngner and whitaker-dowling ; condit ; agol ) . concerning possible interference between mutants of the same virus (the form that may be most relevant to quasispecies behaviour), interference may be associated with multiple, different mutants which, even if present individually at low frequency, may have collectively the effect of a dominant-negative mutant. the latter may act through different mechanisms: for example, in the "rotten apple" hypoth-esis, a defective polypeptide produced by the mutant may enter a multimeric complex, inhibiting its activity. in the "road-block" hypothesis, a defective function (protein or regulatory region) may sequester a factor (or a site) required for replication of both viruses. in the "all things in moderation" hypothesis, imbalances in gene dosage may lead to decreases in efficiency of steps in the life cycle of both viruses. in the "direct competition" hypothesis, both viruses compete for some viral or host factor. according to the "attractive genome" hypothesis, the dominant-negative virus may possess sites that bind required factors, and such sites may be more abundant or show higher affinity than the sites in the wild-type virus. these different hypotheses have been taken from the summary by youngner and whitaker-dowling ( ) , and we propose that at least some of these mechanisms may underlie modulating effects observed within mutant spectra of viral quasispecies (fig. ) . this was proposed to explain the interfering activity of pre-extinction fmdv rna (gonzález-lópez et al. ) and it emphasizes increasing evidence of the critical role that defective genomes may have in dictating virus behaviour ; see also the chapter by escarmís et al., this volume) . within mutant spectra, multiple potentially dominant-negative mutants (when present at high frequency) may have a synergistic effect in preventing standard virus replication by one or several of the proposed mechanisms (youngner and whitaker-dowling ) . these negative effects on replication of individual mutants may be favoured by the multifunctional nature of viral proteins (both in mature and precursor forms), increasingly recognized for many viruses (cornell et al. ; flint et al. ) . parallel concepts can be applied both to modulating effects of mutant spectra (table ) and to the transition to error catastrophe (gonzález-lópez et al. ; grande-pérez et al., in press ; see also sect. ). mutants with complementing or interfering capacity have been referred to as cooperators or defectors, respectively, during processes of competition inside a host (turner and chao ; novella ; novella et al. ) . what we know of quasispecies' capacity to attain different fitness levels suggests that the same altered viral protein may be advantageous or disadvantageous depending on the dominant genomes in the mutant distribution (fig. ) . thus, our proposal is that a mutant spectrum of a replicating viral quasispecies constitutes a "genetic microcosmos" in which interactions among components of a mutant spectrum may include effects similar to those previously characterized between well defined mutants of a virus or between two different viruses. what is observed to occur "between populations" is now extended to interactions among "components of a population". experiments are now in progress to further test the validity of this proposal. ( , , ; shaded box in genome; symbols represent mutations) encode a protein with decreasing biological activity (white to dark grey) that acts as a homopolymeric hexamer (needed to produce viral progeny). b in a mutant spectrum in which and are expressed, increasing proportions of gene will result in a decrease in activity of the hexameric protein, and, consequently of viral yield. c in a mutant spectrum in which and are expressed, increasing proportions of gene will result in a decrease in activity of the hexameric protein, and, consequently of viral yield. note that the same viral subunit can act as a negative modulator (b) or a positive modulator (c) depending on the activity of the accompanying subunit. in real viral quasispecies, modulating effects are far more complex than illustrated here because a range of different activities in trans-acting products may be present, several viral and host gene products may participate in heteropolymeric complexes, viral proteins are often multifunctional, and regulatory signals on the viral rna may be involved viral quasispecies may possess in their mutant spectra minority genomes that reflect those that were dominant at an earlier phase of quasispecies evolution. this has been shown experimentally with fmdv in cell culture (ruiz-jarabo et al. arias et al. arias et al. , baranowski et al. ) and hiv- in vivo briones et al., personal communication) . the experiments with fmdv took advantage of two well-studied classes of mutants: an antigenic variant selected with a neutralizing mab and a very unusual variant that contained in its genome an internal polyadenylate tract, generated upon repeated plaque-to-plaque passage of fmdv clones (escarmís et al. ; see also the chapter by escarmís et al., this volume) . when populations dominated by either one of these mutants were serially passaged in cell culture, revertant viruses with the wild-type sequence (that is, without the amino acid substitution that led to antigenic variation and absence of the internal polyadenylate tract, respectively) became dominant. however, genomes with the initially dominant markers did remain in the mutant spectrum not at a basal level accounted solely by mutational pressure, but at levels -to fold higher. a scheme of implementation of memory is shown in fig. and the main features of quasispecies memory are summarized in table . two types of memory were distinguished during hiv- infections in vivo. one was the "replicative" memory as defined with fmdv, and the other was a "cellular" or "anatomical" memory derived from the retroviral life cycle with table main features of quasispecies memory replicative memory memory genomes derive from genomes that were dominant at an early phase in the evolution of the same viral population. memory genomes are present at higher frequencies (in the cases studied to times) than expected from mere mutational pressure exerted on parental genomes. memory genomes are lost upon subjecting virus to a population bottleneck. memory genomes increase in fitness in parallel with the dominant genomes, as replication proceeds (red queen hypothesis). memory genomes may decrease in frequency as virus replicates. cellular or anatomical memory found when viral reservoirs (displaying no replication or limited replication) are activated and contribute to the composition of an evolving quasispecies. based on ruiz-jarabo et al. , b briones et al. ; arias et al. . schematic representation of implementation, maintenance and loss of memory genomes in a viral quasispecies. genomes of the mutant spectra are depicted as horizontal lines, and mutations as symbols on the lines. the mutant spectra have been divided in three levels: level includes those genomes found most frequently in the mutant distribution; level includes a frequency class potentially occupied by memory genomes; and level gathers the less abundant genomes that occur as a result of mutational pressure and the competitive rating of all components of the mutant spectrum. initially genomes with the genetic marker a belong to level . when genomes with marker a are selected, they occupy the entire quasispecies. upon further replication, when genomes with a display a selective disadvantage with respect to genomes lacking a, genomes with the a marker will eventually become undetectable in the consensus sequence of the quasispecies. however, genomes with marker a may remain as memory genomes in level because in the course of selection their fitness increased (upper right mutant distribution). when bottleneck passages (of sufficient intensity to exclude level genomes) intervene, memory is lost and genomes with a occupy again level as in the initial mutant distribution (bottom right mutant distribution). in real viral quasispecies, mutants are ranked to form many frequency levels (perhaps a continuum of frequency levels) rather than , as assumed here for simplification. experimental evidence and references on implementation, maintenance and decay of quasispecies memory in cell culture and in vivo are given in the text and in table . (based on domingo , with permission) integration of proviral dna in cellular dna, which results in reservoirs that may re-emerge at a later stage of infection briones et al., personal communication) . reemergence (or raise to dominance) of ancestral (minority) genomes in vivo has been documented with several viral infections (borrow et al. ; wyatt et al. ; karlsson et al. ; briones et al. ; lau et al. ; garcía-lerma et al. ; imamichi et al. ; kijak et al. ; charpentier et al. ) , providing additional support for the concept of memory in viruses both in cell culture and in vivo. the experiments to investigate the possible presence of memory in viral quasispecies (ruiz-jarabo et al. ) were motivated by features of virus evolution that fit those of complex adaptive systems (see reviews in gell-mann ; frank ; solé and goodwin ) . a characteristic of such systems is that they vary, yet they have continuity in the form of some built-in (heritable) component, and are endowed with mechanisms to respond to external stimuli. there is a continuous interplay between the components of the system and the interacting environment. examples of complex adaptive systems are neurological activity in memory and learning, and the immune system of vertebrates in which long-lived memory t cells may be expanded when the organism is again exposed to an antigen identical (or related) to the one that triggered the initial response. such potential to act quickly in response to an environmental demand has a functional parallel in the presence of memory genomes in viral quasispecies. indeed, memory in viral quasispecies may serve the adaptive scheme of viruses by providing a molecular reservoir capable of reacting swiftly to a selective constraint which has been previously experienced by the same population, provided no bottlenecks intervened (table ). all evidence suggests that immune or other internal physiological responses in hosts are neither uniform nor continuous, and that memory may help confronting discontinuous or fluctuating challenges that require dominance of related viral genome subsets. minority genomes present at higher than basal levels, associated with mutational pressure, need not be related only to memory. they may occur as a consequence of the fitness values of randomly generated variants that, although not sufficient to drive the genome subset to dominance, may allow the subset to remain at levels similar to those associated with memory. all these possibilities provide an incentive to develop methods to diagnose the presence of minority genomes in viral populations in vivo, for example for a more personalized and adequate antiviral treatment plan (i.e. to avoid antiretroviral inhibitors for which mutations that contribute to resistance are present at memory levels) (reviewed in domingo et al. ) . there are two concepts related to memory in viruses that should be distinguished. one is a form of long-term memory, not erased by population bottlenecks, that has gradually shaped the genetic structure and replication strategy of viruses. a present-day virus can be viewed as a "reservoir of memories" of all influences that have conformed its present organization and biological potential, in constant co-evolution with hosts that themselves are an "outcome of history". in contrast, the quasispecies memory discussed in previous paragraphs is an immediate, short-term consequence of the recent evolutionary history of a virus, independently of the historical events that gradually shaped its current configuration. theoretical studies summarized in the chapters by biebricher and eigen and by wilke et al., this volume, led to the very significant prediction that there must be a limit to the error rate that a replicating system can tolerate if the genetic information is to be maintained (swetina and schuster ; eigen and biebricher ; nowak and schuster ; eigen ) . this limit is termed the error threshold and its value depends on the complexity of the genetic information conveyed by the system. here the term "complexity" means the information encoded by a genome (regulatory regions and number of different open-reading frames), which must be distinguished from the number of different sequences for any regulatory region or open-reading frame represented in a population of genomes; the latter is often referred to as complexity of the mutant spectrum or quasispecies complexity. for rna viruses, which generally encode little or no redundant information, complexity (in its first meaning) can be equated with genome length. it may be expected that the most complex rna viruses (such as the coronaviruses, with - kb genomes) may display higher average copying fidelities than the least complex rna genomes. in this respect, it will be interesting to establish whether a domain found in the sars and other coronavirus genomes, which corresponds to a nuclease activity, can provide a proofreading-repair function during coronavirus replication (snijder et al. ) . the interactions among components of mutant spectra (sect. ) also provide a biochemical basis for impairing viral replication to the point of leading to the information meltdown typical of error catastrophe. interestingly, the proposed negative interactions (for example, among trans-acting products as discussed in sect. and depicted schematically in fig. ) also favour a more likely replicative collapse when the number of open-reading frames is high (higher complexity in the sense of number of genes encoding proteins). un-der mutational pressure, more gene products can be deficient and contribute to replicative incompetence. this is a biochemical counterpart of the genetic basis of error catastrophe. measurements of lcmv rna and infectivity levels during enhanced mutagenesis of steady-state persistent infections of the virus in cell culture have defined two viral extinction pathways. one of them occurs at low mutagenic activities, and it is mediated by defective genomes which drive the infective genomes towards extinction; both experimental results and a computational model support the lethal defection model of virus extinction, with a prominent role of the mutant spectrum . there is considerable experimental evidence that rna viruses replicate with an error rate which is close to the error threshold for maintenance of genetic information. in several viral systems, it has been documented that an increase in mutation rate by added chemical mutagens results in decreases in infectivity, and in some cases it may lead to virus extinction lee et al. ; loeb et al. ; crotty et al. crotty et al. , loeb and mullins ; sierra et al. ; lanford et al. ; pariente et al. ; contreras et al. ; grande-pérez et al. ; airaksinen et al. ; ruiz-jarabo et al. a; severson et al. ; reviews in eigen ; anderson et al. ; domingo b) . two lines of study are particularly promising regarding a possible clinical application of error catastrophe. one concerns the mechanism of antiviral activity of the nucleoside analogue ribavirin ( -β-d-ribofuranosyl- , , -triazole- -carboxamide; rib), a licensed drug extensively used in clinical practice, which shows antiviral activity against a number of dna and rna viruses (snell ; see also airaksinen et al. and references therein). rib is phosphorylated by cellular enzymes to its mono-, di-and triphosphate forms and it exerts its antiviral activity through different mechanisms. one of the mechanisms unveiled by crotty and colleagues, working with poliovirus, is enhanced mutagenesis as a result of incorporation of ribavirin monophosphate (rib mp) into poliovirus rna by the viral polymerase (crotty et al. (crotty et al. , graci and cameron ) . it has also been shown that rib mp can be incorporated by the hepatitis c virus (hcv) and coxsackievirus b polymerase (maag et al. ; vo et al. ; freistadt et al. ). this raises the intriguing possibility that benefits of rib (alone but mainly in combination with interferon α or its derivatives) for treatment of chronic hcv infections (mchutchison et al. , among many other studies) may be partly due to its mutagenic activity (see also the chapter by pawtlosky, this volume, for alternative mechanisms of rib action on hcv infection). if this were established (by many ongoing studies), it would mean that the principle of virus entry into error catastrophe as an antiviral activity would already have been successfully documented without experts being aware of the underlying mechanisms. a second line of promising research was launched by de la torre and his collaborators by showing that the base analogue -fluorouracil (fu) prevented the establishment of a persistent lymphocytic choriomeningitis infection in mice (ruiz-jarabo et al. a) . fu is mutagenic for a number of rna viruses (pringle ; eastman and blair ; sierra et al. and references therein) and is used to treat some types of human cancer (parker and cheng ) . the demonstration that it can be a potent antiviral agent in vivo constitutes a proof-of-principle that is extremely encouraging for the prospect of a clinical application of virus entry into error catastrophe (for recent reviews of error catastrophe as an antiviral strategy, see anderson et al. and domingo b) . this recent field of research illustrates how decisive is to cultivate basic research in its many facets to arrive at potential practical applications, often in unpredictable ways. the studies summarized in this and other chapters of this book emphasize two growing concepts in the studies of infectious diseases that apply not only to viral infections, but also to bacterial, fungal and parasitic infections, as well as to cancer cells. one is the increasing experimental evidence in support of the remarkable statement by theodosius dobzhansky, written three decades ago, that "nothing in biology makes sense except in the light of evolution" (dobzhansky ) . dealing with evolutionary concepts is not rooted in the tradition of many schools of biochemistry and molecular biology. however, recent developments point to a decisive contribution of evolutionary events as part of the interactions between hosts and infectious agents. justification of this statement includes the recognition of pathogen variation and adaptability in disease emergence and reemergence (smolinski et al. ) , as well as in pathogenesis (this volume and many references included in it). evolutionary events underlie selection of viruses resistant to antiviral agents, and of bacteria resistant to antibiotics (for example, welsh ) . the list could go on for any pathogen whose replication cycles last orders of magnitude less than those of the hosts they infect (see the overview of molecular mechanisms of pathogen adaptation in domingo a) . a concept that originated in physics and that is gradually penetrating many fields of science (including biology and infectious diseases) is complexity and emergence (cowan et al. ; solé and goodwin ) . what this concept entails is that certain phenomena whose occurrence depends on interactions among many individual influences cannot be accounted for by the sum of the individual contributions. disease emergence offers an example: although we know (or strongly suspect with a reasonable basis) that human mobility, demographic changes and urbanization favour human-to-human pathogen transmission, that many agents are potentially zoonotic and that in many areas of the world there is close contact between humans and animals, it was not possible to predict the aids epidemic. at most, it can be predicted that because of (at least) thirteen different factors (smolinski et al. ) (each one including, in turn, multiple sub-factors!) it is very likely that in the coming years new infectious diseases will emerge. can studies on complexity be of use to infectious diseases? probably, but the way such help can be translated into practical results is hardly graspable at the moment. this is not so, however, with regard to considering evolutionary concepts in designing antiviral strategies, since important successes (for example, highly active antiretroviral treatments to control hiv infections, or the prospects of lethal mutagenesis as an antiviral strategy) have been a consequence of understanding and trying to counter the evolutionary potential of pathogens. the need of a holistic view of biology with a focus on evolution, emergence and complexity has been emphasized by biologists working in areas other than infectious diseases, such as general evolution and developmental biology (woese ). a point has been reached that encourages a transdisciplinary approach to problems of 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mutagenic and inhibitory effects of ribavirin on hepatitis c virus rna polymerase physical approaches to biological evolution antibiotics. actions, origins, resistance replication at periodically changing multiplicity of infection promotes stable coexistence of competing viral populations natural selection. domains, levels and challenges mathematical models of the epidemiology and control of foot-and-mouth disease immunity in chimpanzees chronically infected with hepatitis c virus: role of minor quasispecies in reinfection encyclopedia of virology acknowledgements work was supported by grants bmc - -c - , cam ( . / / , . / / . ) gr/sal/ / to genetrix, s.l., eu, and fundación ramón areces. ag-p was supported by a postdoctoral contract from cam and a contract ramón y cajal from mcyt, jg-a was supported by a predoctoral fellowship from meyc, and aa by a predoctoral fellowship from cam and an i p contract from csic. key: cord- -ji qq q authors: lagare, adamou; maïnassara, halima boubacar; issaka, bassira; sidiki, ali; tempia, stefano title: viral and bacterial etiology of severe acute respiratory illness among children < years of age without influenza in niger date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: ji qq q background: globally, pneumonia is the leading cause of morbidity and mortality in children, with the highest burden experienced in sub-saharan africa and asia. however, there is a dearth of information on the etiology of severe acute respiratory illness (sari) in africa, including niger. methods: we implemented a retrospective study as part of national influenza sentinel surveillance in niger. we randomly selected a sample of nasopharyngeal specimens collected from children < years of age hospitalized with sari from january through december in niger. the samples were selected from individuals that tested negative by real-time reverse transcription polymerase chain reaction (rrt-pcr) for influenza a and b virus. the samples were analyzed using the fast track diagnostic respiratory pathogens plus kit (biomérieux, luxemburg), which detects respiratory pathogens including viral and bacterial agents. results: among the samples tested, ( %) tested positive for at least one viral or bacterial pathogen; in ( %) sample, only one pathogen was detected. we detected at least one respiratory virus in ( %) samples and at least one bacterium in ( %) samples. respiratory syncytial virus ( / ; %), rhinovirus ( / ; %) and parainfluenza virus ( / ; %) were the most common viral pathogens detected. among bacterial pathogens, streptococcus pneumoniae ( / ; %) and haemophilus influenzae type b ( / ; %) predominated. conclusions: the high prevalence of certain viral and bacterial pathogens among children < years of age with sari highlights the need for continued and expanded surveillance in niger. acute respiratory infections (aris) are responsible for substantial morbidity and mortality globally, especially in children < years of age, and the highest burden is observed in developing nations [ ] . in , approximately . million ari-associated deaths occurred among children < years of age of which % were in africa and southeast asia [ ] . both bacteria and viruses have been identified as the etiological agents of ari. the viruses most frequently detected in children with aris include respiratory syncytial virus (rsv), influenza virus (inf) types a and b, adenovirus (av), parainfluenza virus (piv), human metapneumovirus (hmpv) and rhinovirus (rv) [ ] [ ] [ ] ; however, the clinical presentations of respiratory tract infections are similar, making it difficult to distinguish between etiologic agents without a laboratory diagnosis [ ] . in addition, the interpretation of a viral detection is complicated by the fact that infections with multiple viruses are common in children with ari and many viruses are frequently found in asymptomatic children [ ] . streptococcus pneumoniae and haemophilus influenzae type b (hib) are the most commonly isolated bacteria from ari cases, while, other atypical pathogens such as mycoplasma pneumoniae and chlamydophila pneumoniae are less frequently reported [ ] [ ] [ ] . s. pneumoniae and hib are commonly identified in nasopharyngeal samples from asymptomatic children due to high rates of carriage; however, their identification from the nasopharynx is rarely indicative of invasive disease [ ] . the viral and bacterial etiology of ari has been well documented in countries from the northern hemisphere [ ] [ ] [ ] [ ] ; however, few studies are available from africa [ , ] . in niger, a sentinel surveillance system for influenza viruses was instituted in april ; however, no studies on the etiology of ari have been conducted in the country. we aimed to document the prevalence of selected viral and bacterial infections among children < years of age hospitalized with severe acute respiratory illness (sari) at selected hospitals in niger from january through december . niger is a west african country with a saharan climate characterized by four distinct seasons: the cold season from mid-december to mid-february, the dry and hot season from mid-february to may, the rainy season from june to september, and the hot season from october to mid-december [ , ] . since april , influenza surveillance has been conducted among patients hospitalized with severe acute respiratory illness (sari) at sentinel sites located in of the regions of the country by the centre de recherche médicale et sanitaire (cermes), the national reference laboratory for influenza. the influenza surveillance program in niger has previously been described [ ] . briefly, all patients hospitalized at the participating sentinel sites that met the sari case definition were eligible for enrollment. verbal informed consent was obtained from all cases who were years of age and older. proxy informed consent was obtained from parents or legal guardians of minors. patients who did not meet the case definition or did not provide verbal consent were not included. a sari case was defined as a hospitalized child < years of age with onset of cough or difficulty breathing within days prior to admission, and at least one of the following danger signs: inability to drink or breastfeed, lethargy, vomiting everything, convulsions, nasal flaring, chest indrawing, stridor in a calm child or tachypnea [ ] . a standardized questionnaire was administrated by clinical personnel, to record patients' demographic characteristics and medical history. the questions included information on date of enrollment and symptom onset, sex, age and clinical symptoms. nasopharyngeal (np) swabs were collected from all enrolled cases and placed in cryovials containing virus transport medium (copan kit, italy). the specimens were kept refrigerated at °c at the sentinel site and then transported twice weekly to cermes for testing. samples were aliquoted, screened for influenza a and b viruses by real-time reverse transcription polymerase chain reaction (rrt-pcr), and then stored at − °c. we conducted a retrospective study on the etiology of influenza-negative sari cases among children < years of age enrolled in influenza surveillance during january through december in niger. we randomly selected (using random selection procedures available in stata) a sample of stored np specimens from sari cases which had tested influenza a and b negative. this sample represented % of the total cases aged < years enrolled during the study period. only . % of sari cases in the < year old age group were tested positive for influenza. influenza-positive cases were mainly detected during the cold season [ ] . nucleic acid was extracted using the qiaamp mini kit (qiagen, germany) in accordance with the manufacturer's protocol. an internal control (ic) was added to each extraction tube in order to assess the quality of the extraction at the end of the amplification. the Χ and the fisher's exact tests were used to assess the difference between categorical variables by comparing expected and observed frequencies across evaluated groups. in addition, we compared the characteristics of selected and non-selected children (including influenza-positive cases) using the x statistics. the statistical analysis was implemented using stata version . (statacorp®, texas, usa). in , the national ethics committee of niger approved the national influenza surveillance program (reference no / /ccne of april ). in , prior to the investigation of other respiratory pathogens, the ministry of health provided an extended approval. access to the study samples was granted by the director of cermes. from january through december we enrolled children < years of age hospitalized with sari into the influenza surveillance program, of which ( %) tested negative for influenza virus. of these, ( %) were randomly selected for our study. the age, sex and symptom duration distribution did not differ significantly among selected and non-selected children (including those that tested positive for influenza). however, among the selected group there was a significantly lower proportion of specimens collected during the cold season, when the majority of influenza-positive cases were detected and excluded from randomization (table ) . among the selected children, % ( / ) were < year of age (median age among children age < years: months), % ( / ) were female and % ( / ) had a duration of symptoms ≤ days. most patients presented with a recorded temperature > °c ( %; / ), cough ( %; / ), dyspnea ( %; / ) and chest indrawing ( %; / ). few patients presented with tachypnea or had difficulty in breastfeeding ( %; / ). overall, / ( %) of children included in the study tested positive for at least one pathogen (viral or bacterial). at least one respiratory virus was detected in / ( %) samples and at least one bacterium was detected in / ( %) samples. among the samples positive for any pathogen, ( %) were positive for a single pathogen. of these ( %) were positive for s. pneumoniae, ( %) for rsv and ( %) for rv, while hmpv, cv, piv and hib each accounted individually for < % of the single organisms detected (fig. ) . among the children in whom ≥ organisms were detected, both viral and bacterial pathogens were detected in samples ( %). among the samples tested, rsv (n = ; %) was the most frequently detected virus, followed by rv (n = ; %) and piv types - (n = ; %) ( table ) . cv, hmpv, hbv, ev and av were detected individually in < % of the specimens. no pv was detected in our study. of the samples that tested positive for cv, ( %) were type oc , ( %) were type e, ( %) were type nl and ( %) were type hku . of the samples that tested positive for piv, ( %) were type , ( %) were type , ( %) were type and ( %) were type . even though we selected co-detections with different subtypes were detected in ( %) and ( %) of the cv and piv positive cases, respectively. rsv, piv and hmpv were detected more frequently in infants < year of age compared to children - years of age (table ) . rsv, piv and hmpv were detected more frequently during the hot season (october to mid-december); while rv and hbv were detected more frequently during the rainy season (june to september). the other viruses were detected with similar frequencies across seasons (table ) . among the samples tested, s. pneumoniae (n = ; %) was the most frequently detected bacteria, followed by hib (n = ; %), s. aureus (n = ; %) and c. pneumoniae (n = ; . %) ( table ) . m. pneumoniae was not detected in our study. we report the detection rate of selected viral and bacterial pathogens among children < years of age hospitalized with sari in niger. we detected respiratory viruses in % of our study sample. the high detection rate of viruses found in our study is consistent with results from similar studies conducted in burkina faso ( %) [ ] , kenya ( %) [ ] and brazil ( %) [ ] . however, lower rates of viral detection were reported from other studies from countries such as ghana ( %) [ ] , china ( %) [ ] and egypt ( %) [ ] . these differences can be attributed to different climatic conditions, enrollment criteria, case definitions and testing platforms. in our study, rsv was the predominant virus detected and was most commonly found in children < year of age. rsv has been reported to be an important pathogen in children and especially in young infants in several studies [ , [ ] [ ] [ ] [ ] [ ] . in addition, rsv detection has been reported to be strongly associated with illness from studies comparing symptomatic cases to controls [ ] . rhinovirus was the second most commonly detected virus ( %) with similar rates among infants < year of age and children aged - years, which has been reported in previous studies [ , ] . however, other studies reported rv as the most prevalent virus among children < years of age [ , , , ] . in addition rv has been commonly detected among asymptomatic persons in several studies indicating that rv can act as both pathogen and by-stander, consequently hindering the ability to infer an association between detection and illness [ ] [ ] [ ] . among the piv and cv detected in this study, piv type and cv type oc were the most common virus types, which has been reported in other studies [ , ] . we also found a high detection rate of bacterial pathogens. s. pneumoniae ( %) and hib ( %) were the most common bacteria detected in nasopharyngeal specimens. elevated colonization rates of these bacteria have been reported in children, but only a proportion of colonizations result in invasive disease [ , , ] . the high detection rate of s. pneumoniae in our study is likely due to the fact that s. pneumoniae is a commensal of the nasopharynx [ ] . it has been shown that the prevalence of s. pneumoniae carriage in healthy children < years of age ranges from % to % in low income countries [ ] . the detection of s. pneumoniae from sterile sites like blood or cerebrospinal fluid, reflecting invasive pneumococcal disease, has been shown to be lower ( - %) [ , ] . nonetheless, s. pneumoniae has been reported to be responsible for % of meningitis cases in niger based on cerebrospinal fluid testing; different serotypes were detected among cases of meningitis prior to the introduction of the pneumococcal conjugate vaccine in [ ] . hib and s. aureus, the nd and rd most prevalent bacterial pathogens in our study, have also been shown to be commensal organisms with high nasopharyngeal carriage rates especially in young children [ ] . the substantial hib nasopharyngeal colonization density found in this study should be investigated further as hib vaccine has been available in the niger expanded immunization program since . nasopharyngeal specimens may be used to aid in the diagnosis of certain bacterial respiratory pathogens that do not tend to colonize the nasopharynx, such as m. pneumoniae and c. pneumoniae [ , ] . c. pneumoniae was detected at low rates ( . %), and m. pneumoniae was not detected in our study. using serological methods, prevalence rates as high as % have been reported for c. pneumoniae [ ] ; in contrast, other studies report significant detection of m. pneumoniae (> %) and low detection of c. pneumoniae (< %) [ , , ] . in our study we found an elevated prevalence ( %) of viral-bacterial co-detections, which has been reported in other studies [ , ] . it has been shown that viral infections may predispose to bacterial super-infection by favoring bacterial attachment sites on nasopharyngeal epithelial cells and through increased mucous production that promotes bacterial growth [ , ] . our study has limitations that warrant discussion. first, the small sample size of our study hindered our ability to accurately assess the seasonality of the pathogens included in our study. nonetheless, our results suggest that rsv, piv and hmpv are more commonly detected during the hot season (october to december), while rv and hbv are detected more frequently during the rainy season (june to september). no difference in the detection rate of the other viruses and bacteria was noted across seasons in our study. the small sample size of our study also hindered our ability to detect patterns of co-detection and the association between specific viral and bacterial co-detections. second, we did not keep formal records of the proportion of patients consenting to participate in the sari surveillance. however, a review of the performance of the surveillance system implemented through hospital record review at sentinel sites revealed that only a few patients that met the study case definition were missed by the surveillance program. third, the lack of controls in our study limited our ability to assess the association of pathogen detection with disease. while most of the viral and bacterial pathogens identified in this study have been described by previous studies as causative agents of ari, the assignation of causality remains challenging [ , ] . fourth, influenzapositive samples were excluded from our study. codetection of other viral and bacterial pathogens with influenza is expected and this may have resulted in an underestimation of the prevalence of the pathogens included in this study, especially during the cold season when the majority of influenza-positive cases were detected. last, we did not systematically collect information on progression of illness (including in-hospital outcome) or risk factors for severe disease, which hindered our ability to evaluate pathogen contribution to the more severe spectrum of illness or to identify groups at high risk for severe disease. this study reports the detection rate of viral and bacterial pathogens among children < years of age hospitalized with sari in niger. the high prevalence of certain viral and bacterial pathogens highlights the need for expanded surveillance in niger so as to inform policies and interventions. given the high rsv detection rate observed in this study and the reported association of rsv detection with illness [ ] , rsv should be included in routine surveillance programs in niger. other selected pathogens could be considered for routine surveillance in the country following further assessment to determine association with illness. in addition, information 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molecular epidemiology of human rhinovirus infections in kilifi, coastal kenya genetic diversity and molecular epidemiology of rhinoviruses in south africa the role of multiplex pcr test in identification of bacterial pathogens in lower respiratory tract infections high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya determination of pneumococcal serotypes in meningitis cases in niger surveillance for hospitalized acute respiratory infection in guatemala bacterial and viral etiology in hospitalized community acquired pneumonia with molecular methods and clinical evaluation huamn coronaviruses associated with upper respuratory tract infections in three rural areas of ghana we are thankful to the national ministry of public health for financing this study and our partners: the pasteur institute of paris, the world health organization, and the us centers for disease control and prevention for providing reagents, the rrt-pcr machine and technical supports for influenza surveillance in niger. key: cord- - pui uu authors: gao, chun; zhu, li; jin, cheng cheng; tong, yi xin; xiao, ai tang; zhang, sheng title: proinflammatory cytokines are associated with prolonged viral rna shedding in covid- patients date: - - journal: clin immunol doi: . /j.clim. . sha: doc_id: cord_uid: pui uu since december , coronavirus disease (covid- ) has emerged as a global pandemic. we aimed to investigate the clinical characteristics and analyzed the risk factors for prolonged viral rna shedding. we retrospectively collected data from hospitalized covid- patients in a single center in wuhan, china. factors associated with prolonged viral rna shedding (≥ days) were investigated. forty-nine ( . %) patients had prolonged viral rna shedding. patients with prolonged viral shedding were older and had a higher rate of hypertension. proinflammatory cytokines, including interleukin- r (il- r) and tumor necrosis factor-α (tnf-α), were significantly elevated in patients with prolonged viral shedding. multivariate analysis revealed that hypertension, older age, lymphopenia and elevated serum il- r were independent risk factors for prolonged viral shedding. this comprehensive investigation revealed the distinct characteristics between patients with or without prolonged viral rna shedding. hypertension, older age, lymphopenia and high levels of proinflammatory cytokines may be correlated with prolonged viral shedding. the entire world has been threatened by the outbreak of coronavirus disease caused by severe acute respiratory syndrome coronavirus- (sars-cov- ). - as of may th , , over , , cases were confirmed worldwide with a rough death rate of approximately . %. clinical manifestations of covid- are variable, and they vary from asymptomatic to mild, moderate, severe and critical. in most cases, covid- patients present with symptoms including fever, cough, dyspnea, chest tightness, fatigue, gastrointestinal symptoms, myalgia and headache. laboratory findings featuring lymphopenia and radiographic findings of pneumonia are common evidence of the diagnosis. severe or critical patients might suffer from acute respiratory failure, acute respiratory distress syndrome (ards) and other serious complications involving multiple systems. [ ] [ ] real-time reverse transcription polymerase chain reaction (rt-pcr) for sars-cov- is the standard laboratory examination to confirm the diagnosis of covid- . many studies have found that older patients have a higher risk of having a severe case and they have a higher death rate from covid- , which may be related to a weaker host immune response for antiviral defense and an uncontrolled proinflammatory cytokine storm. , , a recent report by qin et al. showed that severe j o u r n a l p r e -p r o o f journal pre-proof cases of covid- had a profound dysregulation of their immune response. this might be part of the pathological process of covid- and be related to the clinical outcome. viral shedding patterns of sars-cov- have been revealed in several studies, and the dynamics of viral shedding have been reported. according to recent reports of the sars-cov- viral nucleic acid shedding pattern, a certain proportion of patients may experience a longer duration of viral nucleic acid negative conversion. [ ] [ ] therefore, this retrospective study aimed to analyze the clinical characteristics of covid- patients who experienced prolonged viral shedding and to investigate the contributing risk factors. we retrospectively enrolled hospitalized patients ( data were collected from the electronic medical record system. the following information was collected for analysis: . demographic characteristics such as age, sex, j o u r n a l p r e -p r o o f throat swab samples or deep nasal cavity swab samples were collected for extracting rna to confirm the diagnosis of covid- infection as previously reported. the collected swabs were placed into a collection tube with μl of virus preservation solution, and total rna was extracted within hours using magnetic beads (tianlong, xi'an, china). the extraction solution was used for a one-step rt-pcr assay of we present the continuous variables as the mean ± standard error of the mean (sem) or median (interquartile range, iqr) and analyzed them with the chi-square test or mann-whitney u test. we report the categorical variables as whole numbers and percentages. univariate logistic regression was used to evaluate potential risk factors for prolonged viral shedding. only factors with a p-value< . in univariate analysis were included in the final multivariate analysis model. we employed multivariate cox regression to identify independent predictive factors for the length of viral rna shedding. all p values were reported as two-sided with a significance level of . . all statistical tests were calculated using spss version . (ibm, ny, usa). we included patients diagnosed with covid- in this study. the majority of patients ( . %) were classified as moderate. no patient was transferred to the icu or the baseline laboratory and radiological characteristics of patients with and without prolonged viral rna shedding are shown in table . compared with the nonprolonged group, patients with prolonged viral shedding had a significantly lower lymphocyte count ( . × /l vs . × /l, p< . ) and a higher neutrophil count ( . × /l vs . × /l, p< . ). prolonged cases showed significantly elevated serum infection markers, such as c-reactive protein (crp) and ferritin (p< . ). levels of proinflammatory cytokines, such as interleukin (il)- r (p< . ) and tumor necrosis factor-α (tnfα) (p< . ), were significantly higher in patients in the prolonged group. moreover, serum albumin was lower in the prolonged group (p< . ). on admission, the proportion of bilateral pneumonia was similar between the two groups (p= . ). in the multivariate survival analysis, we identified that age (≥ yrs vs. < yrs), preoperative lymphocyte count, serum interleukin- r and hypertension were independent risk factors associated with prolonged viral rna shedding in covid- j o u r n a l p r e -p r o o f table ) . we investigated the differences in treatments between patients with nonprolonged and prolonged viral shedding. as shown in table receiving immunoglobulin and thymalfasin, respectively. compared with the nonprolonged group, patients with prolonged viral shedding were more likely to receive corticosteroids (p= . ) and hydroxychloroquine (p= . ). the differences in the other treatments between the two groups were not statistically significant (p> . ). in addition, we also analyzed the influence of different therapies on laboratory characteristics such as lymphocytes and proinflammatory factors (supplemental the dynamics of the proinflammatory cytokines between the prolonged and nonprolonged groups demonstrated distinct patterns (figure ) . the pooled mean values of il- r and tnf-α were higher with a declining trend during the observation period in patients with prolonged viral shedding ( figure a&b ). il- β and il- showed a fluctuation curve from week to week , and the differences between the two groups were not significant. il- was significantly higher in the prolonged group and continued to decline until week , with a slightly increasing trend thereafter. details of the pooled mean values of proinflammatory cytokines are shown in supplemental table . the fast transmitted covid- has emerged as a global pandemic and has challenged the healthcare system all over the world. until now, no specific treatment has been validated for covid- . the key to limiting virus spread is the diagnosis and quarantine of infected cases. this is a comprehensive report of covid- patients investigating the distinct characteristics and risk factors for prolonged sars-cov- viral rna shedding. in our study, we found that covid- patients with prolonged viral shedding were older (p< . ) and presented with a higher rate of hypertension (p< . ). symptoms such as expectoration, breath shortness, fatigue and chest distress were more frequent in patients with prolonged viral shedding. previous studies have suggested that patients. together with the above evidence, we showed for the first time that elevated levels of proinflammatory cytokines, such as il r, il- and tnfα, may correlate with prolonged viral shedding. prolonged viral shedding in certain cases may arise when the host antiviral defense and immune system are not strong enough to completely block virus replication. in our study, we found that a higher proportion of patients in the nonprolonged group received immunoglobulin ( . % vs. . %). although the difference is not significant, it might provide a clue for the application of immune enhancers in the early stage of disease in selected patients. in addition, inhibitors such as ruxolitinib that block the jak /jak signal transduction pathway induced by pro-inflammatory cytokines may be promising treatments for prolonged viral rna shedding. various reports have revealed the phenomenon of sars-cov- "recurrence" or "repositive". , current guidelines suggest that two consecutive negative rt-pcr test results are one of the criteria for discharge. we argued that due to the high false negative rate of the viral test and an underestimated proportion of patients with prolonged viral shedding, the above patients may experience prolonged viral shedding rather than "recurrence". according to our results, we recommend actively monitoring proinflammatory cytokines in selected covid- patients with a high risk of prolonged viral shedding. the world is now facing a pandemic caused by sars-cov- . this study focused on investigating the risk factors for covid- patients with prolonged viral shedding. we also monitored the dynamics of proinflammatory cytokines in patients with prolonged viral shedding. the present study has several limitations that should be taken j o u r n a l p r e -p r o o f into consideration. first, in this retrospective setting, some data were incomplete. second, the treatments varied among patients. in clinical practice, patients would not receive antiviral treatment when they were first admitted to the hospital. tentative antiviral treatments such as hydroxychloroquine or lopinavir and ritonavir were prescribed in patients with persistent detection of sars-cov- by rt-pcr. therefore, it is difficult to analyze the effects of remaining positive, which might bias the results and not reflect the results of treatment. in summary, in this study, we investigated the distinct dynamics of the inflammatory response in covid- patients with prolonged viral rna shedding. hypertension, older age, lymphopenia and high levels of proinflammatory cytokines were correlated with prolonged viral shedding. we suggest longer observation and monitoring of serum proinflammatory cytokines in selected high-risk patients. all authors declare that there are no conflicts of interest. this study was approved by the ethics committee of tongji hospital, tongji medical college, huazhong university of science and technology. all procedures followed in this study were in accordance with the helsinki declaration and later versions. written informed consent was waived by the ethics commission of the designated hospital for emerging infectious diseases. the database used and/or analyzed during the current study is not publicly available (to maintain privacy) but can be obtained from the corresponding author on reasonable request. zhang table . laboratory and radiological findings of patients with covid- table . multivariate analysis of risk factors for prolonged viral shedding supplemental table tnfα, pg/ml - . . ( . - . ) . ( . - . ) . ( . - . ) < . j o u r n a l p r e -p r o o f outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention epidemiological and clinical characteristics of cases of novel coronavirus pneumonia inwuhan, china: a . lancet clinical features of patients infected with novel coronavirus in wuhan detection of novel coronavirus ( -ncov) by real-time rt-pcr expression of elevated levels of proinflammatory cytokines in sars-cov-infected ace + cells in sars patients: relation to the acute lung injury and pathogenesis of sars dysregulation of immune response in patients with covid- in wuhan, china. clinical infectious diseases sars-cov- viral load in upper respiratory specimens of infected patients factors associated with negative conversion of viral rna in patients hospitalized with covid- dynamic profile of rt-pcr findings from covid- patients in wuhan, china: a descriptive study interim infection prevention and control recommendations for patients with confirmed coronavirus disease (covid- ) or persons under investigation for covid- in healthcare settings china national health commission. diagnosis and treatment of -ncov pneumonia in china. (version ) in chinese clinical characteristics of coronavirus disease in china clinical feature of covid- in elderly patients: a comparison with young and middle-aged patients plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology long-term infection of sarscov- changed the body's immune status viruses and the diversity of cell death expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace + cells in sars patients: relation to the acute lung injury and pathogenesis of sars pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid- patients high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease . open forum infectious diseases inhibition of cytokine signaling by ruxolitinib and implications for covid- treatment positive rt-pcr test results in patients recovered from covid- the authors declare that they have no competing interests and no financial support to declare. we thank ms. cheng chen for english grammar correction of this manuscript. all authors participated in the study design. sheng zhang: conceptualization, writing key: cord- - x on authors: rumlová, michaela; ruml, tomáš title: in vitro methods for testing antiviral drugs date: - - journal: biotechnology advances doi: . /j.biotechadv. . . sha: doc_id: cord_uid: x on abstract despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. a combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. we provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses. viral pandemics remain serious threats to humankind. every year, known viruses such as hiv- and hepatitis b virus newly infect millions of people across the globe. in addition, recent outbreaks of ebola virus, influenza virus, severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome-coronavirus (mers-cov) serve as a reminder of the silent danger. the world health organization reported in that over . million causalities per year are connected with hepatitis b and almost , with hepatitis c. occasional epidemic outbreaks of other viruses are also striking. these include outbreaks of noroviruses, flaviviruses (zika and dengue viruses), new strains of influenza viruses, the re-emergence of west nile virus in italy and the united states. despite relatively long pauses, viruses such as influenza virus can re-emerge and cause global pandemic health problems. unlike cellular genomes, which consist of double-stranded (ds) dna, viral genomes can be formed by a broad variety of nucleic acids (nas), including ds or single-stranded (ss) circular or linear dna or positive-, negative-or sometimes ambi-sense rna. as viral life cycles are dependent on cellular factors and cellular metabolic and signaling pathways, the number of possible antiviral drug targets is limited. however, almost all viruses encode unique proteins and enzymes that may serve as specific targets for antiviral therapy. the overarching goal of modern drug development efforts is to design compounds that specifically inhibit viral targets or cellular targets essential for virus replication. the purpose of this review is to provide insight into the broad variety of cell-based and biochemical assays used for identification and evaluation of antivirotics, including high-throughput screening (hts) methods. an overview of available inhibitors and vaccines, which has been reviewed elsewhere [e.g. in (de clercq and li, ) ], is beyond the scope of this paper. to package their genomes into particles of very limited size, viruses encode few genes. virions consist of an rna or dna genome that is protected by an outer shell called a capsid (also nucleocapsid) formed by a lattice of capsid proteins. in enveloped viruses, the capsid is additionally surrounded by a lipid bilayer spiked with viral proteins. the size of animal viruses ranges from approximately nm to over nm (cohen et al., ) . the capsids and virions of rna viruses adopt various shapes. viral capsids may be icosahedral (e.g. picornaviridae, astroviridae, reoviridae, togaviridae), bullet-shaped (rhabdoviridae), helical (coronaviridae), helical filamentous (filoviridae, orthomyxoviridae, paramyxoviridae), or filamentous (arenaviridae, bunyaviridae). the retroviral capsid core may be conical, spherical, or rod-shaped. the morphology of dna viruses is similarly diverse, ranging from icosahedral (enveloped -hepadnaviridae, herpesvridae; nonenveloped -adenoviridae, parvovirdae, polyomaviridae and papillomaviridae) to rodshaped (baculoviridae) or pleomorphic (poxviridae). the viral life cycle is the process of viral replication in a host cell. first, viruses enter the host cell and replicate their genomes. following translation of viral proteins by the host cell machinery, viruses package their genomic material into protective proteinaceous capsids and exit the cell to infect another host cell. nonenveloped viruses consist only of a protein capsid shell enclosing the viral genome and enzymes, while the capsid shell of enveloped viruses is enclosed in a lipid envelope derived from the host cell membrane. regardless of whether the virus is enveloped, its surface must display (glyco)proteins suitable for specific interactions with host cell receptors. in contrast to the surface proteins of nonenveloped viruses, those of enveloped viruses usually serve another function in addition to host cell recognition and attachment. for example, they may enable fusion of the viral and cellular membranes, usually through an interaction with a secondary receptor(s) or co-receptor(s). the fusion domains of viral surface glycoproteins thus can lower the kinetic barrier for the energetically demanding membrane fusion. the viral particle must be sufficiently stable to protect the genome until its delivery into the host cell. simultaneously, it must be metastable, to allow its disassembly and release of the genome for replication in the infected cell at the appropriate time. the energetic barrier that prevents viral disassembly outside the cell is lowered upon infection by structural changes in the viral components. these changes may be induced by binding of a cellular ligand or by changes in the environment, such as ph change upon entering a specific cellular compartment. numerous viruses preassemble immature particles that undergo irreversible (usually proteolytic) transitions into mature structures of fully infectious virions. mutual interactions of viral capsid proteins are typically different from those of cellular proteins, which predominantly create binary interactions. viral structural proteins interact with multiple neighboring partners to form multicomponent macromolecular structures [reviewed in (cheng and brooks, ; jayaraman et al., ) ]. the economy of the packaged viral genomes due to the limited capsid size implies formation of only a few types of structural proteins, which are usually symmetrically organized [reviewed in (prasad and schmid, ; raguram et al., ) ]. in the initial stage of infection, viruses must overcome several obstacles. the first is the cellular plasma membrane with an actin cortex. then, they must traffic through dense cytoplasm to reach their final destination for replication and assembly. these pathways are specific for different viruses and often are dictated by the size of the particle and its structure. the viral life cycle can be divided into several common stages, including entry, uncoating, genome replication, genome packaging and assembly, release, and maturation ( fig. ) . in general, the first phase of viral infection is specific recognition of the target host cell and binding to a surface receptor displayed on the cell membrane. this process is common to both enveloped and nonenveloped viruses. in enveloped viruses, the binding is mediated by viral surface components, typically oligomers of integral glycoproteins. nonenveloped viruses bind receptors through sites or projections on the capsid surface. viruses can use either a single receptor (e.g. tim- for hepatitis a virus, gm for sv , cd for poliovirus, low-density lipoprotein receptor for human rhinovirus) or multiple receptors with equivalent roles (e.g., nectin- / or hvem for herpes simplex virus / , ace or l-sign for sars coronavirus). for other viruses (e.g. hiv, hcv, adenoviruses, rotaviruses, picornaviruses, and some herpesviruses), the presence of at least two cytoplasmic membrane components is required. for example, hiv- binds to the primary receptor cd and one of two co-receptors (ccr or cxcr ) [reviewed in (cossart and helenius, ; grove and marsh, ) ]. to infect a cell, viruses must overcome the plasma membrane to deliver their genetic material into the cytosol. viruses enter cells by two main mechanisms. a majority of animal viruses, both enveloped and nonenveloped, enter cells by one or two types of endocytosis, such as clathrin-mediated endocytosis (e.g. vsv, influenza a virus, rhinovirus), caveolin-mediated uptake (echovirus, polyoma virus), clathrin/caveolin-independent endocytosis such as caveolar or lipid raft-mediated (e.g. sv , polyomavirus mouse), or macropinocytosis (e.g. vaccinia virus, respiratory syncytial virus, ebola virus, hiv- ) (blaas, ; cossart and helenius, ; fields and knipe, ; kirkham and parton, ; marechal et al., ; mayor and pagano, ; mercer and helenius, ; parton and simons, ; rasmussen and vilhardt, ; saeed et al., ) . these endocytic mechanisms enable the virus to be transported by the cell's machinery through the plasma membrane and to pass through the dense actin cortex. cellular entry of some viruses is coupled with receptor-mediated signaling, resulting in activation of molecules that facilitate virus entry by cytoskeleton reorganization, induction of long-distance transport of the virus-containing vesicles, or actin cortex disassembly. the second type of entry is used by some enveloped viruses (paramyxo-, herpes-, and retroviruses), which upon cell surface receptor binding, infect the cell by direct fusion of viral and plasma membranes at neutral ph. upon internalization, most viruses become trapped and enclosed in vesicles that pinch off the inner side of the plasma membrane and transport their cargo into the cytoplasm. on their way through the cell, these transport vesicles undergo maturation by fusing with other vesicles. to fulfill their task of genome replication, viruses have to escape from these endosomes. the "great escape" is triggered by activation of a fusion or penetration mechanism, such as changes in conditions in the endosomal interior [e.g. ph, ionic environment (e.g. calcium ion concentration), oxido-reducing conditions], changes in membrane composition, and other physico-chemical cues (blaas, ; cossart and helenius, ; inoue and tsai, ) . depending on the requirements for a particular virus, these events can occur in early endosomes (ph . - . ; e.g. hepatitis c virus, vesicular stomatitis virus), late endosomes (ph . - . ; e.g. influenza a virus, dengue virus, sars coronavirus), recycling endosomes, macropinosomes, the endoplasmic reticulum, the golgi apparatus, or lysosomes (ph . - . ) (blaas, ; cossart and helenius, ; grove and marsh, ; inoue and tsai, ) . for the majority of animal viruses, the activation of these fusion or penetration mechanisms occurs through conformational changes and structural rearrangements in viral surface proteins and/or the whole virion shell that may destabilize the capsid core. structural rearrangements in enveloped viruses usually mediate fusion of viral and endosomal membranes. in nonenveloped viruses, the structural changes uncover amphipathic or hydrophobic domains that may induce pore formation or disruption of endosomal membranes. to deliver genetic material to the replication site, these mechanisms ultimately release viral capsid structures from endosomal vesicles into the cytosol either by fusing with the endosomal membrane (enveloped viruses) or by penetrating the endosomal membrane (nonenveloped viruses). uncoating is the partial or complete disassembly of the protective capsid shell and/or lipid envelope to liberate the viral genome. for many viruses, this process is closely connected with conformational changes induced by the virus binding to the cell surface receptor; a low ph environment; or changes in oxido-reducing conditions, ion concentrations, or other factors. for enveloped viruses, uncoating involves a loss of the viral membrane by fusion either with the plasma membrane or with intracellular vesicles, followed by stepwise uncoating of the protective capsid shell. in nonenveloped viruses, the uncoating process typically involves conformational changes that result in the weakening of intermolecular interactions, loss of structural proteins, proteolytic cleavage, and so on (fields and knipe, ) . depending on the virus, uncoating can take place at the plasma membrane, in the cytoplasm, during endocytosis in early or late endosomes, in lysosomes, in the nucleus, or at the nuclear pore complex (npc). the ssrna genome of retroviruses, which is fully enclosed inside a protective capsid shell, must be reverse transcribed into dsdna and released from the capsid. although it is accepted that hiv- uncoating is linked to reverse transcription and nuclear import (ambrose and aiken, ) and is controlled by host factors (e.g. cyclophilin a, trim α), the precise molecular mechanisms that trigger the uncoating remain unknown. several hypotheses have been proposed, including breakage of the capsid shell due to increased inner pressure caused by accumulation of the reverse transcription product (rankovic et al., ) , the requirement of intact microtubules and dynein and kinesin motors (lukic et al., ) , and phosphorylation of capsid shell protein by the host cell kinase melk (takeuchi et al., ) . in contrast to the majority of rna viruses, which replicate in the cytoplasm, most dna viruses (with the exception of large dna viruses including poxviridae, asfarviridae, and mimiviridae) and several negative stranded rna viruses enter the nucleus to replicate their genomes (kobiler et al., ; koonin and yutin, ) . passive diffusion into the nucleus is suitable for molecules smaller than nm in diameter, but larger structures must enter through the nuclear pore complex (npc), which can accommodate molecules of up to nm (pante and kann, ) . translocation through the npc is tightly regulated and requires the presence of a nuclear localization signal (nls) on the passing molecule and nuclear import receptors (importins or karyopherins) (cautain et al., ) . due to the diversity of viral particle sizes (from nm to over nm) and structures, viruses have evolved several strategies to export their dna or rna genome into the nucleoplasm. with regard to the npc, these pathways can be divided into npc-dependent and npcindependent mechanisms. due to size limitations, only a few, very small simplified scheme of common stages of viral life cycle targeted by antiviral drugs. these stages including: ) attachment and entry, ) uncoating, ) genome replication, ) genome packaging and assembly of viral particle and ) virus release and maturation. m. rumlová, t. ruml biotechnology advances ( ) - viral capsid particles, such as hepatitis b virus (hbv; - nm diameter), can pass through the npc (cohen et al., ; rabe et al., ) in an importin-dependent manner. hbv then disassembles at the nuclear side of the npc (the nuclear basket), releasing its genome into the nucleoplasm (fay and panté, ) . the capsid shells or ribonucleoprotein complexes (rnps) of some larger viruses, such as influenza a virus and hiv- , disassemble within the cytoplasm. the viral genome, released from the shell and complexed with nls-containing components, is then translocated through the npc (ambrose and aiken, ; campbell and hope, ; cohen et al., ; fay and panté, ; hutchinson and fodor, ) . another mechanism, used by herpes simplex virus (hsv) and adenoviruses, involves cellular (importins, nup) or viral protein-mediated attachment (docking) of the capsid shell at the cytoplasmic side of the npc. this facilitates the passage of genomic dna into the npc either by ejection from an almost intact particle (through the capsid portal in hsv- ) or upon complete disassembly of the capsid shell (in adenoviruses) (kremer and nemerow, ; ojala et al., ; pasdeloup et al., ) . npc-independent mechanisms are used by some retroviruses (e.g. mlv) that enter the nucleus during the mitotic phase of the cell division cycle when the nuclear envelope is dissolved (matreyek and engelman, ; roe et al., ) . another mechanism, described for parvoviruses, involves partial disruption of the nuclear envelope (cohen and pante, ; fay and panté, ) . viral genomes can be encoded by various types of nas, as summarized in table for dna viruses and table for rna viruses. by convention, ssdna of equivalent polarity to mrna is designated as the positive (+) strand. the complementary ssnas are of minus polarity (−). the majority of dna viruses replicate in the nucleus, where cellular dna replication and transcription also occur. in contrast, rna viruses usually replicate in the cytoplasm. rna viruses are the only "organisms" that store their genetic information in the form of rna. replication of their genomes is accomplished either by rna-dependent rna synthesis ( fig. a , b) [reviewed in (ferron et al., ; lu and gong, ; menéndez-arias and andino, ; pietilä et al., ; tao and ye, ) ] or through rnadependent dna synthesis (reverse transcription) followed by dna integration, replication, and transcription ( fig. c ). as some of these enzymatic activities are not commonly found in uninfected host cells, rna viruses must encode enzymes to aid replication (table ). rna viruses are divided according to the baltimore classification into dsrna viruses (birna-and reoviridae); positive-sense ssrna viruses (corona-, flavi-, and picornaviriade); negative-sense ssrna viruses (filo-, rhabdo-, paramyxo-, and orthomyxoviriade), and ambisense rna viruses (arena-and some members of bunyaviridae) with both positive-and negativesense rnas (nguyen and haenni, ) (see table ). the nature of the genome not only dictates the mechanism of replication, but also has other important consequences. the genome of (+)rna viruses may serve directly as mrna for production of viral proteins. therefore, the mere introduction of genetic material (e.g. in exocytic vesicles) may result in productive infection. the rna polymerases that copy the genetic material of rna viruses are error-prone, which provides considerable genetic flexibility and the propensity to evolve drug-resistant mutants. these features are amplified in viruses with segmented genomes that undergo reassortment. in viruses with segmented genomes (orthomyxoviridae, arenaviridae and bunyaviridae), each segment is transcribed in an autonomous transcription-replication unit by viral rna polymerase that binds to the ′ end cap structure. of note, the genomic rna (grna) is capped by a unique mechanism called cap-snatching (ferron et al., ) , in which the cap is cleaved from cellular mrna and transferred onto the viral grna by a subunit of viral rna polymerase (pflug et al., ) . some dsrna viruses (birnaviridae and reoviridae) also contain segmented genomes. upon replication, these viruses must ensure stoichiometric incorporation of single copies of each grna segment into new particles. this is guided by specific packaging signals on each segment of grna that interact with positively charged domains in the capsid proteins [reviewed in (isel et al., ; pohl et al., ) ]. although different models of packaging have been proposed for various segmented genome viruses, a common feature is co-assembly of the capsid proteins with the grna and rna polymerase. the genomes of dna viruses come in a considerable variety of sizes and shapes, from small ss to large ds molecules that may be linear or circular. the size range of these genomes (from . kb to kb) reflects the necessity for some viruses to encode specific proteins required for viral replication. small-genome dna viruses (polyoma-, papilloma-, and parvoviruses) use only host cell enzymes for replication and transcription. the only exceptions are some hepadnaviruses (e.g. hepatitis b virus) that despite having small genomes (approximately kb), encode their own specific dna polymerase/reverse transcriptase that reverse transcribes pregenomic rna (pgrna) into genomic viral dna (fig. d , ii) (beck and nassal, ) . viruses with intermediate-size genomes (up to kb) (e.g. adenoviruses) encode their own dna polymerase for genome replication, but they usually utilize cellular rna polymerases ii and iii for transcription (fields and knipe, ) . viruses with large genomes (larger than kb) (e.g. herpesviruses) encode proteins for replication, including dna polymerase and dna helicase-primase, as well as some enzymes necessary for biosynthesis of deoxyribonucleotide triphosphates (dntps) and several transcription factors (boehmer and nimonkar, ) . poxviruses (e.g. vaccinia virus), are another type of virus with large dsdna genomes, and their replication, transcription, and translation take place entirely in the cytoplasm within discrete juxtanuclear sites called virus factories (moss, ) , (fig. d , iii). these viruses encode all enzymes and specific factors necessary for genomic replication and transcription. with their own replicative machinery, large-genome dna viruses can replicate in nondividing cells. in contrast, the replication of small-genome dna viruses, which depends on cellular dna polymerases, must occur simultaneously with the s-phase of the cell cycle (e.g. parvoviruses), or must express some viral protein/ oncogene to re-program the host cell-cycle regulatory proteins p or retinoblastoma protein (prb), triggering entry into the s-phase (e.g. polyomaviruses and papillomaviruses). by affecting the g /s checkpoint (controlled by p and prb), the viruses ensure the production of host enzymes required for viral replication (fields and knipe, ) . production of viral proteins of dna viruses with intermediate and large size genomes is divided into early and late phases. in the early (prereplicative) phase, nonstructural proteins required for dna replication are translated. the late phase, during which the late structural proteins needed for assembly are translated, begins after viral dna replication ( fig. d , i). depending on the species, viruses assemble either in contact with the cellular membrane or independent of the membrane in either the nucleus or cytoplasm. the membrane-independent route is used by nonenveloped viruses and a few enveloped viruses (e.g. orthomyxoviruses, herpesviruses, and some retroviruses) that acquire the membrane envelope after intracellular assembly during budding from the cell. the limiting step for nuclear assembly is the size of npcs, which are large enough to transport rna and import proteins required for assembly into the nucleus; however, npc size limits the transport of larger assemblies. therefore, some viruses form assembly intermediates in the nucleus. these structures are then exported to the cytoplasm, where they come together to form viral particles. nuclear export is specific and depends on the presence of nuclear export signals (nes) in the transported proteins. one example of a nonenveloped icosahedral virus that assembles in the nucleus is adenovirus, the assembly of which has been studied intensely due to its potential use in gene therapies [reviewed in (ahi and mittal, ) ]. recent data suggest that upon accumulation of multiple copies of adenoviral dsdna genomes, coordinated assembly and packaging occur by two interlinked mechanisms that involve both capsid proteins and core components (condezo and san martín, ) . the assembly occurs in the so-called peripheral replicative zone with the assistance of scaffolding proteins that facilitate formation of adenoviral particles but are excluded from mature viruses. adenoviruses are finally released upon lysis of the infected host cell. orthomyxoviruses and herpesviruses are enveloped viruses that assemble their nucleoproteins in the nucleus. herpesviruses package their dsdna genome as head-to-tail concatemers and assemble icosahedral procapsids on scaffold proteins in the nucleus [reviewed in (heming et al., ) ]. however, subsequent steps of herpesvirus assembly proceed in the cytoplasm. the preassembled procapsid is too large to pass through the npc, but it exits the nucleus by viral proteindriven vesicular transport across the nuclear inner membrane leaflet. thus, the herpesvirus acquires an initial envelope from the inner nuclear membrane [for review see (fields and knipe, ; hellberg et al., ) ]. next, the herpesviral membrane fuses with the outer nuclear membrane, and the naked particle is released from the nucleus into the cytosol. here, the virus acquires tegument (a protein layer between the capsid and the envelope) and other proteins. final herpesvirus envelopment occurs at the golgi membrane containing the viral glycoproteins (fields and knipe, ) . preassembled orthomyxoviral ribonucleoproteins, upon their export from the nucleus, are driven to the plasma membrane to which they attach through electrostatic interactions of the matrix protein m with membrane phosphatidylserine. the virions then assemble simultaneously with budding during which they also acquire ha, na and m membrane proteins. poxviruses undergo an even more complicated pathway. they are enveloped with multiple membranes acquired from er/intermediate compartments and golgi or early endosomes (moss, (moss, , . these membranes also provide the poxviruses with their membrane proteins. most viruses assemble upon interaction of their structural proteins with cellular membranes. the target membrane for assembly differs according to the virus type. flaviviruses assemble at the surface of the er and then upon their budding into the er lumen, the immature particles are then transported into the tgn. some viruses, such as coronaviruses, assemble at the er-golgi intermediate compartment [reviewed in (ujike and taguchi, ) ]. the assembly of bunyaviruses occurs concurrently with replication of grna segments in virus factories located along the golgi (guu et al., ; strandin et al., ) . the presence of membrane glycoproteins at the golgi membrane determines the sites where assembling bunyavirus particles bud into the golgi lumen, similar to other enveloped viruses. numerous viruses, including paramyxoviruses (cox and plemper, ) , orthomyxoviruses (pohl et al., ) , alphaviruses (jose et al., ) , rhabdoviruses (okumura and harty, ) , and most retroviruses (freed, ) , assemble underneath the cytoplasmic membrane, which facilitates assembly by providing a scaffolding function. the virus then acquires a lipid envelope through budding across the plasma membrane. during this process, it also gains the envelope glycoproteins (env) that are anchored in the plasma membrane by hydrophobic transmembrane domains. env reaches the plasma membrane by a cellular secretory pathway upon synthesis in the er and subsequent glycosylation in the golgi. usually, a specific interaction between viral structural proteins and glycoproteins is required. this may be either direct or mediated through the interaction with matrix protein (e.g. in (-)rna viruses such as ortho-and paramyxoviruses or rhabdoviruses). most retroviruses, including hiv (so-called morphogenetic c-type), also assemble at the plasma membrane of the host cell. the interaction of the structural polyprotein precursor (gag) with the plasma membrane is usually facilitated by a bipartite signal in the n-terminal domain of gag (i.e. matrix protein) comprising both the basic patch and n-terminally linked myristoyl residue (added co-translationally to gag). in contrast to c-type assembly at the membrane, morphogenetic b/d-type retroviruses assemble at pericentriolar sites where gag polyproteins are transported along microtubules by dynein molecular motors (sfakianos et al., ; vlach et al., ) . for both morphogenetic pathways, the packaging of grna facilitates assembly. (+)rna viruses have adopted a mechanism of extensive rearrangement of intracellular membranes to provide a milieu for virus replication and assembly. this mechanism effectively protects dsrna intermediates from degradation by the host cell machinery (delgui and colombo, ; jackson, ) . this process has been well-documented for poliovirus, in which the newly formed membranous structures exclusively serve for virus production. various types of vesicles that are formed upon viral infection have different roles in viral replication (rossignol et al., ) . some nonenveloped mammalian viruses, such as reoviruses, assemble in the cytosol in so-called viroplasms or viral factories into icosahedral particles consisting of three concentric layers encircling segments of genomic dsdna (benavente and martinez-costas, ; jones, ; shah et al., ) . rotavirus seems to be the only exemption in the reovirus family, as it enters the endoplasmic reticulum to gain its outer protein shell (trask et al., ) . numerous viruses assemble from polyprotein precursors that must be specifically cleaved by a viral protease to generate infectious particles. this mechanism, which is an irreversible step in the virus life cycle, ensures equimolar packaging of structural proteins and proportional co-assembly of viral enzymes in the form of precursors. upon proteolytic release, the liberated proteins may undergo different trafficking pathways or fulfill various functions in the virus. maturation changes the energy of interaction forces among those interfaces required for intracellular assembly of immature particles to those suitable for viral stability in the environment and for disassembly and uncoating of genetic material for replication [reviewed in (veesler and johnson, ) ]. in poliovirus, the autocatalytic and subsequent viral proteasemediated cleavage of p precursor protein allows formation of pentamers that interact with grna. additional cleavage of vp , yielding vp and vp , is required to form infectious poliovirus particles. in retroviruses, proteolytic processing is initiated by the autocatalytic liberation of viral protease, which subsequently cleaves the polyproteins to trigger major structural rearrangements in the virus and release of other viral enzymes (reverse transcriptase and integrase) and structural proteins. in herpesviruses, maturation involves proteolytic processing of the scaffolding protein and recruitment of tegument proteins that stabilize the particle and mediate interactions with the membrane during envelopment (gibson, ; tandon and mocarski, ; tandon et al., ) . adenoviruses undergo a maturation process that involves processing of six structural components of the core and one non-structural precursor that initiates replication of gdna (gaba et al., ) . one interesting feature of adenoviral maturation is that dna is required as a co-factor for protease activity. this means that maturation occurs only in particles that have packaged gdna (mangel and san martín, ) . flaviviruses form icosahedral particles upon budding into the neutral milieu of the er. the particles then translocate to the more acidic environment of the trans-golgi network. this ph shift results in disassembly of the immature lattice and extensive rearrangement of the flaviviral particle. this is connected with the exposure of a viral structural glycoprotein (prm) that is specifically processed by the , retroviruses (c) and dna viruses (d) (the schemes a-c were modified from: (ahlquist, ) . a: following endocytosis, the genome of (+)rna viruses may directly serve as mrna for translation of virus encoded proteins. among them, there are proteins of rna-replication machinery that recruit (+)rna into a membrane-associated replication complex. the genomic rna is replicated by using (-)rna template, which is transcribed in a low copy number amplified (+)rna is then packaged into newly assembled virions that exit the host cell either through secretion or cell lysis. b: upon the virus attachment, a core containing both grna and virus encoded rna polymerase is delivered into the cytoplasm by endocytosis. cytoplasmic transcription of the (-)rna template provides (+)rna that serves as mrna for translation of viral proteins. in dsrna viruses, the (+)rna is packaged into new cores which undergo maturation by synthesizing (-) rna (dotted strand) and acquiring surface proteins. in the (-)rna viruses, the (+)rna strand is transcribed into genomic (-)rna in the cytoplasm and then packaged. the new virions egress the cell either through secretion or cell lysis. c: following fusion of viral and cell plasma membrane, the retroviral core is released into cytosol, rna genome is transcribed into dsdna by viral reverse transcriptase concomitantly with uncoating of the viral core, ds dna is transferred into nucleus where it is integrated into host cell genome by viral integrase, following translation of retroviral structural and enzymatic polyproteins, the unspliced grna is packaged into the immature particles that usually assemble at the plasma membrane and the viral particles bud from the cell. d: three mechanisms (i.-iii.) of dna viruses replication are shown: (i): following entry and uncoating, the dna genome is transported to the nucleus; products of early genes (regulatory proteins, transcription factors) regulate the synthesis of viral enzymes (e.g. dna polymerase) required for genome replication; expression of late genes encoding structural capsid proteins in the cytosol, they are then transported into nucleus where packaging and pre-assembly take place; preassembled procapsids exit the nucleus and leave the cell (e.g. herpesviruses). (ii) unique replication cycle of hepatitis b virus (hbv): following entry, the viral particle is internalized by endocytosis and the nucleocapsid is released into the cytoplasm; the genome (relaxed circular rcdna) is transported into the nucleus, where it is converted to a covalently closed circular form (cccdna); which serves as a template for transcription of pregenomic rna (pgrna) for translation of the core protein and the viral polymerase and three subgenomic mrnas used for translation of regulatory and envelope proteins; following viral transcription and translation, the hbv core proteins self-assemble in the cytoplasm into viral nucleocapsid with concurrent incorporation of pgrna and hbv polymerase, pgrna is reversely transcribed into a rcdna within the nucleocapsid; nucleocapsid containing rcdna can either re-enter the nucleus to amplify cccdna, or can be enveloped by hbv envelope proteins in the endoplasmic reticulum. the particles are then secreted from the infected hepatocyte by exocytosis. (iii) upon entering the cell, the replication, transcription and translation take place entirely in the cytoplasm, within discrete juxtanuclear sites called virus factories (e.g. poxviruses) adapted from: ahlquist, p., . parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses. nat rev microbiol ( ), - . cellular protease furin in the trans-golgi network. the liberated globular heads of prm remain attached at the low ph, but are released when the virus enters neutral body fluids (rey et al., ) . one frequently used mechanism of release of nonenveloped and some enveloped viruses is lysis of the infected cell. this is the simplest release mechanism, although the transition to lysis from latent infection is delicately regulated (aneja and yuan, ; levings and roth, ; schmiedel et al., ) . however, viruses that are usually lysogenic may also use alternative release pathways (bird and kirkegaard, a) . these include non-lytic spread of viruses mediated by vesicles, which has been observed for poliovirus (bird and kirkegaard, b; jackson et al., ) , coxsackievirus (alirezaei et al., ) , and hepatitis a virus . another possibility is that vesicles released from a cell infected with (+)rna viruses contain naked viral grna. this (+)grna-containing vesicle functions itself as an infectious agent as it is transferred to another cell (bird and kirkegaard, a) . the standard way for enveloped viruses to leave the host cell is budding, which includes membrane extrusion and separation of the viral and cellular membranes (so-called pinching off). the lipid envelope layer acquired during viral particle budding through the plasma membrane protects the virus particle. there are three basic mechanisms of budding: i) mediated by envelope glycoproteins, such as the e protein of coronaviruses; ii) independent of glycoproteins, in which the viral structural proteins trigger the extrusion of the plasma membrane, such as retroviral budding in which strong interactions between the gag n-terminal domain (matrix protein) and the plasma membrane induce bulging of the membrane; and iii) requiring interaction between the viral glycoproteins and the capsid for membrane extrusion, as in alphaviruses. the final step that results in the separation of virus from the cell surface is pinching off the particle. this is a controlled process that involves both viral protein domains and cellular factors. during this process, viruses apparently use cellular machinery similar to that used during the last step of cell division (the release of the daughter cell) called endosomal sorting complex required for transport (escrt) [reviewed in (campsteijn et al., ; hurley, ; olmos and carlton, ; scourfield and martin-serrano, ) ]. direct interactions of numerous viral domains with escrt complex subunits have been identified (bieniasz, ) . in retroviruses, short specific amino acid sequences (ptap or psap) interact with the escrt components, and the interaction of hiv with the escrt proteins nedd and alix is wellknown (freed, ; fujii et al., ; gomez and hope, ) . however, this mechanism has also been adopted by other viruses that interact with the same escrt components, such as filoviruses (han et al., ; jasenosky and kawaoka, ; liu and harty, ) . interactions with escrt proteins have also been reported for vesicular stomatitis virus luyet et al., ), rhabdovirus (taylor et al., , arenaviruses (ziegler et al., ) , picornaviruses , paramyxoviruses (park et al., ) , and hepatitis c virus, a representative of the flavivirus family (falcon et al., ) . the typical release pathways used by viruses may vary under some conditions. for example, during chronic infection, numerous viruses use alternative cell-to-cell transmission that may help the virus avoid host neutralization (hulo et al., ) . syncytium formation, an hivinduced cell fusion, was recognized in the early s (callahan, ) . another type of cell-to-cell transmission through tight junctions was shown for hiv (hübner et al., ; wang et al., ) and murine leukemia virus (sherer et al., ) . receptors on tight junctions that specifically recognize hepatitis c virus (carloni et al., ; ploss et al., ) and reovirus (barton et al., ) have been identified. some viruses are able to modify cellular pathways to reprogram both the synthesis and metabolism of lipids and membrane compartmentalization for their transmission and to evade cellular defense mechanisms (mazzon and mercer, ) . despite a general understanding that poliovirus spreads through cellular lysis, it was recently found that it may also be transferred between cells by an autophagy-dependent mechanism, called autophagosome-mediated exit without lysis (bird and kirkegaard, b; bird et al., ; lai et al., ; richards and jackson, ) . similar mechanisms have been described for varicellazoster virus and human cytomegalovirus (grose et al., ; meier and grose, ) . poxviruses encode several proteins that block the apoptotic cellular response to the presence of their dsdna in the cytoplasm. this allows cell-to-cell passage of poxviruses by a mechanism known as apoptotic mimicry (amara and mercer, ; nichols et al., ) . in this process, enveloped viruses expose surface phosphatidylserine to mimic apoptotic bodies. these cells are then macropinocytosed by either dendritic cells or macrophages (mercer and helenius, ) . among the plethora of compounds designed to inhibit infectious viruses, only a few (< ) have been approved for clinical use (de clercq, ; de clercq and li, ) . nevertheless, some effective antiviral drugs have been on the market for several decades, such as the anti-influenza a virus drug amantadine, marketed under the trade name symmetrel (by dupont), which was approved in . in , burroughs-wellcome introduced acyclovir as an inhibitor of herpesviruses. its remarkable specificity is connected with its activation by viral thymidine kinase-catalyzed phosphorylation. however, due to development of drug resistance in a number of viruses, especially rna viruses, there is a continuous need to design and test new inhibitors, preferably targeting different steps of viral life cycles. here, we provide insights into the world of biochemical and cell-based assays that were developed to test antivirotics targeting various steps in the viral life cycle. different types of assays, including cell-cell fusion assays, cell-virus fusion assays with pseudotyped viral particles, and in vitro biochemical assays have been developed to screen inhibitors of viral entry. in enveloped viruses, entry is initiated by fusion of the viral envelope with the target cellular membrane. the entry mechanism has been well-described for hiv- , in which it is mediated by env glycoprotein, consisting of transmembrane gp and surface gp subunits. binding of gp to its cellular receptor, cd , and to one of two co-receptors, cxcr or ccr , triggers a refolding of gp that promotes fusion of the viral and cellular membranes. the refolding involves oligomerization of the extracellular n-and c-terminal heptad repeat (hr) domains of gp , which leads to the formation of a -helical bundle [reviewed in (melikyan, ) ]. jiang et al. established an in vitro system to quantify formation of the hiv- gp -helical bundle (jiang et al., ). in their system, the bundle is formed by mixing equimolar concentrations of peptides derived from the n-and c-hr regions of gp . elisa using the monoclonal antibody nc- , which specifically recognizes and binds an epitope formed on the gp -helix bundle but not the individual peptides, enables screening for compounds that prevent formation of fusion-active gp . for hts of hiv- fusion inhibitors, this method was modified to use a fluorescence-linked immunosorbent assay (flisa), in which the c-hr peptide was replaced with fitc-conjugated c-hr peptide (boyer-chatenet et al., ) . cell-based assays are routinely used to screen viral entry inhibitors. high throughput formats have been developed for screening of hiv- fusion inhibitors. cell-cell fusion assays involve two types of cells: effector cells that stably (bradley et al., ) or inducibly (herschhorn et al., ; ji et al., ) express hiv-env glycoprotein and target cells that express cd and either cxcr or ccr . co-cultivation of these cells leads to hiv- env-mediated cell membrane fusion, resulting in formation of multinucleated syncytia. membrane fusion induces expression of a reporter protein such as luciferase (herschhorn et al., ; ji et al., ) or β-galactosidase (bradley et al., ) . one such assay enables determination of both the efficiency and specificity of fusion inhibitors (herschhorn et al., ) . this approach uses effector cells that express both hiv- env and the renilla luciferase (r-luc) reporter protein using inducible tetracycline-controlled transactivator (tta) and target cells that express the hiv- receptor (cd ) and coreceptor (ccr ) and contain the firefly luciferase (f-luc) reporter gene under the control of a tta-responsive promoter. upon fusion of the effector and target cells, tta enters the target cells and activates the expression of the f-luc reporter. the inhibition of fusion of cellular membranes is determined as a decrease in f-luc luminescence, and the inhibitor specificity is measured as the r-luc activity (herschhorn et al., ) . based on an hiv- cell-cell fusion method that uses a computercontrolled digital image analysis system for automatic quantitation , a modified method was developed to screen inhibitors targeting mers-cov s protein (lu et al., ) . to test potential fusion inhibitors, effector cells stably expressing the mers-cov spike protein s s and egfp were used to mediate fusion with target cells expressing the dpp receptor (lu et al., ) . virion-based fusion assays are another category of cell-based fusion assays. one such approach is based on production of chimeric hiv- virions carrying β-lactamase-vpr chimeric protein (blam-vpr). chimeric hiv released into the cell culture media is isolated by ultracentrifugation and used to infect target cells. entry of the virions into the cytoplasm is detected by cleavage of a fluorescent substrate by βlactamase. the fluorescence shift corresponds to the fusion efficacy and is measurable by fluorescence microscopy, flow cytometry, or fluorometric plate reader (cavrois et al., ; cavrois et al., cavrois et al., , . modification of this assay by constructing pseudotyped hiv- virions in which hiv- env was replaced with ebola virus glycoprotein (gp) has also been described (yonezawa et al., ) . tscherne et al. developed an approach to monitor viral entry using the blam reporter (tscherne et al., ) . their assay uses influenza virus-like particles (vlps) bearing the influenza membrane proteins hemagglutinin and neuraminidase. blam tagged with influenza matrix protein (m ) is incorporated into the vlps and delivered into target cells. upon release, blam can be detected by flow cytometry, microscopically, or fluorometrically. a rapid cell-based hts method was developed to assess sars-cov entry inhibitors (zhou et al., ) . this dual envelope pseudovirion (dep) assay employs two hiv pseudoviruses: one encodes an envelope protein from the target virus and firefly luciferase and the second encodes a control, unrelated viral envelope protein and renilla luciferase. reporter expression is determined with the dual-glo luciferase assay system (promega). inclusion of the unrelated envelope protein greatly reduced false positive hits (zhou et al., ) . the method was further used to screen compounds that inhibit entry of filoviruses, including ebola virus . a similar approach employing four pseudotyped hiv viruses, carrying marburg virus glycoprotein, hemagglutinin and neuraminidase isolated from a/goose/qinghai/ / (h n ) influenza virus, ebolavirus zaire envelope glycoprotein, and lassa virus envelope glycoprotein, has been used for entry inhibitor screening . this screening identified multiple compounds with potent inhibitory activity against entry of both marburg and ebola viruses in human cancer cell lines, and confirmed their anti-ebola activity in human primary cells . other pseudotyped viral assays have been used to screen entry inhibitors of sars-cov, ebola, hendra, and nipah viruses. to establish infection, the glycoproteins of these viruses must be processed by the host intracellular lysosomal protease cathepsin l (catl). hts resulted in identification of several inhibitors that block the cleavage of viral glycoprotein but not catl itself (elshabrawy et al., ) . until recently, the development of anti-hbv therapeutics had been limited by the lack of an in vitro infection system. several aspects of the hbv life cycle have been elucidated using in vitro production of hbv particles after transfection of human hepatoma cell lines (hepg ) with recombinant hbv dna and by establishment of hepatoma cell lines with the entire hbv genome integrated, such as the hepg . . cell line (ladner et al., ; sells et al., ; sureau et al., ) . in addition to differentiated human (phhs) (gripon et al., ) and tupaia belangeri (glebe et al., ) hepatocytes, the heparg cell line, which exhibits hepatocyte-like characteristics, also supports hbv replication (gripon et al., ) . the identification of sodium taurocholate cotransporting polypeptide (ntcp) as an hbv receptor by yan et al. opened possibilities to use ntcp-complemented hepg cells not only for studies of hbv replication mechanisms but also for hts of inhibitors (yan et al., ) . in an infection competition assay, hbv particles were isolated and used to infect heparg and phhs cells that had been pre-incubated with hbv envelope protein-derived peptides to test their potential activity to block hbv infection. twelve days after infection, viral rnas were quantified by northern blot (gripon et al., (gripon et al., , . using this assay, researchers identified a peptide that specifically prevents hbv and hdv entry into heparg and phhs cell lines (gripon et al., ) , primary cultures of tupaia hepatocytes (glebe et al., ) , and cells in vivo (lütgehetmann et al., ; petersen et al., ; volz et al., ) . recently, a phase iia clinical trial of a first-in-class entry inhibitor (myrcludex-b) that functions as an ntcp inhibitor was promisingly completed (bogomolov et al., ) . development of cell culture systems producing hcv pseudoparticles (hcvpp) and infectious hcv particles (hcvcc) has shed light on hcv interactions and enabled discovery of antiviral drugs and vaccines (colpitts et al., ) . hcvpp consist of hcv e and e glycoproteins enveloping a retroviral particle that packages gfp mrna during assembly (bartosch et al., ) . the entry efficiency of hcvpp can be quantified by facs analysis as the number of gfp-positive cells. use of this screening system led to discovery of a triazine derivative that blocks the entry of hcv (baldick et al., ) . production of hcvcc is a robust system to generate infectious hcv in naïve cells (kato et al., ; zhong et al., ) . the anti-hcv activity of hundreds of compounds approved for a wide variety of indications was determined immunochemically with anti-hcv e antibody in -well plate format. over thirty compounds displayed anti-hcv activities, most of which were directed against hcv entry (gastaminza et al., ) . the majority of viruses enter cells by endocytosis. unfortunately, there are no suitable experimental techniques for endosome handling, making it difficult to study early steps in the viral life cycle such as uncoating and capsid disassembly. viruses that enter cells by direct membrane fusion, such as hiv- , are an exception. there are several methods available to monitor and quantify hiv uncoating. as some of these were reviewed recently by campbell and hope (campbell and hope, ) , we will discuss them very briefly. two main techniques are used in these assays: ultracentrifugation or utilization of hiv- specific cellular restriction factors. the "in vitro core-stability assay" is based on ultracentrifugation of released hiv- virions through a detergent layer, where the viral membrane dissolves, into a sucrose gradient, where the viral cores are concentrated (shah and aiken, ) . the "fate of capsid" assay uses ultracentrifugation through a sucrose cushion to separate the hiv- core from a whole cell lysate prepared shortly after infection (stremlau et al., ) . the "csa-washout assay" involves specific cellular factors (trim-cyclophilin) that restrict hiv- infection by binding to the capsid core and cyclosporine a (csa), which can effectively turn off restriction (hulme et al., ) . recently, a novel entry/uncoating assay (eurt), an alternative to blam-vpr (described in section . ), was reported (da silva santos et al., ). it quantifies the protein product of a virion-packaged mrna reporter upon uncoating. a method to monitor the uncoating/disassembly of the capsid of influenza a virus, which enters the cell by endocytosis, also is based on ultracentrifugation (stauffer et al., ) . purified virions are separated using velocity gradient centrifugation through a two-layer glycerol gradient. similar to the "in vitro core stability" assay for hiv, the sedimenting viruses migrate through the detergent-containing layer of the gradient, which dissolves the membrane to release the viral core. moreover, modification of the detergent-containing glycerol layer-for example, by lowering ph, changing ionic strength, or adding putative viral uncoating factors-enables study of the factors or compounds that affect viral uncoating in vitro. depending on the virus type, either dna or rna polymerases replicate the viral genome. thus, these enzymes play a key role in viral life cycles. reverse transcriptase, an rna-dependent dna polymerase of retro-and hepadnaviruses, is unique among nucleic acid polymerases. despite the different mechanisms of viral replication, polymerases, which are essential for all viruses, are excellent targets for antiviral therapies. polymerase inhibitors represent the vast majority of clinically approved antiviral drugs, followed by protease inhibitors, immunostimulators, entry inhibitors, and integrase inhibitors [reviewed in (de clercq and li, ) ]. polymerases continue to be a preferred target for newly designed inhibitors (clercq, ; de clercq and li, ) . polymerase inhibitors may be nucleoside and nucleotide analogs, pyrophosphate analogs, or nonnucleosides, such as allosteric inhibitors (de clercq and li, ; Öberg, ) . non-specific approaches such as plaque assays were initially used to monitor the effectiveness of polymerase inhibitors (tino et al., ; tisdale et al., ) . the activity of these inhibitors also can be evaluated based on their ability to prevent the cytopathic effects of the virus (zhang et al., ) . more straightforward assays involve measurement of incorporated radio-labeled nucleotides, which directly reflects the polymerase activity (coates et al., ; hirashima et al., ; joyce, ) ; these include hts methods using homopolymeric polycytidylic acid and [ h]-gtp (amraiz et al., ) . alternatively, the pyrophosphate released during the polymerase reaction can be measured luminometrically by combining the primer extension with the commercial piper assay (malvezzi et al., ) . the pyrophosphate anion also can be detected with dna-attached magnetic nanoparticles (tong et al., ) or quantum dots (chai et al., ) . frequently used fluorescence methods avoid the use of radiochemicals. these methods exploit a fluorescent label, such as dsdna binding dyes like sybr green or pi-cogreen (driscoll et al., ; holden et al., ; zipper et al., ) , or fret between two nucleotides (schwartz and quake, ; weiss, ) . numerous kits for quantification of products of both dna and rna polymerases, including reverse transcriptase, are commercially available (bustin, ) . quantitative real-time pcr is a preferred method to monitor the activity of dna polymerases (cellular as well as viral) in the presence of inhibitors (beadle et al., ; zweitzig et al., ) , and quantitative real-time reverse-transcription pcr has become standard for screening inhibitors of viral rna polymerases okon et al., ; pelliccia et al., ; zhang et al., ) . white et al. described a hts method using a microfluidic apparatus combined with digital pcr for single-cell analysis (white et al., ) . in their method, a fluorescently labeled template also serves as a primer, due to stem formation. it emits a measurable polarization signal both upon binding of the polymerase and extension (mestas et al., ) . the assay has been used for hts of inhibitors of poliovirus rna polymerase (campagnola et al., ) . when available, viral genomes modified with reporter genes can be used for cell-based luminescent or fluorescent screening of viral inhibitors (beadle et al., ; edwards et al., ; fenaux et al., ; feng et al., ; lo et al., ; madhvi et al., ; wang et al., c) . this approach can be extended to screen inhibitors of other enzymes. in the aptamer-based approach, a dna template encodes rna of interest joined to a fluorescence module and ribozyme sequence. when transcribed, the fluorescence module is released and detected (höfer et al., ) . hcv uses an rna-dependent rna polymerase (rdrp). a unique cell-based assay for monitoring hcv rna polymerase (ns b) activity, based on the innate immune signaling molecule retionic acid-inducible gene i product (rig-i), has been developed (ranjith-kumar et al., ) . the rig-i-like receptors are cytosolic proteins that recognize viral rna and induce production of proinflammatory cytokines and interferon-activated genes (jensen and thomsen, ) . rig-i triggers cytokine production stimulated by various viral rnas from different families, including flaviviridae, paramyxoviridae, rhabdoviridae, orthomyxoviridae, and arenaviridae, as well as ebola virus rna (jensen and thomsen, ) . the assay is based on recognition of hcv rna produced by active ns b by rig-i, followed by rig-i-mediated activation of firefly luciferase expression controlled by the interferon b promoter. renilla luciferase expression is used for normalization of transfection efficacy. the viral grnas of some rna viruses are modified at the ′ ends with cap structures, which may be either acquired from the host cell mrna (e.g. in influenza virus) or newly synthesized (e.g. flaviviruses). the methylation of viral grna mimics that of cellular mrna cap structures to enhance the chances of the virus to escape from cellular defense mechanisms and also to increase the efficiency of translation of viral proteins. virus-specific methyltransferases are thus a promising therapeutic target. virus-encoded methyltransferases have been identified and characterized in flaviviruses such as zika virus coutard et al., ; duan et al., ; munjal et al., ; zhao et al., ) , west nile virus, and dengue virus (dong et al., ) ; rhabdoviruses such as vesicular stomatitis virus (vsv) (rahmeh et al., ); coronaviruses such as sars (wang et al., b) ; and roniviruses (zeng et al., ) . in flaviviruses, the n-terminal part of ns methyltransferase catalyzes cap formation via both guanine n- and ribose ′-oh methylations at the ′ end of grna, and the c-terminus of the enzyme is responsible for the rna polymerase activity (ray et al., ; zhao et al., ) . the c-terminal part of the sars nsp protein exhibits guanine n -methyltransferase activity, forming the grna cap, while the ′- ′ exoribonuclease activity of the n-terminus enhances the fidelity of viral replication (case et al., ; minskaia et al., ) . in vsv, the methyltransferase activity resides in the conserved region vi of the multifunctional large polymerase protein (li et al., ; ma et al., ) . some cellular methyltransferases regulate viral infections, as shown for herpes simplex virus, for which epigenetic control is involved in viral latency (cliffe and wilson, ) . inhibition of human histone methyltransferases such as histone-lysine n-methyltransferase (ezh / ) (arbuckle et al., ) or lysine-specific demethylase- (lsd ) induces antiviral signaling pathways (liang et al., ) . the inhibitory effect of histone demethylase activity has been demonstrated for human cytomegalovirus (gan et al., ) and herpes simplex virus (hill et al., ; liang et al., ) . the activity of purified recombinant methyltransferases can be determined by measuring the methylation by-product s-adenosyl homocysteine (sah) using commercially available kits. in one such assay, conversion of s-adenosyl methionine (sam) to sah is monitored luminometrically via luciferase reaction, in which measurable atp is generated through a sequence of reactions with mtase-glo™ reagent (promega). the epigeneous™ methyltransferase kit (cisbio bioassays) is based on competition of produced sah with fluorescently labeled sah (sah-d tracer) for binding to a terbium cryptate-labeled anti-sah antibody. the decrease in fret between the tracer and antibody is then evaluated. elisa using anti- -methylcytosine antibody can be used to quantify methylation of immobilized cytosine-rich dna substrate (e.g. epiquik™ dna methyltransferase activity/inhibition assay kit; epigentek). this method was modified with homogenous time resolved fret (degorce et al., ) to screen a library of inhibitors against sars-cov nsp . flaviviral and human cap n -mtases have been screened with radioactive assays using h-labeled sam and gpppac or m gpppac synthetic rnas. the h methyl transferred onto deae filter-bound rna can be measured by scintillation after multiple washings to remove unincorporated h-sam . alternatively, in vitro transcription can be carried out using h-sam. the radioactively labeled products are then resolved by thin-layer chromatography and developed using a phosphorimager (li et al., ) . a yeast cell-based method was established based on the finding that coronavirus methyltransferase can functionally replace a methyltransferase essential for yeast viability sun et al., ) . in this method, sun et al. constructed recombinant yeasts producing the viral methyltransferase instead of the yeast one (sun et al., ) . this strain was used for a hts of methyltransferase inhibitor activity that negatively correlated with the cell density after h incubation. virus-encoded atpase-driven helicases have been identified in numerous human pathogens. helicases from several (+)rna viruses have been characterized, including ns helicases from flaviviruses such as dengue virus, hcv, west nile virus, yellow fever virus, and japanese encephalitis virus (cao et al., ; gu and rice, ; jain et al., ; lin et al., ; nedjadi et al., ; wu et al., ) . sars and mers coronaviruses encode nsp with helicase activity (adedeji and lazarus, ; hao et al., ; seybert et al., ) . in semliki forest virus, a representative member of the togavirus family, helicase activity is encoded by the n-terminal domain of nsp protein. helicases are also common in some dna viruses, including poxviruses. these include the vaccinia virus helicase-primase d (bayliss and smith, ; hutin et al., ) , e protein of bovine papilloma virus (yang et al., ) , and the large tumor antigen of sv (stahl et al., ) . helicases appear to be attractive targets for antiviral drugs (briguglio et al., ; frick, ; reynolds et al., ) , but development of such compounds is challenged by cytotoxicity and bioavailability issues (kwong et al., ) . traditional methods for monitoring the activity of rna helicases use radioactively labeled dsrna substrates and follow the unwinding reaction by electrophoretic separation (nondenaturing page) of the ss reaction products, which are detected autoradiographically (adedeji et al., ; utama et al., ) . to determine whether the inhibitor affects binding of helicase to nucleic acid, a standard gel mobility shift assay is usually used (adedeji et al., ) . helicase activity is fueled by atp hydrolysis; thus, inhibition of atpase activity became another possible antiviral strategy. atpase activity can be determined either by the decrease in atp or formation of adp (using commercially available fluorescent anti-adp antibodies) or inorganic pyrophosphate. phosphates released by atp hydrolysis can form a molybdophosphate complex that can be measured colorimetrically using malachite green, quinaldine red, or rhodamine b (baykov et al., ; debruyne, ; miyata et al., ) or by lightscattering (oshima et al., ) . the colorimetric methods can be miniaturized for hts (zuck et al., ) . the absorbance-based assay was also converted into a hts method based on fluorescence quenching by a colored quinaldine red complex ). an alternative method employs a europium-tetracycline complex for luminescent determination of inorganic phosphate (schäferling and wolfbeis, ) . a more complex coupled enzyme colorimetric assay (with maltose phosphorylase, glucose oxidase, and horseradish peroxidase) was used for hts of atpase inhibitors (avila et al., ) . assays for luminescent and fluorescent screening of atpase activity, including an immunochemical method using fluorescently labeled anti-adp antibodies, have been reviewed by shadrick and colleagues (shadrick et al., ) . in a radioactive method using [γ- p]atp, the amp product was separated from unreacted atp by thin-layer chromatography on polyethyleneimine-cellulose and visualized autoradiographically (adedeji et al., ) . several research groups have described fluorescence assays to identify inhibitors targeting sars-cov helicase (nsp ) (adedeji et al., ; cho et al., ; Özeş et al., ) . the substrate is usually a dsdna oligonucleotide consisting of a fluorescently labeled strand annealed to the complementary strand carrying a quencher. this approach was also adapted for real-time determination of the rna helicase activity of hcv (tani et al., ) . in this assay, an ssdna capture strand complementary to the strand carrying the quencher was used to prevent reannealing of the unwound duplex. recently, a colorimetric assay for monitoring helicase activity using dna-conjugated gold nanoparticles was developed (deka et al., ) . this method is based on shifts in the optical properties of nanoparticles due to dna unwinding and allows simple screening of inhibitor activity. the dsdna melting curves can be determined spectrophotometrically (at nm and nm) or even by the naked eye. another fluorescence hts of dengue virus ns helicase inhibitors measures the unwinding of a double-labeled molecular beacon (basavannacharya and vasudevan, ) . other approaches involve graphene oxide-based fluorescence monitoring of viral helicase activity [reviewed in (jang et al., ) ]. a gquadruplex-based method for label-free determination of hcv helicase ns activity measures changes in the luminescence of transition metal complexes with dna upon helicase-mediated quadruplex melting (leung et al., ) . both protein tyrosine kinases and protein serine/threonine (s/t) kinases have been found in viruses. tyrosine kinase function is wellunderstood in connection with oncogenic retroviruses, [for review on retroviral oncogenes, see (vogt, ) ]. in contrast to tyrosine kinases from oncogenic viruses, such as rous sarcoma virus src tyrosine kinase, viral s/t kinases share little homology with cellular enzymes. they are exclusively encoded by large dna viruses (e.g. herpesviruses), in which they play important roles in viral virulence, helping the virus to escape defense mechanisms such as those regulated by cytokine signaling pathways (jacob et al., ; sato et al., ) . their autophosphorylation and transphosphorylation activities mimic those of cellular cyclin-dependent kinases such as cdc . for example, viral kinases can phosphorylate translation elongation factor delta (ef- δ) (jacob et al., ; kawaguchi and kato, ) . all herpesviruses encode the s/t protein kinase ul , and us s/t kinases have been described in the alphaherpesvirus subfamily (kato, ; kawaguchi and kato, ) . in addition to protein kinases, hsv encodes a thymidine kinase. unlike cellular thymidine kinase, hsv thymidine kinase has a wide substrate specificity that includes pyrimidine and purine phosphonate analogs (e.g. acyclovir, ganciclovir, penciclovir) (de clercq and li, ) . in the body, ganciclovir is phosphorylated by cellular kinases and penciclovir and acyclovir by virus-encoded thymidine kinases to the active nucleoside triphosphate forms of the drugs that inhibit viral dna synthesis (de clercq and li, ; kokoris and black, ) . some cellular protein kinases appear to support viral replication. for example, polo-like kinases induce early stages in the influenza virus life cycle (pohl et al., ) , and human protein kinase c regulates the assembly of the ribonucleoprotein complexes in influenza virus (mondal et al., ) . inhibitors of abelson tyrosine-protein kinase are active against sars and mers coronaviruses (coleman et al., ) . several in vitro approaches can be used to determine kinase activity as well as the activity of kinase inhibitors. analyses of the cellular phosphoproteome, sometimes accompanied by phosphopeptide enrichment, have become standard to determine kinase activity (lea and simeonov, ; meyer et al., ; vyse et al., ) . these assays can be used to assess the impact of inhibitors on the overall phosphoproteome in mammalian cells. although these methods provide complex information about the overall array of kinases and phosphatases in the cell (olsen et al., ) , they are not applicable to screening inhibitors of a single viral kinase. for these purposes, in vitro assays with recombinant kinases have been developed [for review see ], including some hts fluorescence methods (zegzouti et al., ) that can replace radioactive techniques using [γ p]atp (sanghera et al., ) . mass spectrometric analysis also can be used to identify in vitro kinase inhibitors. for these analyses, synthetic peptides, proteins, or phosphatase-and heat-treated tissue samples (to dephosphorylate the proteins and inactivate all enzymes, respectively) are subjected to kinase treatment in the presence of inhibitors (huang et al., ; meyer et al., ; xue et al., ) . recombinant kinase activity in the presence of inhibitors can be quantified as atp consumption or adp production in the phosphorylation reaction by numerous commercial kits. other methods monitor binding of inhibitors to phage-displayed kinases in ligand competition assays (fabian et al., ) . fluorescence methods including fret, fluorescence polarization or intensity endpoint measurement, and lifetime imaging of fluorescence including fluorescence biosensors (zhang and allen, ) have been reviewed elsewhere. sulfonamido-oxine labeled peptides can be used as chromophores that bind mg + upon phosphorylation and emit chelation-enhanced fluorescence (devkota et al., ; luković et al., ) . kinase-catalyzed phosphorylation of fluorescent peptides promotes their binding to metal-coated nanoparticles, which decreases their mobility and enhances measurable fluorescence polarization (lea and simeonov, ; sportsman et al., ) . tbiii complexes, in which phosphotyrosine induces fluorescence emission (wang et al., a) , may be used to evaluate protein tyrosine kinase activity (akiba et al., ; sumaoka et al., ) . fluorescence polarization methods also can be useful for drug screening [reviewed in (hall et al., ) ]. other alternatives are immunochemical methods that use antibodies specific to the phosphorylated amino acids, such as phosphotyrosine (li et al., ; youngren et al., ) or phosphoserine/ phosphothreonine. these antibodies can be used to detect protein/ peptide phosphorylation by western blotting, elisa, or immunoprecipitation of phosphorylated proteins for further mass spectrometry-based analysis (grønborg et al., ; zhang et al., ) . an elegant approach that limits the false-positive hits in screening of specific kinase inhibitors is based on an in situ proximity ligation assay using both an antibody against the target protein and an anti-phosphotyrosine antibody (leuchowius et al., ) . both antibodies are coupled with oligonucleotides, which when brought together due to antibody binding, can be enzymatically ligated and replicated through rolling circle amplification to form a long linear tandem repeat of sequences detectable by a complementary fluorescent oligonucleotide. in addition to protein kinases, some lipid kinases have been targeted by antivirals. one example is sphingosine kinase , which affects replication of dengue virus (aloia et al., ) . its activity can be determined by measuring the production of p-labeled sphingosine- phosphate from sphingosine and [γ p]atp (clarke et al., ; pitman et al., ) . retroviral integrase inhibitors are a new type approved new type of inhibitors imposed by the emergence of drug-resistant mutants. hiv integrase activities, integrase inhibitors, and drug resistance have been discussed in detail elsewhere (andrake and skalka, ; anstett et al., ; hajimahdi and zarghi, ; liao et al., ; podany et al., ; thierry et al., ) . methods to assess the two major activities of integrase-end processing of the reverse transcription product and its joining to target chromosomal dna-have been reviewed in detail by several groups (engelman and cherepanov, ; marchand et al., ; merkel et al., ) . initial methods used radioactively labeled dna oligonucleotides comprising the terminal cis-acting sequences of linear viral dna required for integration. the joining of the processed strand to the other strand (self-integration) or to supplemented target dnas can be analyzed by page (katz et al., ; katzman et al., ) . a less time-consuming, non-radioactive method involves timeresolved fluorescence anisotropy measurement using a -meric oligonucleotide fluorescently labeled on the terminal gt dinucleotide. this assay monitors the binding of integrase to the substrate as well as the subsequent ′-processing reaction, which both change the anisotropy (guiot et al., ) . alternatively, the yields of both the processing and joining reactions can be measured upon separation of the radioactively labeled product from the rest of the dna molecule using adsorption to pei-cellulose (muller et al., ) . a real-time hts method measures fluorescence emission resulting from removal of the ′-terminal dinucleotide, labeled with a quencher, by integrase (he et al., ) . han et al. described a fluorescence method to screen molecules that inhibit binding of integrase to viral dna . methods evaluating the integrase strand transfer reaction have been modified to a high-throughput format using magnetic beads (he et al., ) or streptavidin-coated microplates (john et al., ) . a method to assess strand transfer by time-resolved fret with a europium-streptavidin-labeled substrate has been optimized for -and -well plate formats (wang et al., ) . the hbv capsid protein is the building block of the viral core, surrounding the viral nucleic acid (pre-genomic rna, pgrna) and reverse transcriptase. the hbv core is icosahedral, formed by copies of capsid protein dimers. in vitro hts of hbv core assembly inhibitors using a modified hbv capsid protein has been described (stray et al., ) . the capsid protein was modified by deleting the nucleic acid binding domain, which is dispensable for capsid assembly, and the nterminal assembly domain alone was used in the assay. to fluorescently label the hbv capsid protein, all cysteine residues dispensable for assembly were replaced with alanines. a unique cysteine residue (c ) was c-terminally joined to the assembly domain and labeled with fluorescent bodipy-fl dye (c bo). during assembly, capsid proteins dimerize, bringing c bo residues close together and resulting in c bo fluorescence self-quenching. following incubation of the labeled hbv protein with inhibitors, fluorescence was measured in black -well microtiter plates. development of in vitro assembly systems has contributed greatly to current understanding of the structure of retroviral particles and mechanisms of virion formation. these systems also became the base for several high throughput assays for screening assembly inhibitors. during the last years, a number of in vitro assembly assays have been established, mainly for hiv- (campbell et al., ; campbell and rein, ; ehrlich et al., ; gross et al., ; lanman et al., ) , mason-pfizer monkey virus (bohmova et al., ; klikova et al., ; rumlova-klikova et al., ; rumlova-klikova et al., ; ulbrich et al., ) , rous sarcoma virus vogt, , ; purdy et al., purdy et al., , , and murine leukemia virus (dolezal et al., ; hadravova et al., ; cheslock et al., ) . hts assays for inhibitors of hiv- assembly include several methods using purified hiv- ca or ca-nc proteins. one of them, the turbidimetric assay, is based on the observation that direct dilution of the hiv- capsid protein (ca) into a high-salt solution ( . - . m nacl) leads to the formation of tubular structures. as the tube formation is accompanied by an increase in light scattering, assembly can be detected as an increase in turbidity, and the rate of turbidity change is proportional to the rate of the assembly (lanman et al., ) . other method published by lemke et al., exploits the affinity of nucleocapsid (nc) to a short tg-rich deoxyribooligonucleotide, d(tg ), which is used as a scaffold (lemke et al., ) . this arrangement enables ca to assemble at much lower protein and salt concentrations than in the turbidimetric assay (lanman et al., ) . biotin-labeled d(tg ) bound on the surface of neutravidin-coated microtiter well plates nucleates assembly of complexes of ca-nc and soluble fluorescein-labeled d(tg ). fluorescence is measured after washing to remove the unbound and unassembled material from the captured assembly products (lemke et al., ) . similarly, the faith assay uses a dually labeled oligonucleotide (tqon). however, in this case, the ssdna oligonucleotide tqon is labeled with the reporter dye fluorescein (fam) as well as black hole quencher (bhq); thus, it does not emit any fluorescence. the assembly reaction is triggered by mixing hiv- ca-nc or a gagtruncated assembly-competent version with tqon. following incubation, during which tqon is incorporated into the particles, exonuclease is added to degrade unbound tqon, while co-assembled tqon is protected from cleavage. degradation of free unbound tqon with exoi results in separation of fam from its quencher, and the emitted fluorescence is measured (hadravova et al., ) . phage display has been employed to screen peptide inhibitors of hiv- assembly (sticht et al., ) . a commercial library of m -derived phages presenting random -amino-acid peptides was analyzed for specific binding to purified ca or ca-nc proteins. the specifically bound phages were sequenced, and corresponding peptides were chemically synthesized and re-tested in an in vitro assembly assay (gross et al., ) . late in the retroviral life cycle, grna is incorporated into the nascent particle during assembly at the plasma membrane. nc contains two zinc-finger domains that are responsible for specific binding of grna. to screen for compounds that would prevent nc-rna/dna interactions, a hts system consisting of two sequential screens was developed (breuer et al., ) . the primary screen uses fluorescence polarization (fp), while the secondary one uses differential scanning fluorimetry (dsf). the combination and order of these two techniques were selected to first identify compounds that disrupt interactions between dna and nc, and then identify the compounds binding to nc during the secondary screen. a rapid and simple turbidimetric method was developed to screen inhibitors of assembly of hcv core protein (fromentin et al., ) . for the in vitro assembly reaction, two components, an n-terminal part of the hcv core protein corresponding to the minimal assembly competent domain and rna corresponding to the full-length ′utr of hcv, were used. assembly of hcv nucleocapsid-like particles was initiated by mixing the purified protein and nucleic acid, and the assembly process was monitored by measuring turbidity at nm. numerous viruses, including picornaviruses, retroviruses, alphaviruses, and flaviviruses, encode proteases that are essential for their virulence. the majority of viral proteases specifically cleave viral polyprotein precursors to liberate the functional proteins of the virion. some viral proteases, such as the papain-like proteases of coronaviruses, also reprogram cellular signaling pathways, including ubiquitination mechanisms and interferon controlled responses, to prevent degradation of viral components (clementz et al., ; frieman et al., ; randow and lehner, ; xing et al., ) . numerous viruses, including papillomaviruses (bronnimann et al., ; buck et al., ) and retroviruses (hallenberger et al., ) , use also host cell proteases, mainly furin, to trim their envelope and surface proteins. this triggers conformational changes required for interaction with cellular receptors and membrane fusion in enveloped viruses. some viruses use proteases other than furin to modulate their infectivity, as shown for viruses entering airway epithelial cells [reviewed in (laporte and naesens, ) ] such as influenza virus (böttcher-friebertshäuser et al., ; kühn et al., ) , newcastle disease virus (gotoh et al., ) , and respiratory syncytial virus (sugrue et al., ) . extensive research efforts have yielded detailed information about hiv- protease and its inhibitors [reviewed in (de clercq, ; konvalinka et al., ; midde et al., ) ]. large amounts of data also are available for hcv (foote et al., ; pawlotsky et al., ; razonable, ) and sars-cov cl protease inhibitors (pillaiyar et al., ) , although there is not yet an inhibitor of the latter target approved for clinical use. numerous approved drugs are synthetic peptides derived from the natural proteolytic substrates of target viruses modified to improve the in vivo effects related to bioavailability, stability, and so on. numerous in vitro assays to monitor the activity of proteases and their inhibitors, including commercial kits, have been developed. classical methods use synthetic peptides that mimic the target sites of the protease. although some of the methods described here were not originally designed to screen the activity of viral proteases in the presence of inhibitors, they can be adapted for this purpose by simply changing the peptide sequence to the target site of the protease of interest. the cleavage yield is usually monitored either colorimetrically (ding and yang, ; zhou et al., ) or as a change in fluorescence triggered by the release of fluorescent labels such as -amido- -methylcoumarin (amc) or rhodamine (grant et al., ) . fluorogenic substrates have been used to determine the activity of coronavirus proteases and screen inhibitors (kuo et al., ; lee et al., ; park et al., ; song et al., ; tomar et al., ; wang et al., ; yang et al., ; zhao et al., ) . some recently described arrangements employ nanoparticles (feltrup and singh, ; khalilzadeh et al., ; udukala et al., ; wang et al., ; zeng et al., ) or quantum dot bioconjugates (lee and kim, ; li et al., ; medintz et al., ) with immobilized fluorescently or luminescently labeled peptide substrates. alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (hu et al., ; joshi et al., ; lathia et al., ; rumlová et al., ) , analytical hplc (teruya et al., ) , or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (gemene and meyerhoff, ; han et al., ) . to study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, x-ray or nmr structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of hiv- [reviewed in (ghosh et al., ) ], hcv (yilmaz et al., ) , and mers . cell-based assays can provide additional information, including the capability of the inhibitor to pass through the cell membrane and its stability in the cytoplasm. a general determination of infectivity, such as a plaque cytotoxicity assay in the presence of protease inhibitor, may be used for confirmation. one elegant example exploits the cytotoxicity of hiv protease. cells are transfected with a protease precursor fused to gfp. in the absence of inhibitor, hiv- protease is autocatalytically activated and cleaves a broad variety of cellular proteins, resulting in activation of apoptosis and cell death (cummins and badley, ; rumlova et al., ) . this toxic effect, when suppressed by active inhibitors, results in production of a gfp signal in surviving cells (lindsten et al., ) . another elegant approach for hiv and coxsackievirus b proteases, which both undergo autocatalytic cleavage, employs constructs in which the protease gene is inserted between sequences encoding the dna-binding domain and the domain that activates transcription of the gal -lacz reporter gene (dasmahapatra et al., ; murray et al., ) . the protease-mediated cleavage separates the dna-binding domain from the trans-activating domain and results in failure of reporter gene transcription. this approach has also been modified with gfp as a reporter gene (hilton and wolkowicz, ) . co-expression of cleavage-activated luciferase substrate and mers-cov protease permits both live-cell imaging and quantification of the enzyme activity (kilianski et al., ) . the need to develop new antiviral compounds will likely persist over the long term, although there has been enormous progress in molecular biology methods, especially rna silencing, bioinformatics, imaging, and structural biology techniques. viruses present challenging targets for drug development due their flexibility and adaptability caused by the error-prone copying of their genomes, which can result in emergence of drug-resistant mutants. viral integration into the host genome and inhibitor toxicity are other obstacles. here, we provide an overview of in vitro methods, including cell-based assays, that may be suitable for screening of antivirotics that interfere with the key steps of viral life cycles and target either virus or cell-encoded proteins required for the infectivity. biochemical characterization of middle east respiratory syndrome coronavirus helicase severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase components of adenovirus genome packaging click conjugation of a binuclear terbium(iii) complex for real-time detection of tyrosine phosphorylation pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo investigation of sphingosine kinase in interferon responses during dengue virus infection viral apoptotic mimicry hiv- uncoating: connection to nuclear entry and regulation by host proteins development of robust in vitro rnadependent rna polymerase assay as a possible platform for antiviral drug testing against dengue retroviral integrase: then and now reactivation and lytic replication of kaposi's sarcoma-associated herpesvirus: an update hiv drug resistance against strand transfer integrase inhibitors toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay inhibitors of the histone methyltransferases ezh / induce a potent antiviral state and suppress infection by diverse viral pathogens highthroughput screening for hsp atpase inhibitors a novel small molecule inhibitor of hepatitis c virus entry junction adhesion molecule is a receptor for reovirus infectious hepatitis c virus pseudoparticles containing functional e -e envelope protein complexes suramin inhibits helicase activity of ns protein of dengue virus in a fluorescence-based high throughput assay format a malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay vaccinia virion protein i r has both dna and rna helicase activities: implications for vaccinia virus transcription -phosphonomethoxy)ethyl]guanine (ode-bn-pmeg), a potent inhibitor of transient hpv dna amplification hepatitis b virus replication early steps in avian reovirus morphogenesis late budding domains and host proteins in enveloped virus release escape of non-enveloped virus from intact cells nonlytic spread of naked viruses nonlytic viral spread enhanced by autophagy components viral entry pathways: the example of common cold viruses herpes virus replication treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study effect of dimerizing domains and basic residues on in vitro and in vivo assembly of mason-pfizer monkey virus and human immunodeficiency virus cleavage of influenza virus hemagglutinin by airway proteases tmprss and hat differs in subcellular localization and susceptibility to protease inhibitors development and automation of a -well cell fusion assay to identify inhibitors of ccr /cd -mediated hiv virus entry identification of hiv- inhibitors targeting the nucleocapsid protein inhibition of rna helicases of ssrna(+) virus belonging to flaviviridae, coronaviridae and picornaviridae families furin cleavage of l during papillomavirus infection: minimal dependence on cyclophilins maturation of papillomavirus capsids absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays hiv- virion-cell interactions: an electrostatic model of pathogenicity and syncytium formation high-throughput screening identification of poliovirus rna-dependent rna polymerase inhibitors hiv- capsid: the multifaceted key player in hiv- infection in vitro assembly properties of human immunodeficiency virus type gag protein lacking the p domain self-assembly in-vitro of purified ca-nc proteins from rous-sarcoma virus and human-immunodeficiency-virus type- in vitro assembly of virus-like particles with rous sarcoma virus gag deletion mutants: identification of the p domain as a morphological determinant in the formation of spherical particles modulation of hiv-like particle assembly in vitro by inositol phosphates novel escrt functions in cell biology: spiraling out of control? molecular mechanism of divalentmetal-induced activation of ns helicase and insights into zika virus inhibitor design hcv infection by cell-to-cell transmission: choice or necessity? mutagenesis of s-adenosyl-l-methionine-binding residues in coronavirus nsp n -methyltransferase m demonstrates differing requirements for genome translation and resistance to innate immunity components and regulation of nuclear transport processes a sensitive and specific enzyme-based assay detecting hiv- virion fusion in primary t lymphocytes fluorescence resonance energy transfer-based hiv- virion fusion assay hiv- fusion assay a reversible fluorescence nanoswitch based on carbon quantum dots nanoassembly for detection of pyrophosphate ion functional screen reveals sars coronavirus nonstructural protein nsp as a novel cap n methyltransferase tsg : a novel anti-hiv- drug target protein-protein interfaces in viral capsids are structurally unique identification of a coumarin-based antihistamine as an anti-filoviral entry inhibitor charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination identification of a novel small molecule inhibitor against sars coronavirus helicase reduction in sphingosine kinase influences the susceptibility to dengue virus infection by altering antiviral responses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases emerging antiviral drugs restarting lytic gene transcription at the onset of herpes simplex virus reactivation (-)- '-deoxy- '-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type and type replication in vitro pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope how viruses access the nucleus abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion structures of ns methyltransferase from zika virus highthroughput approaches to unravel hepatitis c virus-host interactions localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center endocytosis of viruses and bacteria zika virus methyltransferase: structure and functions for drug design perspectives structure and organization of paramyxovirus particles mechanisms of hiv-associated lymphocyte apoptosis: a novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized hiv- infection a genetic system for studying the activity of a proteolytic enzyme antiviral drugs in current clinical use approved antiviral drugs over the past years inorganic phosphate determination: colorimetric assay based on the formation of a rhodamine b-phosphomolybdate complex htrf: a technology tailored for drug discovery -a review of theoretical aspects and recent applications dna-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors a novel mechanism underlying the innate immune response induction upon viral-dependent replication of host cell mrna: a mistake of + srna viruses' replicases. front high-throughput screens for eef- kinase quantitative serine protease assays based on formation of copper(ii)-oligopeptide complexes functional and structural characterization of novel type of linker connecting capsid and nucleocapsid protein domains in murine leukemia virus ′-o methylation of internal adenosine by flavivirus ns methyltransferase a quantitative fluorescence-based steadystate assay of dna polymerase the crystal structure of zika virus ns reveals conserved drug targets high-throughput minigenome system for identifying small-molecule inhibitors of ebola virus replication assembly of recombinant human immunodeficiency virus type capsid protein in vitro identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay retroviral integrase structure and dna recombination mechanism a small molecule-kinase interaction map for clinical kinase inhibitors ultrastructural and biochemical basis for hepatitis c virus morphogenesis nuclear entry of dna viruses development of a fluorescence internal quenching correction factor to correct bont/a endopeptidase kinetics using snaptide antiviral nucleotide incorporation by recombinant human mitochondrial rna polymerase is predictive of increased in vivo mitochondrial toxicity risk a pathogenic picornavirus acquires an envelope by hijacking cellular membranes inhibition of hepatitis c virus replication by gs- , a potent c-nucleoside monophosphate prodrug transcription and replication mechanisms of bunyaviridae and arenaviridae l proteins fields virology boceprevir: a protease inhibitor for the treatment of chronic hepatitis c viral late domains hiv- assembly, release and maturation helicases as antiviral drug targets severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf and nf-κb signaling a method for in vitro assembly of hepatitis c virus core protein and for screening of inhibitors beyond tsg : the role of alix in 'escrting' hiv- proteolytic cleavage of bovine m adenovirus -encoded pviii epigenetically repressing human cytomegalovirus lytic infection and reactivation from latency in thp- model by targeting h k and h k histone demethylases unbiased probing of the entire hepatitis c virus life cycle identifies clinical compounds that target multiple aspects of the infection detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode recent progress in the development of hiv- protease inhibitors for the treatment of hiv/aids structure and formation of the cytomegalovirus virion pre-s antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis b virus mapping of the hepatitis b virus attachment site by use of infection-inhibiting pres lipopeptides and tupaia hepatocytes the ins and outs of hiv replication mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/pace to pc or pc / pc development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates hepatitis b virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide infection of a human hepatoma cell line by hepatitis b virus efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein a mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, frigg, as a protein kinase a substrate varicella-zoster virus infectious cycle: er stress, autophagic flux, and amphisome-mediated trafficking a conformational switch controlling hiv- morphogenesis the cell biology of receptor-mediated virus entry the spring α-helix coordinates multiple modes of hcv (hepatitis c virus) ns helicase action relationship between the oligomeric status of hiv- integrase on dna and enzymatic activity bunyavirus: structure and replication in vitro assembly of virus-like particles of a gammaretrovirus, the murine leukemia virus xmrv faith -fast assembly inhibitor test for hiv progress in hiv- integrase inhibitors: a review of their chemical structure diversity fluorescence polarization assays in high-throughput screening and drug discovery: a review inhibition of furin-mediated cleavage activation of hiv- glycoprotein gpl selective monitoring of peptidase activities with synthetic polypeptide substrates and polyion-sensitive membrane electrode detection development of a fluorescence-based hiv- integrase dna binding assay for identification of novel hiv- integrase inhibitors alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp : a role for alix in ebola virus egress crystal structure of middle east respiratory syndrome coronavirus helicase highthroughput real-time assay based on molecular beacons for hiv- integrase '-processing reaction a novel highthroughput format assay for hiv- integrase strand transfer reaction using magnetic beads nuclear egress of herpesviruses: the prototypic vesicular nucleocytoplasmic transport herpesvirus capsid assembly and dna packaging an inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of hiv- entry inhibitors inhibition of lsd reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes an assay to monitor hiv- protease activity for the identification of novel inhibitors in t-cells benzimidazole derivatives bearing substituted biphenyls as hepatitis c virus ns b rna-dependent rna polymerase inhibitors: structure − -activity relationship studies and identification of a potent and highly selective inhibitor jtk- universal aptamer-based real-time monitoring of enzymatic rna synthesis factors affecting quantification of total dna by uv spectroscopy and picogreen fluorescence peptide code-on-a-microplate for protease activity analysis via maldi-tof mass spectrometric quantitation a systematic ms-based approach for identifying in vitro substrates of pka and pkg in rat uteri quantitative d video microscopy of hiv transfer across t cell virological synapses complementary assays reveal a relationship between hiv- uncoating and reverse transcription the ins and outs of eukaryotic viruses: knowledge base and ontology of a viral infection escrts are everywhere transport of the influenza virus genome from nucleus to nucleus domain organization of vaccinia virus helicase-primase d how viruses use the endoplasmic reticulum for entry, replication, and assembly experimental approaches to study genome packaging of influenza a viruses poliovirus-induced changes in cellular membranes throughout infection subversion of cellular autophagosomal machinery by rna viruses viral serine/threonine protein kinases structure of the ns helicase from zika virus a new helicase assay based on graphene oxide for anti-viral drug development filovirus budding oligomeric viral proteins: small in size, large in presence sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion development of a novel dual ccr -dependent and cxcr -dependent cell-cell fusion assay system with inducible gp expression a screening assay for antiviral compounds targeted to the hiv- gp core structure using a conformation-specific monoclonal antibody development and application of a highthroughput screening assay for hiv- integrase enzyme activities avian reovirus infections. revue scientifique et technique a structural and functional perspective of m. rumlová alphavirus replication and assembly the rational design of therapeutic peptides for aminopeptidase n using a substrate-based approach techniques used to study the dna polymerase reaction pathway molecular mechanism by which us protein kinase regulates the pathogenicity of herpes simplex virus type- cell culture and infection system for hepatitis c virus the avian retroviral in protein is both necessary and sufficient for integrative recombination in vitro the avian retroviral integration protein cleaves the terminal sequences of linear viral dna at the in vivo sites of integration protein kinases conserved in herpesviruses potentially share a function mimicking the cellular protein kinase cdc reduced graphene oxide decorated with gold nanoparticle as signal amplification element on ultra-sensitive electrochemiluminescence determination of caspase- activity and apoptosis using peptide based biosensor assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and c-like proteases using luciferase-based biosensors clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers efficient in-vivo and in-vitro assembly of retroviral capsids from gag precursor proteins expressed in bacteria virus strategies for passing the nuclear envelope barrier characterization of herpes simplex virus type thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity retroviral proteases and their roles in virion maturation origin and evolution of eukaryotic large nucleo-cytoplasmic dna viruses adenovirus tales: from the cell surface to the nuclear pore complex the proteolytic activation of (h n ) influenza a virus hemagglutinin is facilitated by different type ii transmembrane serine proteases characterization of sars main protease and inhibitor assay using a fluorogenic substrate viral and cellular rna helicases as antiviral targets inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication the autophagic machinery in enterovirus infection kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type capsid protein assembly in vitro airway proteases: an emerging drug target for influenza and other respiratory virus infections multiplexed protease assays using element-tagged substrates fluorescence polarization assays in small molecule screening fluorescent and bioluminescent nanoprobes for in vitro and in vivo detection of matrix metalloproteinase activity identification of novel drug scaffolds for inhibition of sars-cov -chymotrypsin-like protease using virtual and high-throughput screenings characterization of the activity of ′-c-methylcytidine against dengue virus replication distinct effects of two hiv- capsid assembly inhibitor families that bind the same site within the n-terminal domain of the viral ca protein high content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation label-free luminescence switch-on detection of hepatitis c virus ns helicase activity using a g-quadruplex-selective probe †electronic supplementary information (esi) available: compound characterisation and supplementary data immunity to bovine herpesvirus : i. viral lifecycle and innate immunity small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity vesicular stomatitis viruses resistant to the methylase inhibitor sinefungin upregulate rna synthesis and reveal mutations that affect mrna cap methylation fluorescence detection techniques for protein kinase assay quantum dots based molecular beacons for in vitro and in vivo detection of mmp- on tumor inhibition of the histone demethylase lsd blocks α-herpesvirus lytic replication and reactivation from latency a novel selective lsd /kdm a inhibitor epigenetically blocks herpes simplex virus lytic replication and reactivation from latency authentic hiv- integrase inhibitors single-molecule imaging reveals the translocation and dna looping dynamics of hepatitis c virus ns helicase cellbased fluorescence assay for human immunodeficiency virus type protease activity viral and host proteins that modulate filovirus budding rapid and automated fluorescencelinked immunosorbent assay for high-throughput screening of hiv- fusion inhibitors targeting gp gs- and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses. sci. rep. a structural view of the rna-dependent rna polymerases from the flavivirus genus automatic quantitation of hiv- mediated cell-tocell fusion with a digital image analysis system (dias): application for rapid screening of hiv- fusion inhibitors structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor hiv- uncoating is facilitated by dynein and kinesin recognition-domain focused (rdf) chemosensors: versatile and efficient reporters of protein kinase activity humanized 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ribose ′-o-methyltransferase encoded by the ronivirus branch of nidovirales fret-based biosensors for protein kinases: illuminating the kinome phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs cell-based high-throughput screening assay identifies ′, ′-difluoro- ′-deoxycytidine gemcitabine as a potential antipoliovirus agent structure of the main protease from a global infectious human coronavirus, hcov-hku molecular basis for specific viral rna recognition and ′-oribose methylation by the dengue virus nonstructural protein (ns ) structure and function of the zika virus full-length ns protein robust hepatitis c virus infection in vitro inhibitors of sars-cov entryidentification using an internally-controlled dual envelope pseudovirion assay a new colorimetric strategy for monitoring caspase activity by hrp-mimicking dnazyme-peptide conjugates protease inhibitors targeting coronavirus and filovirus entry the lymphocytic choriomeningitis virus matrix protein ppxy late domain drives the production of defective interfering particles investigations on dna intercalation and surface binding by sybr green i, its structure determination and methodological implications miniaturization of absorbance assays using the fluorescent properties of white microplates characterization of a novel dna polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes this work was supported by ga Čr (cz) ga - s to mr, ga - s to tr and the ministry of education, youth and sport of the czech republic(oppc cz. . / . . / ), through its "national program of sustainability i" npu lo . key: cord- - jthjf authors: gupta, akanksha; kumar, sanjay; kumar, ravinder; choudhary, ashish kumar; kumari, kamlesh; singh, prashant; kumar, vinod title: covid‐ : emergence of infectious diseases, nanotechnology aspects, challenges, and future perspectives date: - - journal: chemistryselect doi: . /slct. sha: doc_id: cord_uid: jthjf wuhan, a city of china, is the epicenter for the pandemic outbreak of coronavirus disease‐ (covid‐ ). it has become a severe public health challenge to the world and established a public health emergency of international worry. this infectious disease has pulled down the economy of almost all top developed nations. the coronaviruses (covs) known for various epidemics caused time to time. infectious diseases such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), followed by covid‐ , are all coronaviruses led outbreaks that scourged the history of mankind. covs evolved themselves to more infectious, transmissible, and more pandemic with time. to prevent the spread of the sars‐cov‐ , many countries have ordered the complete lockdown to combat the outbreak. this paper briefly discussed the historical background of covs and the evolution of human coronaviruses (hcovs), the case studies and the development of their antiviral medications. the viral infection encountered with present‐day challenges and futuristic approaches with the help of nanotechnology to minimize the spread of infectious viruses. the antiviral drugs and their clinical advances, along with herbal medicines for viral inhibition and immunity boosters, are described. elaboration of tables related to covs for the compilation of the literature has been adopted for the better understanding. in last two decades, entire world faced three major outbreaks of coronaviruses like severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and novel coronavirus disease i.e., covid- . the first case of recent outbreak of covid- was recognized at wuhan, hubei, china five months ago. later, it spread to the whole world and affects health as well economy globally in a very short span. although, the world is well equipped with technological advances and well aware of the complete structural details to deal with the outbreak of coronavirus (covs), still all are struggling to find out its cure. since ancient times, appearance or reappearance of several diseases have affected the human life significantly. [ ] from the early s to date, around viruses species have been evolved, and these keep on increases in the coming years ( figure ). [ ] historical background starting from the tobacco mosaic virus ( ), foot-mouth disease virus ( ), yellow fever virus ( ) followed by a deadly virus created influenza epidemic during world war-i and took around million lives. it is an all-time high in the mankind history [ ] and this virus probably one of the influenza virus species. [ ] coronavirus disease (covid) was first reported in , and the first coronavirus (hcov- e) isolated from humans in . these infect various birds, animals, and mammals, including humans. the isolation of the prototype murine coronavirus strain of coronavirus was reported in . [ ] the pathogenesis and replication mechanisms of various coronaviruses have been elaborated in details since the s. human coronaviruses (hcovs) were first identified in and caused acute upper respiratory infection (uri). [ ] covs affected the humans worldwide and are the pathogenic agents for both mammals and avian. these viruses can infect respiratory, hepatic, central nervous, and gastrointestinal systems of humans, bats, birds, mice, and some wild animals. [ ] these viruses are highly pathogenic to humans. previously, cov causes an epidemic of sars in humans and infected thousands viruses belong to family coronaviridae, which shows crown-like appearances under an electron microscope. the covs contain large, positively charged strands of rna associated with nucleocapsid phospholipid protein and the genetic material is surrounded by viral glycoproteins. the covs have three types of membrane proteins, which include the spike glycoprotein, membrane protein, and small hydrophobic membrane protein. in addition to these proteins, other proteins like hemagglutinin esterase (he) has been isolated from a different group of coronaviruses which are not present in the sars-cov genome. [ ] the spikes of coronavirus cause infection in host cells. the structure of spike is clove shaped and has trimeric s head and s stalk, respectively (figure b ). during infection, s (head) of virus spike binds to host cell receptor for viral attachment and s (stalk) allow viral genome to enter into host cell through fusion of viral and host membrane surface. [ ] apart from these four principle structural proteins, various coronavirus also encodes some special structural and accessory proteins like a/b protein, a/b protein and hemagglutininesterase (he) protein. [ ] the subgenomic rnas (sgrnas) of coronaviruses encodes all the structural and accessory proteins. [ ] the translation of coronavirus genomic rna is controlled by the replicase-transcriptase protein genes. these replicase-transcriptase proteins encoded by two open-reading frames (orf) namely, orf a and orf b, respectively. these two orfs are synthesized two large polyproteins like pp a and pp ab. the first orf is / rd of the entire genome length from the '-terminal and they encode a pp ab polyprotein. [ b] the replication-transcription complexes formation. [ ] interestingly, the nsps or viral replication processes are found to be potential antiviral drug targets which might be helpful in the development of drugs against coronavirus. [ ] these nsps regulate genome transcription and replication processes. [ b, ] for example, nsp has role in viral replication/transcription complexes formation whereas the viral rna replication and transcription are controlled by nsp . [ ] it is essential to understand the function of all nsps for the development of promising drugs/vaccines against the covid- pandemic. the function of nsps of coronaviruses are shown in the table that helps to interpret covs for effective design of drugs. human coronavirus is spreading rapidly among people via viral respiratory infections like cough and sneeze. coronavirus disease is most contagious and lethal. various stains of coronaviruses have been reported so far ( table ). the incubation periods and clinical symptoms of each coronaviruses are varying significantly. the symptoms and incubation periods of human coronaviruses play very important role in the identification and diagnosis of coronavirus diseases, shown in the table . the viral spike glycoprotein is dimer or trimer and has two functions: attaching the virus to receptor sites located on the host surface and activate the virion membrane to fuse with host membrane so that rna genome released inside the host cell. [ ] it may be noted that coronaviruses attack inside the cell membrane rather than acting on the surface of host cells. covs genome is non-segmental, and the two-third portion consists of overlapping orfs responsible for translating the polypro- promotes degradation of cellular mrna as well as blocks translation of host cell, obstructive distinctive immunity reaction, inhibit interferon (ifn) signals [ ] nsp some functions are not known, holds to prohibitin proteins [ ] nsp multi-domain large transmembrane protein, functions include: * n protein interact with ac and ub domains * cytokine expression promote due to adrp activity * viral polyprotein cleaved by plpro/deubiquitinase domain which blocks host innate immune response * unknown function of nab, sud ubl , g m, and y domains [ ] nsp potential transmembrane scaffold protein, role in (dmvs formation [ ] nsp chymotrypsin-like protease ( clpro), main protease (mpro), cleaves viral polypeptides, inhibit interferon (ifn) signals [ ] nsp restrict expansion of autophagosome, potential transmembrane scaffold protein, double-membrane vesicle (dmv) formation [ ] nsp formation of hexadecameric complex (cofactor) with nsp and nsp , role as a processivity clamp and primase for rna polymerase [ ] nsp forms hexadecameric complex (cofactor) with nsp and , may act as primase as well as processivity clamp for rna polymerase [ a,c, ] nsp rna binding protein, dimerization [ ] nsp stimulates -o-mt and exon activities, scaffold protein for nsp and nsp , [ ] nsp unknown functions [ , d] nsp primer and rna-dependent, rna polymerase [ ] nsp rna helicase, ' triphosphatase [ ] nsp exoribonuclease activity for viral genome proofreading, n -mtase activity adds ' cap to viral rnas, '- ' exoribonuclease, [ b, ] nsp nsp endoribonuclease, evasion of dsrna sensors [ ] nsp '-o-methyltransferase ( '-o-mtase); avoiding mda recognition, negative regulation of innate immunity [ a,c, ] fever, coughing, cold, sore throat, nasal congestion and rhinorrhea, diarrhea, asymptomatic, organ function damage, acute kidney and cardiac infection, liver dysfunction, pneumothorax - days but in some cases extended to days [ , ] teins. remaining orfs of genomes contained for structural proteins. depending upon the protein sequences covs are subcategorised into four genera (α-, β-, γ-, and δ-covs). [ ] the two genera αand βwere primarily associated with rodents and bats for their genes source, although birds are the key reservoir for γand δ-covs. so far, there are seven human coronaviruses (hcovs) reported in literature where two of the coronaviruses ( e and nl ) are α-covs, while remaining five coronaviruses namely, sars, mers, oc , hku , and sars-cov- are β-covs. [ ] however, past few years, studies are underway to explore these viruses and develop potential drugs to cure the people from the infection. [ b, , c, a, ] there are various natural hosts of coronaviruses have been reported so far. natural hosts are mainly mammals, birds, dog, man, pig, cat, mouse, bat, mouse, tree sparrow, chicken, etc. (table ). these hosts are very important in the identification of specific coronavirus strains. several coronaviruses are classified as species in the coronaviridae. [ a, ] a total of coronaviruses and their natural hosts are shown in table , which provides information about specific genera like α, β, δ and γ and acronym of each covs. woo et al. analyzed birds species in which only . % of species found positive for cov as mentioned in table . [ b] bats and birds are reservoirs of distinct types of covs. the evolution model of hcovs is shown in figure . [ ] according to the evolutionary model (figure ), the origin of first covs might be in the bat and transfer to birds or vice-versa. based on the figure , the bat coronavirus family transfer the virus to other bat species and then, further transfers to mammals, including humans. similarly, the bird covs transfer to another bird species and further transfer in birds and mammals like pigs and whale. [ d] the human coronavirus first emerged in and was obtained from human embryonic tracheal culture from adults suffering from the common cold by tyrell and bynoe. [ ] similar findings were carried out by hamre and procknow [ ] on common cold specimens and isolated a new category of virus known as e from wi- lung cell line culture. these two human respiratory viruses were sensitive to ethers and get multiplied in the presence of inhibitors of dna synthesis. [ ] later, mcintosh and his group at the nih, bethesda isolated six morphologically related viruses grown in organ culture, oc and oc . the term "coronaviruses" described in based on crown-like projections on the surface for the new generation of viruses. [ ] human and animal coronaviruses were classified under three main categories, category-i has e and similar type of other viruses, category-ii has oc and similar viruses, while in category-iii, avian infectious bronchitis viruses (ibvs) were included. [ ] both e and oc coronaviruses are impacting globally in the winter season, and incubation time is less than one weak. [ ] another epidemic was caused in emerged from guangdong province, china caused by sars-hcov. the virus spread in almost countries, and more than patients were reported to suffer from this viral infection. the sars-hcov was isolated from the himalayan palm civets and was believed to originated from a live animal market in guangdong province, china. [ ] the worldwide spread of the virus was accompanied by the incubation period of - days and the highest viral load on the th day. [ ] patients infected with this virus show myalgia, headache, fever, and respiratory difficulties. severe conditions present with lymphopenia, liver disfunction, diffused alveolar damage, an increased number of macrophages. [ e, ] the virus was re-emerged in late because of favourable condition available for amplification of virus and it was isolated from horseshoe bats. [ ] the fourth human coronavirus strain, hcov-nl was identified by hoek and co-workers [ ] in netherland and was isolated from seven months old baby in . it was observed that about . % of common respiratory problems in children due to hcov-nl . [ ] in again, fifth human coronavirus strain, hcov-hku was isolated by patrick and co-workers [ ] from years old man suffering from pneumonia and bronchiolitis in hong kong. the old person was returned from shenzhen, china. the virus causes acute asthmatic exacerbation and it was different from common pneumonia. [ ] in saudi arabia , mers-hcov ( th hcov) reported in an old aged patient who suffered from acute pneumonia and renal failure. mers-hcov sequenced and resemble with sars-cov group and placed in the same category with a new lineage. mers-hcov, unlike sars-cov, observed with severe symptoms along with more epidemic and sporadic nature. [ ] recently, emergence of th hcov reported in late december from wuhan of china. the pandemic outbreak of the novel coronavirus is most devastated to date resulting in . million infected and thousands deaths so far until this paper was written up to rd june . sars-cov- , which shares % nucleotides similarities with sars-hcov.. [ ] the novel coronavirus shows an incubation period of - days but in some cases extended to days and spread rapidly through inanimate surfaces (metal, plastic, and glass) where it persists up to days. the pandemic outspread can only be controlled through surface disinfestation procedures by applying ethanol ( - %), -propanol ( %), -propanol ( %), h o ( . %), and sodium hypochlorite ( . %). [ ] coronavirus enters into host cells through viral spike (s) glycoproteins. [ b, ] spike (s) contains two subunits, namely, s and s . the s subunit contains bind to host cell receptors via the receptor-binding domain. receptor binding is a crucial step to determine the host range for a coronavirus. some human coronavirus has receptors like angiotensin-converting enzyme (ace ) for sars-cov and hcov-nl , aminopeptidase n (apn) for hcov- e, -o-acetylated sialic acid hcov-hku , . unbroken arrows represent confirmed transmission between the two species in question, and broken arrows represent suspected transmission (copyright permission @ elsevier ltd. [ ] ). and hcov-oc , and dipeptidyl peptidase (dpp ) for mers-cov. [ ] in the case of novel covid- , the receptor-binding domain (rbd) of s protein has a strong affinity to angiotensinconverting enzyme (ace ) receptors of the bat and human. [ ] while s subunit comprises specialized fusion machinery that mediates fusion of viral and host cell membranes. [ a,b, , ] later, s is cleaved at s of fusion peptide by proteases activities of the host in all coronaviruses. [ ] after entry, viruses get uncoated and start cap-dependent translation of orf a from viral genomic rna to produce polyprotein pp a, and further translation of orf b encodes longer polyprotein pp ab. [ ] the pp a and pp ab produce - nonstructural proteins (nsps) through autoproteolytic cleavage. significantly, nsp encodes the rna-dependent rna polymerase (rdrp) activity, [ ] whereas nsp and nsp encode papain-like protease (plpro) and main protease (mpro) activities, respectively. [ ] the nsp , nsp , and nsp from dmvs or spherules via also induce the rearrangement of the cellular membrane. [ a, ] further, the coronavirus replication transcription complex (rtc) is anchored and assembled at dmvs. the entire life cycle, replication and pathogenicity of sars-cov- is shown in figure . [ a, ] hcovs have different symptoms for various host, and accordingly dissimilar disease caused as mentioned in table . dromedary camels (saudi arabia) were responsible for the spread of different hcovs in the middle east, including south korea ( ). [ ] several countries such as the united states of america, the united kingdom, saudi arabia, kenya, france, china, and other countries are profoundly affected by different types of hcovs before wuhan outbreak (table ). in , a novel β-cov mers was found in an old aged patient in jeddah, saudi arabia suffered from pneumonia, renal failure, and respiratory infection who linked to jordon. later on, doctors and nurses were examined for the cov, but they found negative, which proposed that it was not scattered easily. the mers-cov caused lower respiratory tract illness (rti) in almost all over the globe, predominantly in the middle east, and took presently, the entire world is fighting againt covid- pandemic which is caused by sars-cov- . this disease was first reported in the hubai, china, in december and spreaded throughout the globe in a very short time. [ , a] according to "who" as of rd june , the total number of confirmed covid- cases is approximately . million and thousand deaths throughout the countries in the world. at present, there is no effective drug available in the market to control the spread of covid- . the researchers are working for the development of therapeutic drugs to treat infections. in silico methods offer a way to methodically and rapidly yield additional repurposing candidates. three-dimensional structure for the main protease of sars-cov- in complex with n (pdb id: lu ) available on the rcsb protein databank is being use in insilico study. bioinformatics analysis is used to virtually screen the molecules to get the promising candidate against the main protease of sars-cov- . a recent study published, relied on this approach, using the predicted structure of all sars-cov- proteins based on their homology with other known coronavirus protein structures, and identified several compounds with potential antiviral activity. [ d, ] since the outbreak of sars-cov in and mers-cov during , evoked scientists to explore and elaborate the hcovs. [ b, b] complete information at molecular level of any disease causing agents (virus/bacteria) may helpful in the development of the medicine. there are many approved drugs to cure the patients suffering from different diseases. these durgs are approved based on the efficacy and safety (pharmacology, formulation and toxicity) of medicines to humans. subsequently, then many therapeutic and defensive agents have been discovered. more than patents have been filed to combat these viruses in various authorities worldwide, so far. [ ] the treatment regimen of this epidemic appearance hard as the world is eyeing for the finding of effective drugs and vaccines. the pioneer of the pathophysiology of covid- sacked the world to discover the drug/antiviral. [ a, d, ] many of the broad-spectrum antiviral drugs are in the trial stage at different levels to combat the effect of covid- by blocking the rna polymerase, rdrp. [ , ] some of the drugs are in trial stages such as quinine, chloroquine, lopinavir, ritonavir, alferon ldo, poly-iclc, streptokinase, arbidol and glucocorticoid alone or in a combination of hydroxychloroquine, ritonavir/ribavirin, asc /ritonavir, daunavir/lopinavir, interferon α- b/ribavirin, camrelizumab/thymosin, etc. [ , a, ] till date, none of the compounds is approved drugs for curing from covid- infection in satisfactory way. in most of the countries on the trial basis, hydroxychlroquine with azithromycin have been used to cure the infected people. further, it is reported that hydroxychlroquine is less effective when administered alone than in combination with the azithromycin. although, hydroxychlroquine and azithromycin have toxicity to heart and hydroxychloroquine showed toxic effect on eye. [ , ] a biological preparation provides active acquired immunity against particular infectious disease like covid- [ , ] shenzhen, china sars-cov, nl , hku isolation of sars-cov from civet cats, the study revealed that it might be originated from animals [ ] kenya hcov e, nl , oc , hku out of samples, . % hcov-nl , ∼ % oc , ∼ % hku , % e. covs are spreaded globally [ a] still, we are strugling to find out rapid detection kit, effective antiviral and drug to make potent theranostics against sars-cov- . the peripheral protein sequence adaptation and genome modification of virus lead to the resistance toward conventional therapies. with the advent of nanotechnology in recent years, nanomedicine have emerged to be an alternative for conventional therapeutics. [ ] professor chan emphasis on the development of nanotechnological tools as best weapon to deal with covid- and focussed on rapid point-of-care diagnostics, surveillance and monitoring, therapeutics and vaccine development. identification, treatment and prevention using nanotechnology tools would escalate the development to fight against covid- as frontline tools. [ ] kerry et al. explored the nano-based technique to combat nipah virus and a similar method may be explored to combat hcovs where nanosystems could be developed and used to treat viral diseases. [ , ] in the last few years, nanomaterials have also been investigated against various viral strains, and promising results were attained. a prominent approach, development of iron-based magnetic nanoparticles (imnps) as nanomedicine would help immensely to deal with covid- . [ ] the movement of imnps can be controlled by the external magnetic field and targeted to affected organs invaded by covid- , i. e., lung, throat, etc. without going to other organelles, as shown in figure . [ ] imnps alone could interact with the nanovirus, covid- [ ] as of similar size regime and destroy or inhibit it. additionally, in combination with photodynamic therapy, it deforms the structure of nanovirus and inhibit the growth. [ ] except magnetic nps, other nanomedicine can be utilized as antiviral agents such as ti-based nps known for influenza virus, [ ] carbon-based nps elaborated for ebola, respiratory syncytial virus, etc. [ ] inhibition of norovirus and pseudorabies virus through viral replications or by attacking histo-blood group antigens using carbon dots, [ ] graphene oxides based nanomaterials preventing viral spread (coronavirus, respiratory syncytial virus) on the damaging virus by interacting with negative charge present over it [ ] and similarly other nps such as se nps, solid lipid nps, ag nps, au nps, etc. are also active in fighting against viruses. [ ] huy et al. reported the electrochemical synthesis of agnps and explored their activity against non-enveloped viruses. the concentration of agnps up to ppm or less was not cytotoxic, while antiviral activities were found in . ppm for minutes of incubation with poliovirus. [ ] viruses inactivation were carried out with different nanoparticles such as tio nps, [ b] au nps, [ d] and silica nps. [ ] the composite of various nanomaterials also exhibits promising results that may comprise the delivery of target species through nanocarrier. the nucleic acid-based antiviral drugs face problems of cell penetration. the fragments of nucleic acid, therefore, introduced with the help of nanomaterials. reviews doi.org/ . /slct. low concentration. [ , ] cdte quantum dots (cdte qds) mixed with sandwiched complexes, nh -reporter, and biotin receptor and target dna accelerating the research for developing a sensor for human t-lymphotropic virus- (htlv- ) identification. [ ] au nps based materials are well known for the detection of various viruses such as dengue virus, influenza virus, bovine viral diarrhea coronavirus, etc. [ ] recently, seo et al. [ ] reported the field-effect transistor (fet) based biosensor for sars-cov- . the novel biosensor was fabricated by immobilizing the sars-cov- spike antibody on the graphene surface through -pyrenebutyric acid n-hydroxysuccinimide ester (pbase) as probe linker. the fet based biosensor exhibited a good response for clinical samples obtained from nasopharyngeal swabs and cultured sars-cov- with a limit of detection of fg/ml. further, thiol modified antisense oligonucleotides capped aunps showed better response and sensitivity against rna sequence of sars-cov- ( . ng/μl virus load) by modification in surface plasmon resonance behavior. [ ] till today, there is no single vaccine developed for effective treatment of deadly coronavirus disease covid- . on exposure to the deadly virus, the recovery rate of the patient mainly depends upon the strength of the immune system. according to who, % population of the developing country make use of herbal plants for successful treatment of various disease as well as for boosting up the immunity of individuals. the best preventive measures under practice to minimize the rate of infection includes primarily controlling the source of infection along with maintaining proper hygiene, avoiding crowded places, isolation, early diagnosis, and supportive treatments (boosting the immune system using herbal medicine). the herbal drugs are designed using specific parts of plants (roots, stem, bark, flowers, seeds, and fruits) to increase the resistance against the emerging and re-emerging viruses and bacteria (used in ayurveda, as mentioned in charaka samhita and susruta samhita). various plants produce beneficial ingredients such as oregano oil, fennel, garlic, peppermint, echinacea, sambucus, licorice, astragalus, ginger, tulsi (ocimum tenuiflorum), turmeric, etc. which contain different flavones, alkaloids, polyphenols, etc. these substances release interferons and antibodies which play an essential role to fight against various viral disease. it also increases the quantity of phagocytosis that protects the body from harmful particles. one of the herbal drugs 'fifatrol', a natural antibiotic, is a combination of vital phytoconstituents, immunomodulatory and antioxidants. it is advantagenous against viruses, allergens, and bacteria, thereby providing multi symptoms relief. the essential oil of garlic is a source of organosulfur compounds and allicin is a reactive sulfur species found in it. the organosulfur in the essential garlic oil inhibit the ace (host-receptor site of the virus) and main protease of the virus as well as to treat the infection due to sars-cov- . docking simulation and synergistic interactions show that the essential garlic oil has the ability to inhibit ace and resist cov- . [ ] this oil is also beneficial in treatment of antithrombotic, hypoglycemia, hypotension, and immunomodulation. natural products present in common vegetables are explored due to its tendency to effectively bind the active sites of covid- protease and hinder the viral replication. turmeric (curcuma longa), coriandrin, apple peels (ursolic acid), cucurbit vegetables (hederagenin), olive oil (oleanolic acid), rosemary, mint family plants (sageone), red pepper (apigenin), glycyrrhiza glabra (glabridin) are some of the natural compounds that have better insight into the mode of inhibition of viruses and have the potential for further research. [ ] further, anthocyanins represent a class of glycosylated polyphenol, that can act as a potential therapeutic antiviral compounds. it produces anthocyanidins whose nanoformulation are used in drug delivery systems and can reveal antiviral effects through a number of ways, i. e., by binding to host cells, inhibiting viral life cycle, or stimulating host immunity. [ ] arundina gramnifolia belongs to orchid family and produces gramniphenol (orange gum) and others phenolic compounds that can unveil anti-tobacco mosaic virus activity, in addition to anti-hiv- activity. [ ] codiaeum peltatum bark extract is used as an anti viral, attributed to its structural novelty and antiviral potential against zika, chikungunya and dengue. [ ] similarly, zingiber officinale (ginger) was used as an antiviral for the treatment of chikungunya virus. [ ] the anti-infectives is highly dependent on natural products and their structures. combinatorial chemistry techniques have emerged as a successful approach for optimizing structures and have been used effectively in the optimization of various drugs. [ ] few natural products that can act as starting materials for host-targeting antiviral drugs show a broad spectrum in treating antiviral problems. tannins that are normally found in plants acts as antimicrobial secondary metabolites. hydrolysable tannins i. e. chebulagic acid and punicalagin possess inhibitory mechanism against bacteria, viruses, and eukaryotic microorganisms. [ ] mycalamide is used for treatment of polio, hsv- influenza and covÀ a (mice survival days after a cov infection under . mg/kg of % mycalamide a) and griffithsin exhibits inhibitory effects against sars-cov virus. [ ] finally, there is a urgent need to develop natural antiviral medicines which can possess high chemical diversity and biochemical specificity to control coronavirus pandemics in future. * controlling and development of vaccines to prevent the outbreak of infectious diseases such as covid- , needs fast action. till date, no efficient vaccine/drug for its treatment is developed. clinically patients treatment is mainly supported by emphasizing on the strength of their immune systems. in case of respiratory failure, patients are kept in the intensive care unit. various nonspecific drugs such as hydroxychloroquine, ribavirin, lopinavir etc. are used for treating covid- ; however, their actual effectiveness is still not apparent. * since nanotechnology has improvised therapeutic advancements in recent years and is an advanced combating tool in drug designing as well as drug targeting. scientists could be encouraged towards the use of nanomaterials for targeting viral structures and depriving the impact of such novel viral infections. * the traditional diagnostic methods for viral disease sometimes are bulky, expensive and require skilled staff to operate, as it requires cell culture and sample preparation and thus, they are limited to centralized laboratories. but, the application of nanosensors, new tools offers massive advantages in the diagnosis of viral diseases; it requires only suitable nanoparticles. the development of nanosensors will be beneficial due to low cost, rapid detection tool and can help in the epidemics such as covid- . moreover, the existing toxicity evaluations of the nps, though, cannot eliminate health risks. * to encounter the pandemic situation in the current scenario is herculean. however, it could be possible by undergoing strong preventive major like social distancing, avoiding crowded places, wearing a good quality mask, maintaining personal hygiene like washing hands regularly, and early diagnosis of infection is of utmost crucial if symptoms of disease appear. * the herbal drugs designed using specific parts of plants (roots, stem, bark, flowers, seeds, and fruits) increase the resistance against the emerging and re-emerging viruses and bacteria (used in ayurveda, as mentioned in charaka samhita and susruta samhita). oregano oil, fennel, garlic, peppermint, echinacea, sambucus, licorice, astragalus, ginger, tulsi, turmeric, etc. have flavinoids, alkaloids, polyphenol's plays an essential role against various viral infections. these herbs release interferons and antibodies against viruses. it also increases the quantity of phagocytosis that protects the body by harmful particles. one of the herbal drugs 'fifatrol' natural antibiotic combination of vital phytoconstituents, immunomodulatory and antioxidants is helpful against viruses, allergens, and bacteria, thereby providing multi symptoms relief. [ ] * since covid- outbreak is a global concern, therefore, confronting similar future situations needs uncompromising regulations. zoonotic origin and genetically crossed species barriers need to be seriously encountered through the scientific database. since -ncov probably originated from bats and there are many future chances to get more viruses like this, which may prove more deadly and transmissible. therefore, scientists must take fast action to recognize the origin of this deadly virus, discover active and safe drug and therapeutics to prevent its escalation and to combat any upcoming pandemic. proc. natl. acad. sci coronaviruses with special emphasis on first insights concerning sars the coronaviridae the lancet proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci proc. natl. acad. sci drug discoveries ther proc. natl. acad. sci proc. natl. acad. sci b) v. surveillances, china cdc weekly proc. natl. acad. sci proc. natl. acad. sci. usa - ; b) b. fadeel, k. kostarelos, nat. nanotechnol. , , ; c) ; d) , ; b) prepr. doi . /preprints . . v the authors declare no conflict of interest.keywords: sars-cov- · antiviral agents · coronavirus · herbal · biosensors submitted: april , accepted: june , key: cord- - qj ri authors: roux, simon; matthijnssens, jelle; dutilh, bas e. title: metagenomics in virology date: - - journal: reference module in life sciences doi: . /b - - - - . - sha: doc_id: cord_uid: qj ri metagenomics, i.e., the sequencing and analysis of genomic information extracted directly from clinical or environmental samples, has become a fundamental tool to explore the viral world. against the background of an extensive viral diversity revealed by metagenomics across many environments, new sequence assembly approaches that reconstruct complete genome sequences from metagenomes have recently revealed surprisingly cosmopolitan viruses in specific ecological niches. metagenomics is also applied to clinical samples as a non-targeted diagnostic and surveillance tool. by enabling the study of these uncultivated viruses, metagenomics provides invaluable insights into the virus-host interactions, epidemiology, ecology, and evolution of viruses across all ecosystems. historically, viruses have been primarily explored using laboratory cultivation: new viruses were obtained from clinical or environmental samples through propagation and isolation on cell cultures. this process is, however, biased and challenging to apply at large scales because (i) many viruses depend on host cells that are difficult to maintain as clonal culture in the laboratory, and (ii) even if the cells are available, propagating viruses may require specific conditions distinct from those used to cultivate the cells. these considerations are especially meaningful for viruses with microbial hosts, the vast majority of which remain uncultivated to date. metagenomics bypasses this requirement for cultivation and instead relies on the sequencing of viral genomic material extracted directly from a sample (see box and fig. ). thus far, the history of viral metagenomics has seen two major phases. initially, entire communities of viruses were assayed by analyzing and comparing short sequencing reads obtained from diverse environments. because of the fragmented nature of these data, most of these studies had to be conducted at the community scale, and identifying and distinguishing individual viruses in these datasets remained challenging. more recently, bioinformatic advances have enabled the reconstruction of individual viral genome sequences from metagenomes, allowing naturally occurring viruses to be identified and studied at high, genomic resolution. using a metagenomics approach, entirely new types of viruses can now be discovered, surveyed, and characterized even without cultivation. the unique ability offered by metagenomics to study uncultivated viruses led to the emergence of two parallel and interconnected fields: a clinical one, where metagenomics promises to be a catch-all method for the unbiased surveillance and diagnosis of viral pathogens, and one focused on natural biomes. that aims to describe the diversity of the viral world and understand its ecological and evolutionary drivers and impacts. when the field of shotgun environmental metagenomics was pioneered in by the laboratory of forest rohwer at san diego state university, the first datasets consisted of three viral metagenomes (viromes) that, together comprised just under , short genomic fragments derived from two natural marine viral communities and one human feces sample. while limited in scope and resolution, these and other early viromes provided an unprecedented view of complex viral communities in nature. both oceanic and human fecal viromes pointed toward the existence of an extensive virus diversity. this diversity of the virosphere was estimated by comparing the sequencing reads within each metagenome, and observing that almost every fragment was unique. moreover, comparing the short sequencing reads to a reference database of known viral genomes sequences revealed that up to % was not similar to any known virus, suggesting that most of the virosphere was yet to be discovered. this uncharted genomic biodiversity became popularly known as "viral dark matter". in the years that followed, a broader range of environments was progressively surveyed using viromics including freshwater lakes, hot springs, agricultural soils, or human skin, saliva, and gut samples. improvements in dna sequencing technologies, especially the advent of the popular pyrosequencing platform, that has since been surpassed and discontinued, increased the scale of these datasets by providing hundreds of thousands of short genomic fragments for each sample. by directly comparing the sequences across these datasets, several studies indicated that virus genes tend to structure by environment rather than by sample location, implying that some of these genes may be globally distributed. in addition, when sampled from the same freshwater and hypersaline ponds across several days, weeks, and months, viral metagenomes revealed that the genetic composition of viral communities was coherent at a broad level, but some individual viral genes experienced rapid changes in relative abundance. while the analyses outlined above were foundational for our current understanding of virus diversity, they were limited by the short length of next-generation sequencing reads which fragmented the view of viral genomes. these limitations were progressively overcome through an increase in sequencing throughput associated with improvements in sequence assembly and analysis tools. the first large-scale assemblies of viral genomes from short metagenomic fragments were published around , and quickly became a standard analysis in the viral metagenomic field so that by the year b , complete or near-complete virus genomes were routinely reported and analyzed in viromics studies. in only a couple of years, metagenomics has thus transformed the way scientists can identify and study viruses in the environment, as illustrated by the quick rise of virus genomes and genome fragments assembled from metagenomes available in public databases (fig. ) . in , only viral genomes (fragments) assembled from metagenomes were publicly available, while this number reached , in , and , in . genome sequences of uncultivated viruses are frequently obtained not only from viral metagenomes, i.e., metagenomes from virus-targeted samples, but also from "bulk" metagenomes in which virus particle were not enriched and viral and microbial sequences are mixed. combined with genome sequences obtained from isolates, these "uncultivated virus genomes" represent the foundation of an extensive mapping of the viral sequence space. over the past few decades, a number of molecular techniques, such as (q)pcr or elisa, have been developed and used to detect pathogenic viruses in clinical samples. however, these techniques can only detect previously known viruses, and often require box use of complementary methods to target different types of viruses a number of approaches have been developed to specifically select and survey the genetic material contained by virus particles in a given sample. alternatively, viral genomes can also be analyzed from "bulk" metagenomes which include both virus particles and microbial cells. virus sequences obtained from "bulk" metagenomes will typically reflect viruses infecting their host cell at the time of sampling, either actively replicating or not, while viromes enables a deeper and more focused exploration of the virus diversity in a specific site or sample. regardless of the type of sample, viromes are most often generated through a combination of centrifugation, filtration and dnase/rnase treatment, aiming at removing as much of the cellular genomes as possible (fig. ) . a typical protocol will notably include a filtration through . , . , or . µm membrane filters to remove bacteria and larger cells. depending on the initial concentration of virus particles, a concentration step using e.g., iron chloride (fecl), polyethyleneglycol (peg), or tangential flow filtration step(s), may be necessary to obtain enough material for sequencing library preparation. cesium chloride density gradients ultracentrifugation can also be used to further separate viruses from extracellular dna and large particles in complex samples, although this step can also lead to a substantial loss of viral material. finally, the virus particles obtained are typically treated with dnase or rnase to remove free dna and/or rna. depending on the type of virus studied, the corresponding protocols for rna or dna extraction and sequencing library preparation are then applied, after releasing the genetic material from the virus particle through e.g., a heat shock if necessary. a critical step in this process is to recover enough material for sequencing. while micrograms of dna were initially needed, several protocols are now available which only require b ng of dna. in addition, a dna/rna random amplification step, called "whole genome amplification", can also be conducted in order to gather enough input material. this type of approach was initially used in almost every virome study, and revealed important information for example on the unsuspected diversity of ssdna genome viruses in the environment (see below). however, the whole genome amplification process is inherently biased, and these datasets are not quantitative, i.e., one cannot draw any conclusion about the relative abundance of the viruses identified in these amplified metagenomes. thus, whole genome amplification methods have now been often replaced by advanced library preparation protocols which require nanogram-scale input but enable quantitative datasets well suited for ecological studies. alternatively, for cases in which target viruses represent a minor part of the templates, targeted sequence capture approaches have been used, mainly in a clinical framework as they can only be applied to viruses with known genomes but can detect these viruses with a very high sensitivity. the recovery of virus genomes from bulk metagenomes and from viromes each have their own limitations. for bulk metagenomes, viruses typically represent only a minor fraction of all sequences compared to cellular genomes. this means that the virus genomes obtained this way will tend to be restricted to abundant viruses found in their host cells, while viruses that are not infecting at the time of sampling, viruses with a low frequency of infected hosts, or viruses infecting rare hosts will likely be missed. viromes provide a deeper description of the virus community, since most of the sequencing data will be obtained from virus genomes. in addition, virus particles will not represent only current infections but a more integrated sampling of all recent successful infections, the timing of which depending on the type of sample and the individual virus decay rate. yet viromes still suffer from several biases. notably, the size-based selection of virus particles excludes most of the larger viruses such as the "giant viruses", and viromes also tend to be dominated by viruses with high burst size while under-sampling viruses with low burst size and long infection time. all metagenomes (bulk and viromes) will struggle with very rare viruses, as well as hypervariable viruses which genome will not assemble well. hence complementary approaches such as targeted capture approach for the former, and long read sequencing for the latter, are being developed (fig. ) . overall, the different methods developed over the last decade to sequence genomes from uncultivated viruses are mostly complementary and can be individually tailored for specific applications. virus discovery can be achieved through bulk metagenomes or viromes, while viral ecology studies will tend to rely more on viromes as a reflection of virus activity and transport, and metagenomics used as a diagnostic tool in the clinic would be the most likely to use sequence capture. nevertheless, all these complementary approaches will be needed for achieving a comprehensive picture of viral diversity. specific assays for each pathogen. metagenomics instead offers the possibility to detect known and novel viruses without prior knowledge from a single analysis, and is thus well suited and already applied to study emerging and/or rare viruses, as well as cases which remain negative using the available diagnostic tests (see below). a number of challenges remain however for viral metagenomics to become a standard clinical procedure. first, given the current cost associated with sample processing and sequencing, metagenomes are still more expensive and slower than elisa's or qpcr assays. second, there is no generally validated bioinformatics pipelines than can perform a rapid, sensitive, and specific analysis of the obtained data on a bench top computer. finally, physicians will have to be trained and guided to deal with the obtained breadth of data. specifically, it is becoming clear that each individual is chronically infected with a dozen or more eukaryotic viruses (many of which have not been associated with any disease, e.g., anelloviruses), and that many known viral pathogens can also cause asymptomatic infections. therefore, a physician might get a list of viruses (and other potentially pathogenic or unknown organisms), and it will be a challenge to identify the actual cause of a particular disease. nevertheless, the price of sample preparation and high throughput sequencing has declined enormously in the last decade including with the development of smaller and faster machines, while automatic virome fig. overview of the viral metagenomics workflow. the overall process used to generate and analyze viral metagenomes can be divided into four major steps: (i) collection of environmental and/or clinical sample, (ii) sample preparation, (iii) library preparation, and (iv) sequence analysis. the sample preparation step can target either the cellular fraction (left) or the viral fraction (right) in which case viral particles are often further concentrated and purified to remove free nucleic acids. *targeted sequence capture can be applied to the extracted dna/rna to enrich for a specific virus. **while whole genome amplification was initially used routinely for viral metagenomes, it has now been supplanted by methods enabling the preparation of more quantitative libraries from low input (b ng), hence whole genome amplification is now primarily used in single-cell or single-virus-particle experiments. ***the genome assembly can be bypassed if using long-read sequencing technologies, although these long-read datasets require a more careful error correction. ****genome binning, i.e., the identification of multiple contigs assembled from a metagenome and corresponding to the same genome, is typically only used for large genomes (e.g., kb), and individual contigs are directly analyzed instead for most viruses. analysis pipelines are being actively developed, so that metagenomics will likely be available in the near future as a routine test allowing physicians to get a viral diagnosis from a biological sample in a matter of minutes to hours in their home office or on the clinic bedside. currently, metagenomics is most often used in a diagnostic context when both conventional and enhanced molecular testing fail to identify a causative agent in a sample. these cases can represent a significant fraction of patients for diseases such as acute diarrhea, for which an etiological agent is identified in only b % of cases. in this framework, metagenomic analysis can lead to the discovery of unexpected or novel viruses that are associated with a specific set of symptoms. first, metagenomics can successfully identify known viruses in unexpected sample types. these studies include the detection of enterovirus d in clinical samples (rectal, throat, and oral swabs as well as blood samples) in cases of acute flaccid paralysis, the detection of herpes simplex virus (hsv- ) in cerebrospinal fluid samples of a patient with encephalitis, and the detection of mumps vaccine virus from the brain biopsy of a patient with chronic encephalitis. in addition, new human pathogens only distantly related to known viruses have also been discovered with metagenomics. these include the bas-congo virus, a rhabdovirus that was associated with a hemorrhagic fever outbreak in the african congo, as well as novel rhinovirus, bocavirus, arenavirus, and parechoviruses. finally, entirely novel types of potentially pathogenic viruses have been described through metagenomics, including previously unknown cycloviruses, cosaviruses, and klasseviruses. diagnostics through viral metagenomics has also been applied to non-human animals as well as plants, and similarly revealed new potential viral pathogens in organisms showing unexplained symptoms. multiple new virus types including novel parvoviruses, polyomaviruses, sapoviruses, and picornaviruses were for example identified in livestock samples (porcine and bovine), while a large diversity of persistent rna viruses were newly identified across several groups of plants. however, it is important to note that the detection of a (novel) virus in a sample from a patient with an illness of unknown etiology does not prove causation, even in cases of a demonstrated significant association between the presence of the virus sequence and the observed symptoms. hence, metagenomics will often be the first step of a longer process involving attempts to propagate the virus in culture, or monitoring healthy individuals exposed to the suspected pathogen (see below "future of viral metagenomics: major challenges and upcoming innovations"). in parallel to the diagnosis application, metagenomics is also very well suited for environmental surveillance. species representing important reservoirs of viruses with high zoonotic pandemic potential such as mosquitoes, rodents, and bats have been specifically targeted in this context. a recent study investigating the virome of more than invertebrate species (a fraction of known invertebrate species), identified more than , novel rna viruses, exemplifying that the diversity of unknown eukaryotic viruses a. the total number of genomes from isolates was based on queries to the ncbi nucleotide database portal, while the number of uncultivated virus genomes was estimated by compiling data from the literature and from the img/vr database. the number of sequences is displayed on a log scale. b. comparison of complete viral genomes assembled from viral metagenomes sampled from the indian, pacific, and atlantic oceans, through the tara oceans expedition. these sequences were identified and analyzed as part of the "global ocean virome" dataset (gov). predicted genes are colored by functional annotation. c. overview of the host predictions available for uncultivated virus genomes in the img/vr database. host prediction was based on signals including sequence similarity with isolate viruses, prophages, and crispr spacers derived from known bacterial and archaeal genomes. in the environment is enormous and only poorly characterized. since the majority of human pandemics have a zoonotic origin, one hope is that such metagenomic surveillance will allow a faster identification of novel pandemic viruses during outbreaks, as well as identify their natural reservoirs. this knowledge is crucial for an appropriate and fast response from a medical and global health perspective. as an example, in the last two decades zoonotic coronaviruses were able to jump from bats to humans and pigs. both the sars (severe acute respiratory syndrome virus) and mers (middle east respiratory syndrome) viruses caused large-scale disease outbreaks in humans, whereas sads (swine acute diarrhea syndrome) caused an epidemic in the swine industry. ongoing efforts to characterize the virome of such reservoir animals will facilitate the implementation of control measure to prevent epidemics or enforce appreciate actions to stop ongoing epidemics. in an ideal situation, obtained environmental virome data in combination with biochemical experiments could help with the early identification of candidate viruses with the potential to transfer to a human host. for instance, a combination of metagenomics and dna synthesis-based experiments revealed that a novel coronavirus (wiv -cov) initially detected in bat samples could be prime for transfer and emergence into human hosts. metagenomic analysis can also be leveraged in response to viral outbreaks, for example to rapidly determine viral subtypes in a novel infection source. this has been applied to cases of influenza infections as well as for a novel wild type ebola virus outbreak, for which metagenomic approaches could correctly identify the causative agent, even in cases where traditional methods were unsuccessful because the wild type virus was too distantly related to known ebola viruses. a correct and rapid identification of these viruses could enable the application of the correct therapeutics and guide preventive efforts against potential epidemics. while viruses of humans, animals, and plants may have direct clinical or economic relevance, the vast majority of the (estimated) virus particles on earth infect micro-organisms, including bacteria, archaea, protists, fungi, and other environmental microbes. initial studies of environmental viral diversity focused on human feces, coastal and open ocean, freshwater lakes, as well as hypersaline and hot geothermal ponds, because protocols for efficient separation of virus particles from microbial cells were first developed for aquatic samples. importantly though, recent innovations and technology improvements now enable application of viromics to more complex samples such as soil, groundwater, or ice cores, helping to expand our view of global viral diversity both in the human microbiome and in the environment. a striking example of a viromics discovery is that of a highly abundant bacteriophage, named "crassphage", that was assembled from a set of human fecal viromes in . the crassphage genome was identified by combining information from individual viromes, which yielded a high-confidence kb sequence with matching ′ and ′ ends, suggesting that it represented a complete circular genome. this crassphage genome was mostly unrelated to any isolated phage genome known at the time: from the predicted proteins, less than half ( ) were even remotely similar to known proteins or domains, and only had a predicted function, such as "phage structural protein" or "dna helicase". while clearly novel, crassphage was also found to be uniquely abundant and ubiquitous: its genome was detected across metagenomes, primarily from human feces, at average levels that were six times higher than all other known phages combined. by applying several independent computational host-prediction approaches, a bacterial host (bacteroides) was predicted. thus, in this instance, metagenomics revealed what remains to date the most abundant and widespread phage associated with the human gut microbiome, which had until then evaded detection through classical approaches like laboratory cultivation and pcr. assembling genomes of uncultivated viruses can not only identify some of the most abundant and widespread viruses in an ecosystem, but these sequences also represent foundational data for targeted follow-up experiments aimed at further characterizing these novel viruses. in the case of crassphage, two major studies leveraged this initial genome sequence to better understand the diversity and host of these phages. first, predicted proteins from the original crassphage genome were used as "bait" to identify related phages in a broad range of metagenomes. this revealed an extensive and diverse group of "crass-like" phages predicted to represent a new family within the caudovirales order, that may be related to podoviridae. genome comparisons within this new family also enabled the identification of conserved structural and replication gene modules. meanwhile, another study was able to isolate a member of the crassphage-like family through broth enrichment on bacteroides intestinalis strains isolated from human gut samples, confirming the computational predictions from bioinformatic analyses that these phages were likely infecting bacteroidetes hosts and had a podoviridae-like morphology. in , a comprehensive effort to chart viral diversity across the global oceans yielded a similar observation. this study detected more than , viral genome fragments, and grouped them into clusters of closely related viruses, approximately consistent with genera in the viral taxonomy. two out of the four most highly abundant and ubiquitous clusters were entirely novel and had not been described before, while the other two were similar to known bacteriophages. with viral metagenomics being applied to a larger set of samples and environments, and with bioinformatic analyses including genome assembly and interpretation constantly improving, novel groups of dominant and widespread viruses may thus be progressively revealed across many environments. another group of viruses whose known diversity has been vastly expanded through metagenomics are the so-called "giant viruses", dsdna viruses with a uniquely large virion (b . - µm) and genome (often mb), blurring the boundaries between "simple" viruses and "complex" cellular life. following the isolation and characterization of the first giant virus in ("acanthamoeba polyphaga mimivirus"), around other members of this group have been isolated, the vast majority by using an acanthamoeba host. however, metagenome analyses suggest that the true diversity of giant viruses vastly exceeds the number of isolates. as early as , an analysis of metagenomes revealed that giant viruses could be found in the ocean at concentrations of b genomes/ml. these initial studies were based on the detection of marker genes, since the technologies available at the time did not enable the assembly of complex and large genomes like those of giant viruses. more recently, four complete or nearcomplete giant virus genomes could be assembled from metagenomes of a wastewater treatment plant. this revealed a new subgroup of giant viruses named klosneuviruses which comprised some genomes with the largest set of translation system components found at the time in any virus, including aminoacyl transfer rna synthetases with specificity for all amino acids. undoubtedly, as our collective ability to assemble large genomes from metagenomes increases, the giant virus diversity will keep expanding. while most sequencing technologies are designed for dsdna templates (see box ), our knowledge of single-stranded dna (ssdna) and rna viruses has also been transformed by metagenomics. in both cases, specific sample processing steps are required to access these genomes, however their relatively short length (usually o kb) means that complete genomes are routinely assembled from total community shotgun metagenomes that target all the nucleic acids in an environment. as for dsdna viruses, metagenomics revealed that these ssdna and rna viruses were much more diverse and broadly distributed than previously inferred from isolation and cultivation approaches. enrichment for circular ssdna viruses can be achieved through phi -based whole genome amplification, which is known to over-amplify small circular ssdna templates. pragmatically, this translates into viral metagenomes that are dominated by ssdna viruses with circular genomes, which helped shed a new light on the diversity of two major groups: bacteriophages from the microviridae family, and eukaryotic viruses from the cress dna (circular rep-encoding ssdna) supergroup. the latter saw the more striking expansion: until , these viruses were known exclusively in plants and vertebrates, specifically pigs and birds, yet in less than a decade, cress dna viruses were detected in metagenomes sampled from primates, arthropods, and unicellular protists, as well as diverse aquatic, terrestrial, and man-made ecosystems. hence, while the exact host range and impact of these viruses remain to be fully characterized, metagenomics already revealed that ssdna viruses are ubiquitous and can be found associated with all types of cellular hosts. for rna viruses, several additional sample processing steps have to be performed to preferentially sequence viral rna, typically including reverse transcription and random amplification. the most comprehensive study of rna virus diversity to date included samples from invertebrate species across phyla, and led to the discovery of nearly , novel viruses across the major clades of rna viruses. in addition, the assembly of complete genomes provided new insights on the recombination patterns of these viruses, highlighting a remarkable propensity of rna viruses to exchange or acquire genes horizontally, both with other viruses and with their host. rna viruses were also detected in a much broader host range than currently known from isolates, although these host associations now have to be confirmed through laboratory experiments since virus detection in metagenomes does not equate active infection. improvements in metagenomics protocols post b enabled the analysis of dozens of samples in parallel. in the field of viral metagenomics, this increased capacity has been leveraged specifically to analyze viral signal along time series and thus investigate virus-host dynamics in nature. such datasets have notably been obtained from freshwater lakes, for which recurrent sampling across months or years can be done, and which usually harbor a high concentration of viruses. these first explorations of environmental viral diversity across months and seasons indicated that viruses display a large range of relative abundance patterns, from "ephemeral" ones with a single peak in abundance to "constitutive" ones detected in virtually all samples. some of these patterns were seasonal and possibly linked to similar patterns of abundance for their microbial hosts, while other viruses displayed drastic changes from one year to the next. for instance, although longitudinal virome studies of the human gut are scarce, available data suggests a rather stable population of gut viruses (almost exclusively phages) in adults over time, whereas the infant gut virome is much more variable and may be dominated by eukaryotic viruses at particular time point coinciding with an acute enteric infection. time series metagenomes are especially interesting to discover and predict virus-host associations, and to analyze dynamics of known virus-host pairs. the former approach already provided host prediction for several giant viruses that are so far known exclusively from metagenome assemblies, and suggested that these may be linked to uncultivated protist hosts. the latter raised the intriguing possibility of complex and diverse virus-host relationships occurring in nature: while the expected patterns would be a strong correlation between virus and host abundances with possibly a short lag in the virus signal in a typical predator-prey fashion, these large-scale metagenome time series instead suggested that some of the viruses could peak prior to a peak in abundance of their host, while other virus-host pairs showed no similarity in relative abundance at all. these conflicting results likely reflect the complex interactions at play between viruses and microbes in nature, including variable host ranges, from viruses infecting a unique host strain to others infecting multiple host species sometimes across different genera, as well as the spectrum of infection dynamics from fast-acting lytic viruses to slower, temperate, and even chronic ones, and the development of resistance to the virus among the host population. despite these numerous challenges in their analysis, time series metagenomes are poised to become a key approach to complement laboratory experiments and untangle the intricate relationships between viruses and their hosts. metagenomics has quickly become a major tool for exploring viral diversity, yet several challenges need to be addressed in order to fully leverage the potential of these methods. first, metagenomes built from limited input material are still difficult to reliably obtain and interpret, and do not yet provide a comprehensive and quantitative view of the viral community present in the sample. this includes environments with low biomass such as some human tissues, hydrothermal vents, ice cores, or ancient samples, but also samples with a thick substrate or matrix to which cells and virus particles tend to adhere such as human lung mucus or coral samples. improvement in the recovery of cells and virions from this type of substrates and in the generation of quantitative libraries from sub-nanogram input will help better survey these viral communities. the second major challenge lies in the absence of direct host information for genomes assembled from metagenomes. in a clinical context, this means that one of koch's postulates, which requires that the candidate etiological agent be isolated from a diseased organism and grown in pure culture, cannot be fulfilled. already, several smacoviruses which had been detected in human samples metagenomes and suspected to represent new human viral pathogens have been found to likely infect prokaryotic cells from the human microbiome instead. in a similar way, evidence is emerging that picobirnaviruses, which are believed to be eukaryotic viruses, might actually infect bacterial cells. these examples should thus serve as a cautionary tale when trying to detect entirely new viral pathogens from mixed samples containing both human and microbial cells. a modified koch's postulate for the metagenomic era has been proposed in which potential new pathogens must first be present and more abundant in the diseased subject compared to matched control. then, experiments using either a sample from a disease subject or an artificial virus obtained through dna synthesis and expression in cell cultures must be performed to demonstrate that this agent induces disease in another healthy subject. while not trivial, these additional experiments based on metagenomic results could still lead to the identification of viral pathogens much more quickly than classic culture techniques. in an ecological context, associating uncultivated viruses to their host is also critical to understand their impact on microbial communities and to meaningfully integrate viruses into ecosystem models. because viral ecology studies typically include hundreds to thousands of viruses of interest, these host associations are typically derived from in silico approaches based on various types of genome sequence comparison. while methods for in vitro confirmation of these metagenome-derived virus-host pairs are currently being developed, they will need to improve both in terms of scale and resolution to provide meaningful host association for the vast diversity of uncultivated viruses. among the expected technological improvements, two stand out as likely to benefit the field of viral metagenomics in the near future. first, long-read sequencing technologies are progressively amenable to the sequencing of environmental viral communities. pragmatically, this means that instead of having to assemble virus genomes from short reads, a process which can yield potentially erroneous and/or incomplete genome sequences, a complete viral genome could be sequenced as a single read. once broadly available, these long-reads metagenomes will not only bypass assembly issues but also provide valuable information about virus genome evolution by enabling whole-genome phasing of polymorphisms. meanwhile, in an epidemiological context, long-read sequencing technologies associated with miniaturized devices, streamlined sample preparation, and live scanning of the sequencing results offers unique possibility for real-time surveillance or diagnostics. this is especially the case for the minion sequencer based on nanopore sequencing technology, allowing the identification of viral pathogens from a patient sample in less than h, compared to more than h for other sequencing technologies. the computational framework to analyze and share these types of data in a timely, safe, and meaningful way remains to be built, however it is likely that metagenomics through portable genome sequencers will become a major component of the epidemiological toolkit in the near future. complementarily, the throughput of sample preparation protocols and short-read sequencing approaches is likely to keep increasing at a fast pace. concretely, these technological improvements will translate into a lower cost per sample, and an increased ability to process hundreds of samples in parallel in a timely fashion, in particular through laboratory robotics automation. for the detection of viral pathogens as well as the exploration of viral diversity and virus-host interactions in nature, this increased throughput will provide the opportunity to generate e.g., high-resolution time-series, possibly including paired cellular and viral size fractions with multiple replicates per sample, enabling more robust and sensitive data analyses. eventually, a fully developed virus metagenomics toolkit will enable the accurate identification of viruses in natural, clinical, and biotechnological samples for monitoring and diagnostics purposes. moreover, as bioinformatics analysis tools advance, the reconstruction of full viral genome sequences will allow predictions to be made for the most important viruses in different environments, leading to the reconstruction of environmental virus-host networks and, when combined with other 'omics' approaches, the comprehensive evaluation of viral activity across an entire ecosystem. collectively, these studies should lead to a life sciences deeper understanding of viral impacts on ecological, evolutionary, and metabolic processes as well as information on potentially new viral pathogens and putative molecular virus-host interactions which could then be further characterized through targeted metagenomic identification of viral pathogens phage puppet masters of the marine microbial realm modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis virus discovery by metagenomics: the (im)possibilities real-time digital pathogen surveillance -the time is now a decade of rna virus metagenomics is (not) enough beyond research: a primer for considerations on using viral metagenomics in the field and clinic metagenomics and future perspectives in virus discovery moving beyond metagenomics to find the next pandemic virus a field guide to eukaryotic circular single-stranded dna viruses: insights gained from metagenomics minimum information about an uncultivated virus genome (miuvig) a viral reckoning: viruses emerge as essential manipulators of global ecosystems bacteriophages of the human gut: the 'known unknown' of the microbiome the phage metagenomic revolution viruses in soil ecosystems: an unknown quantity within an unexplored territory using metagenomics to characterize an expanding virosphere vr -collection of viral genomes assembled from metagenomes key: cord- -v u mr i authors: bonnin, paul; miszczak, fabien; kin, nathalie; resa, cecile; dina, julia; gouarin, stephanie; viron, florent; morello, remy; vabret, astrid title: study and interest of cellular load in respiratory samples for the optimization of molecular virological diagnosis in clinical practice date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: v u mr i background: respiratory viral diagnosis of upper respiratory tract infections has largely developed through multiplex molecular techniques. although the sensitivity of different types of upper respiratory tract samples seems to be correlated to the number of sampled cells, this link remains largely unexplored. methods: our study included upper respiratory tract specimens of which negative and positive for viral detection in multiplex pcr. all samples were selected and matched for age in these groups. for the positive group, samples were selected for the detected viral species. results: among the factors influencing the cellularity were the type of sample (p < . ); patient age (p < . ); viral positive or negative nature of the sample (p = . ); and, for the positive samples, the number of viral targets detected ( . < p < . ) and viral species. conclusion: the cellular load of upper respiratory samples is multifactorial and occurs for many in the sensitivity of molecular detection. however it was not possible to determine a minimum cellularity threshold allowing molecular viral detection. the differences according to the type of virus remain to be studied on a larger scale. associated with particular diseases (respiratory syncytial virus and bronchiolitis, parainfluenza virus and laryngitis, rhinovirus and common cold, influenza virus and flu syndrome), there is no evidence for a clinical specificity, and only the virological diagnosis provides an accurate identification of the ari [ , ] . detection of respiratory viruses is of little interest in general practice, in that the infection does not present a risk of severity for the patient. however, virological confirmation of ari is needed in severe clinical presentations, requiring hospitalization in intensive care units and occurring in vulnerable subjects [ , ] . the goal of early virological diagnosis would be an optimization of patient care, which could lead to reduction in length of hospital stay, a saving of antibiotics, and complementary examinations [ ] . virological tests allow for the establishment of accurate diagnosis of infection, assessment of evolving risks (bacterial infection, acute respiratory distress syndrome), and the establishment of measures to limit its spread (isolation, wearing gloves and masks). pandemics of severe acute respiratory syndrome (sars, (sars, - and influenza a-h n ( ) lead to the development of molecular biological techniques applied to virological diagnosis, mainly based on pcr (polymerase chain reaction). performances of molecular methods in respiratory virology are so significant that they have replaced conventional techniques (culture, detection of viral antigens) as a reference method [ ] [ ] [ ] [ ] . multiplex pcr techniques are particularly suited to medical diagnosis because they can detect multiple viral targets in the same time, avoiding the virologist a selection of viral targets to search. there are now many commercial kits for the detection of a range of to respiratory viruses and some intracellular bacteria [ , , , ] . molecular techniques (real-time pcr) also make it possible to achieve a semi quantification of the viral molecular material present in the sample, giving additional information about the respiratory viral load (interest in therapeutic monitoring and infection transmission risk) [ ] . a normalized viral load can be obtained by adding a cell quantification step. the primary site for replication of respiratory viruses is the ciliated airway epithelium. the sample must be taken as soon as possible after the onset of symptoms. this is usually a nasal swab or nasopharyngeal aspiration (especially realized in children under ) [ ] . these samples are easily accessible and especially adapted to upper ari. if a rich cell collection appears to be an important prerequisite for the quality of respiratory viral diagnosis, there is currently no information on a possible cellularity threshold that would validate the result of the viral molecular detection. the main objective of this work is the study of cellularity in respiratory specimens previously characterized virologically. the results should help to define the concept of "cellular richness" and determine the factors that influence it. eight hundred respiratory samples were included in this study. all were collected between september , and february , in different departments of the university hospital of caen (france), and immediately sent to the virology department for a respiratory viral diagnosis. respiratory samples were divided into nasal swabs corresponding to nasopharynx sampling (posterior nares), collected on universal viral transport medium (utm) and nasal aspirates. after receipt in the laboratory, each sample underwent a pre-analytical step including division into aliquots: one was immediately used for the viral detection and the other two were stored frozen at - °c. one of its two frozen fractions was used for this study. this complementary diagnostic study was then conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l - . these aliquots were selected in the laboratory samples bank according to their results in virological diagnosis: positive and negative for molecular detection of respiratory panel using the respifinder ® smart_ _fast technique (pathofinder, maastricht, netherlands). this kit allows for the detection of rna and dna viral targets and intracellular bacterial targets in respiratory specimens (tables and ). a total of age groups reflecting the distribution observed in practice in the laboratory were indicated as follows: infants (age < ; %; n = ), children (aged from to ; %; n = ), adults (aged from to ; . %; n = ) and elderly (age ≥ ; . %; n = ). each group is composed of half positive and half negative in molecular viral detection, and so as to be matched for age. within the group of positive samples, the distribution of detected viral species was modeled on that observed in the routine activity of the laboratory (tables and ) . after thawing the samples, the extraction of nucleic acids was performed using the plc qiasymphony® (qiagen, hilden, germany). the extraction was performed with μl of sample using qiasymphony_dsp_virus/ cell quantification was achieved by amplification and detection of a human household gene in real-time pcr (hypoxanthine phosphoribosyl transferase- ) [ , ] . cell quantification was performed on a lightcycler ii® platform (roche, meylan, france). the reaction mixture included μl of amplification premix cell_control r-gene® (argene/biomerieux, lyon, france) and μl of nucleic acid extract. each manipulation included two negative controls: one undergoing all the analytical steps, called ec (extraction control) and one introduced prior to the pcr reaction, called "negative control". cell quantification standard (qs : × cells/pcr and qs : × cells/pcr) was ready to use in the kit. an external standard curve was performed using these two standards and additional dilutions, respectively containing × and cells/pcr. the reading of the results was carried out directly from the plc software, which displays the ct values (cycle threshold) obtained and the corresponding number of cells/pcr-reaction (i.e. μl of extract). this number was converted to "cells/ml" and the final results were expressed in logarithmic scale (log /ml). the quantification kit performances were verified in our laboratory by quantifying mrc cells (human embryonic fibroblasts) of known concentration (rd-biothech, besancon, france). a range of -fold serial dilutions was made from initial mrc cell suspension in viral transport medium (utm). the nucleic acids were extracted from these cell suspensions, and cell quantification reaction was carried out under the same conditions as for the respiratory samples. descriptive statistics were used to show the characteristics of the different variables. quantitative variables were described using means and standard deviation. qualitative variables were described using frequencies and percentages. the relationships between qualitative variables were studied using the chi-square test or fisher's exact tests. the anova was used to compare the means of quantitative variables in two or more independent groups with the bonferroni post hoc test. the relationship between two quantitative variables was assessed using the spearman correlation coefficient (ρ). to look for a diagnostic threshold to divide positive and negative samples, a receiver operating characteristic curve (roc curve) was used. all the tests were two-tailed and their level of significance (p) was defined as p < . . ibm®-spss® . for windows® was the statistical software used. the -fold serial dilution range from initial mrc suspension had expected cellularity values between log/ ml (concentration given by the manufacturer) and . log/ml (last dilution). the measured cellularity values were consistent with those expected for the concentrations between and . log. the last dilutions deviated more than . log of the expected value, corresponding to concentrations between . and . log ( table ). the mean deviation from expected values was . log/ml. regarding our samples population, cell quantification was negative (no hprt- dna detection) for of the positive and for of the the negative in viral detection. comparative study of sample "swabs" and "nasal aspirates" among the cell-quantified respiratory samples, were nasal aspirates and were nasal swabs. cellularity these results are presented in table . regarding nasal swabs only, average cellularity were not significantly different between the age groups except for children compared with adults (p < . ). comparison of cellularity among the positive (n = ) and negative (n = ) samples in viral detection as the subjects were matched for age, the age distribution is identical in the two groups positive and negative (p = . ). these two groups are comparable, as expected. the average cellularity was . (+/- . ) log/ml for the positive group and . (+/- . ) log/ ml for the negative group. this difference was significant (p = . ). the results of comparison between the age groups according to the result of the viral detection (positive or negative) are presented in fig. b . within a single age group (infants, children, adults, elderly), the differences between positive and negative samples were not significant (p = . , p = . , p = . and p = . respectively). based on the results of the comparison between positive and negative samples, a roc (receiver operating characteristic) curve was performed. no minimum cellularity threshold could be defined for molecular viral detection (fig. ) . aspirates swabs fig. average cellularity of respiratory specimens depending on the sample type and age group. a nasal aspirate was the sample type that provides, on average, more epithelial cells for patients under the age of ( . log/ml for aspirates versus . log/ml for swabs). b although having the same shape, the average cellularity curve of positive samples was always located above the negatives among the selected samples, viral detection was negative in , were positive for viral target, were positive for targets and were positive for targets. the average cellularity was . (+/- . ) log/ml, . (+/- . ) log/ml, . (+/- . ) log/ml, and . (+/- . ) log/ml for these groups respectively. the average cellularity in negative samples was significantly lower than in cases of mono (p = . ), bi (p = . ) or tri-detection (p = . ). a significant tendency was observed between positive samples for one viral target and those positive for or virus (p = . ), this trend was confirmed by a spearman correlation (ρ = ) indicating a strong correlation between sample cellularity and the number of viruses detected. molecular detection, including multiplex techniques, is currently the gold standard for viral respiratory diagnosis. we have very powerful molecular tools, ensuring a quality respiratory viral diagnosis, available for all clinicians supporting hospitalized patients. one factor limiting this diagnosis is represented by the collected respiratory specimens. the main objectives of this work have been to study the cellularity of these clinical respiratory specimens, to propose a possible definition of what is commonly called "cellular richness," and to measure the impact of this marker on the molecular viral diagnosis. very few published studies have been completed in this area. however, a number of facts are commonly accepted within the medical community: respiratory specimen should be "rich" to allow for "good" viral diagnosis, the "good" samples are obtained almost exclusively in infants and children. in total we identified studies published in international journals between and , whose objective was to compare the various upper respiratory samples, in terms of sampling equipment (flocked swabs versus rayon swab), in terms of sampling site (nasal, oropharyngeal, nasopharyngeal, combining sites), and sampling modality (swab, wash, aspiration) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the number of target cells and/or the number of extracellular viral particles collected could define the respiratory sample quality during sampling. it is not unreasonable to consider that the majority of the viral sequences detected by molecular techniques are mainly located in the intracellular compartment. however, several factors could modulate this distribution: the viral species, the cytopathic effect induced in vivo, the inflammatory responses, and the sampling delay from the onset of the symptoms. this study is retrospective; it was therefore not possible to collect data from the time between sampling and clinical symptoms in a standardized way. this matter is nonetheless very interesting and remains to be explored. for this study, we considered cell quantification, or cellular richness, as the main marker of diagnostic efficacy for a sample. the studies referenced above used indirect measurement of sample richness through the virus detected in it: used molecular detection methods, and one was an antigen detection using rapid diagnostic test evaluate the number of infected and uninfected cells deposited on the slide [ ] . overall, the results are consistent and show a superiority of nasopharyngeal swab versus oropharyngeal and a superiority of flocked swab versus classical ones [ , , , ] . the superiority of the "wash" versus the "swab" is not found by all authors, and comes well counteract the widespread idea that washing is always greater than swabbing [ ] . alsaleh et al. ( ) performed a molecular cell quantification using real time pcr in order to validate viral detection on nasal swabs. the cellularity of these samples was assessed using the quantification of a human endogenous retrovirus (erv ) known to be present in two copies per diploid cell [ ] . the authors report their results by comparing the ct obtained upon erv detection and not in terms of cellular load [ ] . in our study, we made the choice to use a direct detection method for assessing the number of cells in respiratory samples. to the extent that we needed a large number of samples characterized by many markers for statistical analysis (age of the sampled patient, detected viral target, etc.), a prospective study on freshly sampled respiratory specimens could not be performed. we therefore used previously characterized and stored frozen (- °c) respiratory samples. the definition of cellular richness is not affected by freezing or thawing samples since it theoretically does not change the amount of hprt nucleic acid, i.e. copy per haploid cell, whether or not lysed. similarly, no distinction between living and dead cells was made before the freezing process because such a distinction does not affect the quality of molecular detection by pcr (detection of viral genome into infected cells, living or not). the analysis of samples, divided into positive and negative in viral detection, failed to establish a minimum cellularity threshold to invalidate the negative results in viral molecular detection. the roc curve shows that the cellularity is not a quantifiable predictor of the outcome of the virus detection. however, our work has yielded interesting results concerning the factors influencing the cellularity of samples and the impact of cellularity on the result of molecular detection of respiratory viruses. we have clearly demonstrated that nasal aspirates allowed us to collect more cells than with nasal swabs and this only in the age group under years old. this result must be tempered by the fact that % ( / ) of the nasal aspirations of the study were from children under years of age, since the distribution was random at inclusion of samples in the study. this reflects a common practice of sampling methods in clinical departments: nasal aspirates are rarely performed in adults and the elderly over years old. this gesture is considered invasive and unpleasant. our results support the idea that, de facto, it would not be appropriate to perform this type of sampling in this group. for all types of samples (swabs and/or nasal aspirates), the age of the sampled patient remains an important marker influencing cellularity, with a maximum average cellularity under the age of , a minimum average cellularity in adults group, and an intermediate average cellularity in the elderly group. the reasons for this lack of cellularity in respiratory samples from adults remain obscure. regarding the influence of cellularity on the result of viral detection, it should be noted that negative samples present, all age groups combined, an average cellularity lower than that obtained for the positive samples, even though it has not been possible to establish a predictive relationship. this difference was tenuous for the children group. this observation leads to two possible explanations: either in the positive group the infection increases the sample cellularity by promoting epithelial desquamation and, to a lesser extent, the mucus capture, containing free viral particles; or, in the negative group, there are false-negatives in virus detection, induced by a lack of cells in the sample. this result was obtained from all samples (aspirates and swabs). insofar as aspirates are evenly distributed in both positive and negative groups, we think that the result of comparison is not biased given that aspirates are richer samples. considering viral detection in its entirety, without analysis of the viral species detected, it is interesting to note that the two factors, "cellularity" and "viral codetection" (detection of or viruses) are associated positively. this suggests that the detection of several viruses is facilitated in the context of a rich sample. in cases of viral co-detection, the question of whether these are the same cells that are infected or not is not resolved, even if it is conventionally accepted that an already infected cell is less permissive to a second viral infection. it should also be noted that viral co-detection can be either a co-infection, or two or more sequential infections. such phenomena have already been discussed in the works of alsaleh et al. which showed that the positive samples in viral detection had, on average, a greater amount of genetic material of human origin than negative ones. similarly, samples where the gene erv was not detected had lower viral detection. finally, they also found that the positive samples for several viruses were also those in which cellular loads were highest [ ] . the analysis of the potential impact of cellularity on the specific detection of various viruses included in the "respiratory panel" showed results that should be confirmed with larger numbers in each group. indeed, on the one hand, the highest average cellularity was obtained in the hmpv positive samples, equally detected among adults and children groups; on the other hand, the lowest average cellularity is obtained in the rsv positive samples, mostly detected in infants and children groups. these results are surprising in that the two viruses, rsv and hmpv, belong to the same virus family (paramyxoviridae) as well as to the same virus subfamily (pneumovirinae), and are genetically close. they have many similarities in the circulatory mode and in the pathophysiology of the infection they cause. yet the cellularity of hmpv positive samples is significantly greater than that of the rsv positive specimens. the quality of samples dramatically affects the quality of results provided to clinicians. it is important that a better understanding of the sample characteristics goes along with technological developments. this work is uncommon. he tries to give answers to a trivial scientific question: respiratory specimens should they be « rich in cells » to ensure optimal virological molecular diagnosis? the cellular load is multifactorial and occurs for many in the sensitivity of molecular detection. however, it was not possible to determine a minimum threshold allowing molecular viral detection. adv, adenovirus; ari, acute respiratory infection; ct, cycle threshold; dna, deoxyribonucleic acid; ec, extraction control; erv , human endogenous retrovirus ; flu, influenzae virus; hbov, human bocavirus; hcov, human coronavirus; hmpv, human metapneumovirus; pcr, polymerase chain reaction; piv, parainfluenza virus; rhv/ev, rhino/entero virus; rna, ribonucleic acid; roc, receiver operating characteristic; rsv, human respiratory syncytial virus; sars, severe acute respiratory syndrome; utm, universal transport medium department of virology epidemiology of viral respiratory infections bronchiolitis viruses the underrecognized burden of influenza in young children viral epidemiology and severity of respiratory infections in infants in : a prospective study techniques actuelles de diagnostic des infections virales respiratoires en réanimation cost analysis of multiplex pcr testing for diagnosing respiratory virus infections superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms quantitation of respiratory syncytial virus rna in nasal aspirates of children by real-time rt-pcr assay molecular diagnosis of respiratory viruses comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections identification of respiratory viruses in adults: nasopharyngeal versus sampling development of an efficient qrt-pcr assay for quality control and cellular quantification of respiratory samples guideline to reference gene selection for quantitative realtime pcr comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control swabbing for respiratory viral infections in older patients:a comparison of rayon and nylon flocked swabs comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays evaluation of the quidel quickvue test for detection of influenza a and b viruses in the pediatric emergency medicine setting by use of three specimen collection methods a quantification of human cells using an erv- real time pcr assay not applicable. this study was supported by national reference laboratory for measles and respiratory paramyxoviridae. we have no additional data to communicate and have incorporated article all data, tables and figures necessary for the understanding of the study.authors' contributions av and rm designed the study. pb and av wrote the main manuscript text and prepared figures. rm was responsible for statistics analysis and wrote the statistics part of the methods. cr provided protocoles for the use of cell quantification kit. av, fm, nk, jd, sg and fv participated in the successful conduct of experiments, construction of the methodology and the reading of the manuscript. pb conducted all experiments. all authors have read and approve of the final version of the manuscript. the authors have declared that no competing interests exist. not applicable. this is a complementary diagnostic study conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l - . this provision permits to work on the remaining clinical specimen sampled for diagnostic test (in our study, samples were upper respiratory specimen). it was possible to perform an additional analysis (in our study, it is the cell quantification of sampling) unless the patient, previously informed, does not express its refusal. the approval of an ethics committee was therefore not necessary for this kind of study. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -qk xb a authors: hanada, shigeo; pirzadeh, mina; carver, kyle y.; deng, jane c. title: respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qk xb a influenza and other respiratory viral infections are the most common type of acute respiratory infection. viral infections predispose patients to secondary bacterial infections, which often have a more severe clinical course. the mechanisms underlying post-viral bacterial infections are complex, and include multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. studies over the past years have demonstrated that unique microbial communities reside on the mucosal surfaces of the gastrointestinal tract and the respiratory tract, which have both direct and indirect effects on host defense against viral infections. in addition, antiviral immune responses induced by acute respiratory infections such as influenza are associated with changes in microbial composition and function (“dysbiosis”) in the respiratory and gastrointestinal tract, which in turn may alter subsequent immune function against secondary bacterial infection or alter the dynamics of inter-microbial interactions, thereby enhancing the proliferation of potentially pathogenic bacterial species. in this review, we summarize the literature on the interactions between host microbial communities and host defense, and how influenza, and other acute respiratory viral infections disrupt these interactions, thereby contributing to the pathogenesis of secondary bacterial infections. influenza and bacterial pneumonia are the leading cause of morbidity and mortality from infectious diseases worldwide. influenza and other respiratory viral infections predispose patients to secondary bacterial super-infections, which are frequently associated with a more severe clinical course. it is estimated that the so-called "spanish flu" pandemic of h n influenza a virus from to resulted in more than million deaths, with many caused by bacterial superinfection leading to secondary pneumonia ( ) ( ) ( ) ( ) ( ) ( ) ( ) . even in the antibiotic era, over half of patients with severe infections in the h n and h n pandemics had bacterial complications ( ) ( ) ( ) . bacterial co-infection was also detected in ∼ % of cases in the h n pandemic, with high mortality rates despite administration of appropriate antibiotics ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . thus, it is evident that a better understanding of the pathogenesis of secondary bacterial pneumonia following viral infections is needed in order to make therapeutic strides for this devastating complication. the mechanisms of post-viral bacterial infection are complex, comprising multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. the pathogenesis of super-infection has been attributed to direct mucosal/epithelial damage by influenza virus, increased bacterial colonization of the upper and lower respiratory tracts (urt and lrt, respectively), and dysregulation of immune responses, which all lead to increased susceptibility to secondary bacterial infections. however, emerging evidence suggests that our microbial communities residing on our mucosal surfaces likely shape the rigor of our immune responses and shape the ecological relationships between host and pathogens. over the past years, intense interest has focused on examining how the microbial communities which inhabit our bodies-which some consider to be a separate "organ system" given the sheer physical bulk, number of genes, and metabolic activities-govern the balance between health and susceptibility to diseases, including infections. this raises the possibility that disruptions in the normal microbial communities by an acute viral infection might contribute to the development of post-viral bacterial pneumonia. the recent development of culture-independent methods of microbial identification has enabled the study of microbial communities on mucosal surfaces of the human body, referred to as "microbiota." the microbial communities of mammalian hosts are diverse, comprised of bacterial, viruses, archaea, parasites, and fungi. the human microbiome project (hmp) and other similar large-scale sequencing projects worldwide have characterized the distinct microbial communities that have adapted to the unique environmental niches within our bodies, such as the gut, skin, airways, genitourinary tract, and oral cavity. the gut microbiome, in particular, has been shown to play an integral role in shaping the immune system starting early in life, with continued influence on priming the nature and robustness of immune responses throughout one's lifetime. the respiratory tract also harbors distinct communities of microbes, with multiple discrete ecological niches (e.g., nasal cavity, oropharynx, upper airways) that vary in terms of temperature, ph, oxygen tension, mucus production, and other factors. the effects of viral infections on both the gut and respiratory microbiome have recently undergone examination. surprisingly, influenza infection has been found to result in significant changes in the gut microbiome, despite the lack of detectable virions in the gi tract. by comparison, the effects of viral infection on the respiratory microbiome appear to be relatively modest, but detectable. while the effects of these alterations on risk of secondary bacterial pneumonia have not been studied, potential mechanisms by which these changes might modulate susceptibility to secondary bacterial infections include alterations in the nature and magnitude of the immune response in the host (microbiome on host effects) and facilitating growth of pathogens in the absence of normal commensals (inter-microbial effects). in this article, we review the current understanding of how alterations in the microbiome following viral infection might alter host immune responses and increase susceptibility to secondary bacterial infections. although the term "microbiome" encompasses all microbial communities, there is currently a paucity of studies on how the mycobiome (fungal microbiome) and the virome (viral microbiome) affect host defense against respiratory infections and vice-versa; thus, this review will focus on the bacterial microbiome literature. of the niches in the body, the gut microbial community has been the most intensively studied, with over , publications to date. while the virome and mycobiome (fungi) are also being analyzed, the bulk of the literature has focused on the bacterial component of the microbiome, and thus most of our understanding of the relation of the gut microbiome to host immunity and pathogenesis of chronic diseases comes largely from studies of the bacterial community. during health, the human gut bacterial community is diverse, with each individual harboring over trillion bacteria, comprised of over different species. the gastrointestinal microbiota is dominated by firmicutes (e.g., lactobacillus, bacillus, and clostridium) and bacteroidetes (e.g., bacteroides), with lower abundances of proteobacteria (e.g., escherichia) and actinobacteria (e.g., bifidobacterium) ( , ) . wild-living mice exhibit more diverse microbiomes, with significant abundance of proteobacteria as well as firmicutes and bacteroidetes ( ) . the gut microbiome, in addition to its metabolic functions in the host, plays an integral role in the development, instruction, and priming of the immune system. germ-free (gf) mice (which lack microbiota) have markedly underdeveloped gut-associated lymphoid tissues, decreased number and smaller-sized peyer's patches and mesenteric lymph nodes, and defects in antibody production, compared to specific pathogen free (spf) mice. not surprisingly, germ-free animals exhibit increased susceptibility to multiple types of infections, including viruses, bacteria, and parasites ( ) ( ) ( ) ( ) ( ) ( ) . however, compared to free-living mice or laboratory animals exposed to gut flora from wild mice, spf animals have a more limited microbial community and are also more susceptible to inflammatory diseases, with a reduced immune repertoire including deficits in memory responses ( , , ) . although an extensive discussion of the healthy gut microbiome and its impact on host immunity is beyond the scope of this review, we will highlight a few important aspects of how the intestinal bacterial community microbiome maintains a healthy host immune environment. first, bacterial metabolites generated by gut commensals contribute to the maintenance of intact epithelial integrity, regulatory t-cell development, and a relatively anti-inflammatory immune state. in particular, short-chain fatty acids (scfas) such as acetate, propionate, and butyrate are fermentation products of dietary fiber and carbohydrates by large intestinal bacteria ( ) . in addition to being a major energy source for intestinal epithelial cells, scfas promote the development of naive cd + t cells into regulatory t cells ( , ) , induce "tolerogenic" dendritic cells in the intestinal mucosa ( ), and limit autoimmity ( , ) . at the same time, microbial metabolites are integral for promoting immune responses in the gut against pathogens, including inducing secretion of il- ( ) and defensins ( , ) . thus, the products of microbiome metabolism are integral to the appropriate regulation of mucosal barrier integrity and immune homeostasis. in addition, specific members of the bacterial community have been shown to foster the proper maturation and development of the immune system. while this is still an area undergoing intense investigation, one notable example is the discovery that segmented filamentous bacteria are critical promoters of intestinal mucosal iga production ( , ) and th cell induction ( , ) . dysbiosis, or an imbalance in the normal composition of the microbiome, is associated with a variety of chronic diseases, many of which are characterized by chronic inflammation or abnormal metabolism, including inflammatory bowel disease, cardiovascular disease, and diabetes. thus, fostering appropriate levels of diversity and composition of the gut microbial community is critical for promoting health and immune homeostasis. during health, the composition of the microbiome is governed by a number of selective pressures unique to each anatomic niche, including temperature, nutrient availability, ph, oxygen tension, and the local immune environment. shortterm perturbations in the gut microenvironment caused by illness, antibiotic usage, or dietary changes (e.g., starvation) can alter the gut microbiome and subsequently lead to transient alterations in immune responses. thus, investigating whether influenza and other respiratory viruses alter the gastrointestinal microbiome could have mechanistic implications for viralmediated suppression of antibacterial immune responses. although the composition of the gastrointestinal microbiome is largely influenced by dietary patterns, respiratory viral infections could also contribute, along with other stress inducers such as broad-spectrum antibiotics exposure and chronic inflammation. using animal models of pulmonary infections by influenza and respiratory syncytial virus (rsv), multiple groups have shown that the gut microbiome is clearly impacted by respiratory viral infections, despite the lack of detectable respiratory virus in the gut ( ) ( ) ( ) ( ) ( ) . in a murine model of influenza infection, the investigators found that although the total numbers of bacteria in the gut did not decrease, there was a reduction in the quantities of segmented filamentous bacteria (sfb) and lactobacillus/lactococcus, accompanied by increases in enterobacteriaceae. interestingly, although sfb have previously been shown to induce th cells ( , ) , flu-infected mice had increased il- a levels and numbers of th cells in the small intestine and colon, which appeared to contribute to intestinal injury ( ) . in this study, antibiotic treatment prior to influenza infection ameliorated the degree of intestinal injury, but not lung injury, suggesting that gut dysbiosis contributed to local but not systemic inflammation. other groups have similarly reported increased proteobacteria (the phylum of which enterobacteriaceae are members) ( , ) , decreased firmicutes (which include sfb, lactobacillus and lactococcus species), and increased bacteroidetes ( ) following infection by flu or rsvs but not after administration of live attenuated influenza vaccine (laiv), indicating that live viral infection is required for these changes ( ) . the increase in proteobacteria appears to be mediated by type i interferons (ifns) ( ) , which not only depleted anaerobic bacteria but also increased susceptibility to secondary salmonella colitis. however, caloric restriction also figure | shifts in the mouse gut microbiome in the setting of influenza infection. during an acute respiratory viral infection, changes in the bacterial composition of the gut microbiome can be observed despite the absence of detectable virus in the gastrointestinal compartment. this suggests that systemic immune signals, physiologic changes (e.g., weight loss), and other still unknown factors are disrupting the normal ecology of the gut, thereby leading to dysbiosis. however, the majority of these studies have been conducted in laboratory animals housed under spf conditions. it remains to be determined whether human patients and mammalian hosts with more diverse baseline gut microbiota (i.e., mice in the wild), exhibit similar qualitative or quantitative changes. results in increased relative abundance of proteobacteria and increased bacteroidetes to firmicutes ratio, raising the possibility that decreased oral intake during influenza may contribute to changes in the microbiome ( , , , ) . it has also been shown that influenza infection alters intestinal microbiota composition through type ii ifn produced by lung-derived t cells recruited to the intestine ( ) . thus, changes in the gut microbiome appear to result not from direct viral effects but from systemic inflammatory signals that travel from the lung and trigger local inflammatory responses in the gut (figure ). interactions between respiratory tract infections and the gut microbiome are bidirectional. while respiratory viral infections can change the gut microbiome, the gut microbiome also shapes the adaptive immune responses against respiratory pathogens. mice pretreated with an antibiotic cocktail showed increased morbidity and mortality during influenza infection ( , ) . the severity of infection was associated with reductions in dendritic cell migration rate and the number of local t cells. mice given a week oral course of broad-spectrum antibiotics before respiratory viral infection mounted an attenuated anti-pr antibody response, were incapable of inducing cd + t cell-mediated ifn-γ response to pr antigen, and had fewer influenza-specific cd + t cells ( , ) . these mice also had higher viral titers in their lungs ( ) . germ-free mice and antibiotic-treated mice also exhibit impaired antibody responses to seasonal influenza vaccination, which was restored by oral administration of flagellated e. coli, demonstrating a dependence on tlr -mediated sensing of the host microbiota ( ) . the gut microbiome is essential for priming innate immune responses against pulmonary infections as well. during viral infections, the degree of macrophage response to respiratory viruses depends on the presence of gut microbes. macrophages from animals treated with antibiotics exhibited defective responses to type i and ii ifns and impaired capacity to limit viral replication, suggesting that intestinal microbiota provide immune stimulation that establishes an "activation threshold" for innate antiviral immune responses ( ) . a comparison of c bl/ mice from the jackson laboratory (which lack sfb in the stool) and taconic biosciences (which are sfb positive) revealed that sfb-deficient animals have increased lung bacterial burdens and more severe pneumonia when challenged with methicillin-resistant staphylococcus aureus (mrsa) ( ) , which was associated with decreased il- -mediated responses in the lung. another study using broad-spectrum antibiotic treatment followed by intranasal administration of s. pneumoniae in mice demonstrated that microbiome depletion led to decreased survival, increased lung bacterial burden, and increased systemic dissemination of bacteria ( ) . antibiotic-pretreated animals displayed altered cytokine profiles in the lung compared to untreated controls following s. pneumoniae infection, including significantly decreased tnf-α levels at and h after infection. additionally, in the microbiota-depleted group, alveolar macrophages and blood neutrophils exhibited decreased phagocytic activity, and decreased inflammatory cytokine production following ex vivo stimulation by toll-like receptor (tlr) ligands such as lipoteichoic acid (lta) ( ) . these effects might be mediated in part by decreased nod sensing of meso-dap (diaminopimelic acid)-containing peptidoglycan found in gut microbiota, which previously was shown to be essential for priming innate immune responses to s. pneumoniae ( ) . thus, antibiotic-induced disruptions in the normal gut microbial community alter multiple aspects of normal host defense against acute respiratory pathogens (figure ). collectively, the studies above suggest that modulation of the gastrointestinal tract microbiome plays an important role in acute respiratory infections, but precisely how the microbiome should be manipulated to promote appropriate immune responses during acute respiratory infections is unclear. currently, clinical studies have shown that although probiotics do not influence the incidence of respiratory tract infection, they do reduce the severity of symptoms and duration of the illness ( , ) . pinpointing which members of the gut microbial community are essential for proper immune priming is challenging, but necessary for guiding further microbiome-based therapies. clostridium orbiscindens, a member of the human gut microbiome, has been found to produce desaminotyrosine (dat) from metabolism of flavonoids and amino acids. antibiotic-treated mice exhibited markedly decreased fecal and serum dat levels, which was associated with attenuated type i ifn responses to influenza infection and increased mortality ( ) . thus, identification of dat-producing microbiota might serve as a modality for priming type i ifn responses against viral infections. another group demonstrated that oral administration of lactobacillus plantarum enhanced the type i ifn response and lowered viral titers in the lungs in a murine model of influenza infection ( ) . other lactobacillus strains are known to enhance tnf-α and ifnγ production by nasal lymphocytes upon influenza infection ( ) . oral administration of a probiotic cocktail containing lactobacillus restored the immune response and enhanced the activation of signaling pathways associated with recognition of single-stranded rna virus ( ) . an alternative approach to administering probiotics is to alter the local metabolic environment to regulate immune responses. a recent report demonstrated that animals fed a high fiber diet had increased generation of scfas, leading to enhanced antiviral cd + t cell immune responses and attenuated neutrophil-mediated lung injury during influenza infection, resulting in improved survival ( ) . thus, one strategy for decreasing the incidence of post-viral bacterial infections is to limit the severity of the primary viral infection. however, activation of antiviral immune responses, including type i and type ii ifns, have been associated with increased susceptibility to secondary bacterial pneumonia ( , ) . thus, another strategy is to enhance immune responses against common bacterial causes of pneumonia. one group re-colonized antibiotic-treated or germ-free mice with groups of cultivatable commensal bacteria, and found that administration of lactobacillus reuteri, enterococcus faecalis, lactobacillus crispatus, and clostridium orbiscindens, which are strong stimulators of nod (i.e., cytosolic receptor for muramyl dipeptide, which is found in cell walls of certain bacteria), are able to protect against bacterial pneumonia by enhancing gm-csf production ( ) . whether viral-induced changes in the gut microbiome is associated with immune defects that promote secondary bacterial pneumonia, or whether the impaired antibacterial defenses observed in virally-infected hosts can be restored by augmenting certain components of the microbiome are important areas to be investigated. the microbiome of the respiratory tract has also been investigated in the context of viral infections. its role in the development of secondary bacterial pneumonia following influenza and other acute respiratory viral infections is unclear. the respiratory tract is the main site of continuous contact with frontiers in immunology | www.frontiersin.org figure | effects of antibiotic pre-treatment on immune responses to influenza, streptococcus pneumoniae, and lipoteichoic acid (lta). the effects of the gut microbiome on immune responses to respiratory pathogens have been investigated by administration of oral antibiotics to generate alterations in the gut flora, followed by acute infection, and analyzing host immune responses compared to non-antibiotic-pretreated animals. multiple aspects of innate and adaptive immune responses are altered in antibiotic treated animals, including decreased antibody production, decreased phagocytic activity, and decreased inflammatory cytokine production by innate immune cells (e.g., alveolar and peritoneal macrophages) following ex vivo stimulation with tlr ligands. exogenous microbes. as is the case with the gut, immunity at the mucosal interface of the respiratory tract is a constant balance of tolerance of commensal and non-invasive microbes and immune activation against pathogens. the urt and lrt have similar microbial community compositions, although microbe densities are much higher in the former in healthy hosts. several factors are known to influence airway microbiome composition including infection history, age, genetics, and structural lung disease. the urt is an interconnected system consisting of the anterior nares, nasal cavity, nasopharynx, sinuses, eustachian tube, middle ear cavity, oral cavity, oropharynx, and larynx, each of which serve as distinct niches with their own microbial communities. in healthy adults, bacteria present in the nasal cavity are typically those associated with skin, predominantly members of the actinobacteria (e.g., corynebacterium spp., propionibacterium spp.), followed by firmicutes (e.g., staphylococcus spp.), and proteobacteria ( ) ( ) ( ) . the oropharynx contains members of firmicutes, proteobacteria, and bacteroidetes, including streptococcus, neisseria, haemophilus, and lachnospira spp. ( , , ) . skin and oral cavity lineages are represented in the nasopharynxe.g., streptococcus, staphylococcus, corynebacterium, and prevotella ( , , ) . a limited number of pathogens including streptococcus pneumoniae, neisseria meningitides, and haemophilus influenzae are commensal bacteria of the urt. in healthy individuals, the microbial community richness (i.e., the total number of bacterial taxa) is lower in the lrt than that in the urt ( , ( ) ( ) ( ) . contrary to dogma that normal healthy lungs are a sterile environment, a distinct, and somewhat dynamic lung microbiome can be identified using sequencing technology, with microaspiration serving as the primary route of microbial immigration from the urt to the lrt ( , ) . the major phyla in healthy lungs are bacteroidetes and firmicutes, which mainly include prevotella, veillonella, and streptococcus ( ) ( ) ( ) . individuals with chronic airway diseases (e.g., cystic fibrosis, copd) have increased bacterial populations in the lungs ( ) and differences in the relative abundance of certain species ( ) . impaired airway clearance due to intrinsic or extrinsic factors leads to the proliferation of bacterial species that can exploit this growth opportunity ( ) . how respiratory viral infection affects the diversity of microbial communities and whether viral-induced dysbiosis influences immune functions is being examined. nonetheless, bacterial colonization of the urt is generally considered as the first step in the development of invasive bacterial infections ( , ) , including secondary bacterial infections following respiratory viral infection. bacterial abundance, species diversity, and factors that shape the immune response to subsequent infections are discussed in greater detail below. respiratory viruses enter the human body through the urt and are the most common type of acute infections of the respiratory tract. one possible mechanism by which influenza and other viral infections might predispose infected hosts to secondary bacterial pneumonia is by altering the microbial composition of the upper respiratory tract, fostering enhanced growth of pathogens, and facilitating the subsequent entry of large bacterial loads into the lrt ( ) . this section will examine recent literature on how acute respiratory viral infections have changed the urt microbiome. given the effects of viruses on enhancing bacterial adherence to the epithelium ( ) ( ) ( ) , it is perhaps not surprising that multiple studies of human subjects as well as in animal models have shown that viral infections are associated with increased colonization by potentially pathogenic bacteria (known as "pathobionts"). a comparative analysis using qpcr to detect specific bacteria in adult patients with or without influenza a infection showed that staphylococcus aureus, s. pneumoniae, and h. influenzae were present in , , and % of infected patients, respectively as compared to , , and % of uninfected patients ( ) . in experimental in vitro models, viral infections increase the colonization rates of various bacteria in the urt ( - ), including s. pneumoniae and h. influenzae ( ) ( ) ( ) . in children, influenza is associated with a -fold increase in nasopharyngeal titer of s. pneumoniae ( ) . animal models have similarly confirmed that viral infection, particularly influenza, increases bacterial colonization rates in the urt, enhancing the risk of secondary bacterial infections ( ) ( ) ( ) . higher pneumococcal colonization density has been linked to respiratory virus coinfection and invasive pneumococcal pneumonia, after adjusting for age and sex ( ) . another case-control study comparing nasopharyngeal bacteria with and without pneumonia also found an association between nasopharyngeal load of s. pneumoniaebut not of h. influenza and m. catarrhalis-and viral coinfection and pneumonia ( ) . in addition, viral infections potentially may enhance transmission of bacteria. in a study of mice colonized with s. pneumoniae and then infected with influenza a virus days after, s. pneumoniae transmission occurred only when all mice were infected with influenza and was blocked by an influenza-neutralizing antibody ( ) . however, while specific bacteria might gain a competitive advantage during viral infections, this does not universally translate to all bacterial taxa. a recent study of subjects with and without respiratory viral infections demonstrated lower overall bacterial reads from nasopharyngeal samples in virally-infected subjects compared with uninfected controls ( ) . the relationship between acute viral infections and bacterial colonization appears to be bidirectional. bacterial carriage or their ligands can increase or decrease viral infectivity rate, thereby positively or negatively influencing the subsequent host immune response to viral infection. viral replication in the respiratory tract can be enhanced by exposure to s. pneumoniae ( ) . patients harboring s. pneumoniae are more likely to experience subsequent acute respiratory illness episodes than those without colonization ( ) . in addition, bacteria present in the airways can modulate host responses against viral infection. the presence of a nasopharyngeal commensal protected mice against rsv-induced airway hyperresponsiveness. rsv-infected mice who underwent antibiotic-mediated depletion of streptococcus viridans in the nasopharynx exhibited increases in number of inflammatory lymphocytes and airway hyperresponsiveness, and decreases in regulatory t cell number and transforming growth factor-β production ( ) . others have shown that colonization of the urt with s. aureus drastically reduced influenza-induced acute lung injury and mortality in mice by recruiting a c-c chemokine receptor type + cluster of differentiation (cd) b + monocyte subset to the lungs and inducing an m macrophage phenotype ( ) . with the availability of next-generation s rrna sequencing, microbiome-based studies have attempted to discern global patterns of change in the bacterial community of each anatomic niche during viral infections, such as changes in diversity. diversity can be assessed using a variety of indices, such as total number of unique species of the microbiome (i.e., richness) or other measures that account for both richness and the evenness of relative abundance of the members of the community (e.g., shannon index). results from microbiome analyses have not demonstrated consistent changes in diversity when comparing virally infected subjects with healthy controls. this is not surprising given the variability of the subjects sampled, differences in type and severity of viral infections, type and timing of sample collection, and analysis methodology. in some studies, increased bacterial diversity appeared to be associated with influenza severity. a french study of children admitted to the hospital with influenza revealed increased diversity of the nasopharyngeal microflora with increased influenza severity ( ) . children with severe influenza showed decreased relative abundance of s. aureus and increased abundance of prevotella, streptobacillus, porphyromonas, granulicatella, veillonella, fusobacterium, and haemophilus. a recent chinese study in patients with h n avian influenza demonstrated significantly increased diversity in the oropharyngeal microbiome of h n -infected patients compared to healthy controls, particularly h n patients with secondary bacterial pneumonia ( ) . conversely, a french study of nasopharyngeal samples and a south korean study of oropharyngeal samples from patients with acute respiratory viral infections both displayed decreases in diversity indices during viral infections compared to healthy controls ( , ) . both studies included subjects ranging from infants to adults > years of age, limiting conclusions about age-related effects. longitudinal studies conducted in healthy volunteers who underwent experimental self-innoculation with rhinovirus also failed to demonstrate significant changes in diversity of the urt microbiome, while administration of laiv vaccine to healthy adults led to increases in diversity measures following viral challenge ( , ) . thus, unlike other diseases where decreased diversity is considered deleterious to the host, the effects of viral infections on diversity per se are variable and not presently considered a good indicator of risk for complications, including secondary bacterial pneumonias. microbiome sequencing studies also enable investigators to identify changes in abundance among multiple bacterial taxa simultaneously, beyond just what can be cultured individually. this allows investigators to determine what groups of bacteria are changing in unison during viral infection and which are existing in competition with one another. this information may have implications for the development of probiotic therapies (as discussed below). a recent metagenomics-based study in france reported enrichment of s. aureus, s. pneumoniae, h. influenzae, moraxella catarrhalis and klebsiella pneumoniae in nasopharyngeal samples of subjects with confirmed respiratory viral infections compared to healthy controls ( ) . an examination of the oropharyngeal microbiome of pneumonia patients with and without influenza a h n pandemic viral infection showed that firmicutes (which include staphylococcus and streptococcus spp.) and proteobacteria (mainly pseudomonas amygdali, p. fluorescens, pseudomonas sp. uk , acinetobacter baumanii and a. junii)-were significantly enriched in patients with influenza ( ) . another study of patients with pandemic h n influenza infection revealed that the predominant phyla of the upper respiratory tract (nasal and nasopharyngeal samples) in patients harboring pandemic h n were actinobacteria, firmicutes, and proteobacteria although normal controls were not included; however, the authors suggested that flu is associated with an expansion of proteobacteria ( ) which is generally less abundant in healthy hosts. these findings are supported by another group who found that moraxella and enterobacter spp. (which are classified as proteobacteria) were the most highly represented bacteria in nasopharyngeal samples obtained from patients with pandemic h n influenza ( ) . however, these studies demonstrated that there was considerable inter-subject variability, highlighting the need for longitudinal studies to decipher changes following viral infection. investigators have also sought to determine whether specific viruses are consistently linked to enrichment of certain bacterial taxa. in the nasopharyngeal compartment of aboriginal and non-aboriginal children in australia, positive associations were detected between hrv and s. pneumoniae, h. influenza, and moraxella catarrhalis carriage as well as between adenovirus and m. catarrhalis ( ) . another study examining the presence of respiratory viruses by pcr panel and prevalence of bacterial carriage in the nasopharynx of children found a strong positive association between s. aureus colonization and influenza virus ( ). moreover, s. pneumoniae colonization was positively associated with the presence of hrv and enteroviruses; h. influenzae was positively associated with hrv and rsv; and m. catarrhalis colonization was positively associated with coronaviruses and adenoviruses. a s rrna sequencing-based study conducted in infants with acute rsv or hrv respiratory infections reported that infants with rsv had significantly higher abundance of staphylococcus spp. compared to hrv-infected infants ( ) . an analysis of the urt bacterial content of healthy asymptomatic individuals and patients with influenza virus, parainfluenza, hrv, rsv, coronavirus, adenovirus, or metapneumovirus by culture-independent pyrosequencing revealed six distinct bacterial profiles-i.e., streptococcus + prevotella + veillonella, streptococcus + haemophilus + neisseria, streptococcus, moraxella, haemophilus, and klebsiella. these profiles, however, were not associated with virus type but were linked to the age of subjects ( ) . given that many human studies are cross-sectional in nature, it remains unclear whether post-viral bacterial pneumonias might be the result of viral infections enhancing bacterial colonization or acquisition, colonizing bacteria influencing host susceptibility to respiratory viral infections, or a combination of both. another complicating factor particularly in cross-sectional studies examining the microbiome during viral infections is that the groups are not well-controlled and the sample numbers are relatively small considering the number of variables that could affect the respiratory tract microbiome-such as age, gender, oral hygiene and nose-picking habits, healthcare-based employment status, smoking status, medication use, exposure to small children, etc. the underlying type of viral infection, sampling timepoint after onset of infection, severity of infection, and concomittant antimicrobial usage are other confounding factors. this may underlie the highly variable and sometimes discrepant observations from microbiome studies in patients with viral infections. there have been few clinical studies comparing baseline pre-and post-infection microbiomes in otherwise healthy individuals with acute viral infections due to the difficulty of sampling before infection. however, the relatively few studies available provide insights into the dynamicity and stability of bacteria colonization patterns over time, and whether and how perturbations brought on by acute viral infections alter these patterns. in healthy children, the major phyla among nasopharyngeal microbiotas are proteobacteria, firmicutes, bacteroidetes, actinobacteria, and fusobacteria, with moraxella, haemophilus, streptococcus, flavobacteria, dolosigranulum, corynebacterium, and neisseria as predominant genera. changes in nasopharyngeal microbiome diversity were observed across seasons, with a predominance of proteobacteria and fusobacteria in fall-winter and bacteroidetes and firmicutes in spring; these differences were independent of recent antibiotics and viral co-infection ( ) . however, another analysis of two nasopharyngeal washes collected . - . months apart from children and adolescents with asthma showed no significant differences in nasopharyngeal microbiome diversity across seasons, although mean relative abundances of haemophilus, moraxella, staphylococcus, and corynebacterium varied significantly between summer and fall samples and between age groups. moreover, in . % of patients, operational taxonomic units (otus) in patients varied significantly between time points ( ) . an investigation of the frequency and seasonal variation in bacterial and viral load in asymptomatic healthcare professionals during the winter and summer months showed that of the subjects tested during the winter, were colonized with at least one bacterial species and tested positive for at least one virus. the most frequently detected pathogens were methicillinresistant staphylococcus aureus (mrsa), m. catarrhalis, and coronavirus. in contrast, of the subjects tested during the summer, harbored at least one bacterium (mainly mrsa and k. pneumoniae) and four tested positive for one virus ( ) . several larger scale surveillance studies of mainly pediatric populations have examined the natural temporal patterns in bacterial colonization during viral infections. one clinical investigation assessed the presence and density of s. pneumoniae, h. influenzae, and m. catarrhalis in the nasopharynx of children during urt infection and in the healthy state, and reported that the proportion of children colonized with these bacteria was higher during infection than during asymptomatic surveillance visits. mean density of all bacterial species was significantly higher at each visit when a virus was detected. interestingly, the percentage of colonized children and bacterial density were also higher at asymptomatic visits in which virus was detected than at those in which virus was not detected ( ) . another study of families with small children using longitudinal nasal swab sampling demonstrated that rhinovirus infection was associated with increased acquisition of s. pneumoniae from the community as well as increased transmission of s. pneumoniae within the family ( ) . other groups have examined the effects of experimental innoculation of hrv into the urt (nares) (figure ) . these studies reported no significant changes in total read counts or of the main phyla (e.g., actinobacteria, firmicutes, and proteobacteria) over time in nasopharyngeal samples ( ) or throat swabs ( ) . in the oropharyngeal compartment, rhinovirus infection was associated with a strong trend toward transient increases in the relative abundances of h. parainfluenzae, neisseria subflava and a weak trend toward an increase in s. aureus ( ) . by days, abundance of these bacteria had returned to baseline. nasopharyngeal sampling showed completely opposite results, with decreased relative abundance of haemophilus and neisseria spp., but an increase in the normal nasal commensal, propionibacterium, in subjects following hrv infection ( ) . no differences in staphylococcus were observed. however, the number of subjects were small in both studies, limiting the power to detect changes over time. nasopharyngeal microbiota composition has been shown to be altered by influenza vaccination (figure ) . administration of live attenuated influenza vaccine (laiv), which is nasally instilled, to healthy children increased the nasal colonization density of s. pneumoniae in subjects who harbored this bacterium at the time of vaccination, and transiently increased rates of colonization by h. influenza ( ) . in healthy adult volunteers, it was demonstrated that intranasal laiv administration induced an increase in the diversity of the nasopharyngeal microbiome, figure | changes in the human upper respiratory tract microbiome following viral exposure. given that bacterial pneumonia frequently arises as a result of aspirated bacterial pathogens, a potential mechanism by which viral infections might increase the risk of secondary bacterial infections is through increased colonization of the upper respiratory tract by bacterial pathogens. in human subjects, live attenuated influenza vaccine (laiv) and human rhinovirus (hrv) have been shown to disrupt the local host bacterial community, with increased relative abundance of potential pathogens (or pathobionts), such as staphylococcal and neisseria species. the major changes in the upper respiratory tract microbiome are highlighted here. as well as relative abundances of staphylococcus and bacteroides ( ). these changes were not observed in subjects given saline nasal spray. in a mouse model, bacterial density in the nasopharynx after laiv administration was increased as much as , times compared to influenza-naive hosts, and the duration of carriage of s. pneumoniae or s. aureus was also increased to -fold ( ) . however, systemic vaccination can also alter the urt microbiome. a longitudinal study of healthy subjects found a significant association between the presence of lactobacillus helveticus, prevotella melaninogenica, streptococcus infantis, veillonella dispar, and bacteroides ovatus and influenzaspecific h and h iga antibody response ( ) . thus, it is remarkable that a relatively mild viral stimulus such as flu vaccine can lead to detectable changes in the urt microbiome. although the data are still preliminary, animal studies have suggested that antiviral immune activation contributes to changes in the urt microbiome and facilitate colonization by potential pathogens, such as s. aureus. in a mouse model of s. aureus nasal colonization, the absence of type i ifn receptor was associated with decreased persistence of bacteria ( ) . type iii ifn, which is also induced during influenza infections, led to changes in the nasal microbiome, including increased numbers of culturable bacteria. increased upper respiratory tract persistence of s. aureus as well as increased risk of s. aureus pneumonia was observed in flu-infected wildtype mice compared to mice lacking the type iii ifn receptor ( ) . currently, however, is it unclear to what extent viral-induced changes in the urt microbiome alter subsequent immune responses against secondary bacterial infections. compared to studies of the urt microbiome, studies of the lrt microbiome following viral infections are relatively scarce due to the difficulty of obtaining uncontaminated samples from the lung. samples of convenience, such as sputum, suffer from oral contamination, but bronchoscopic samples are invasive and expensive to obtain on a regular basis. moreover, it is unclear whether outside of patients with chronic lung disease (e.g., copd), the lung microbial burden is of sufficient magnitude to exert robust effects on immune responses and risk of secondary bacterial infection during viral infection. data from a mouse model of influenza infection seem to indicate that flu infection has only a modest effect on bacterial counts, diversity and composition of the lung microbiome ( ) . in subjects with chronic obstructive pulmonary disease (copd) after hrv infection but not in healthy individuals, there was an increase in bacterial burden and growth of bacteria present at baseline, particularly h. influenzae ( ) . the researchers observed that the growth of bacteria seemed to arise from the existing community. s. pneumoniae intranasally inoculated into mice pre-infected with influenza virus first colonized the nose, followed by the trachea and lungs several days later with purulent inflammation. however, this effect was not observed in uninfected animals. this suggests that pneumococcal infection may sequentially develop from the urt to the lrt in influenza virus-infected subjects ( ) . thus, it is possible that some individuals with influenza infection might develop changes in their lung microbiome as a result of changes in their urt microbial communities. respiratory viruses not only alter the bacterial community in the urt, but also promote bacterial colonization of the lrt by a variety of mechanisms that impair bacterial clearance. first, mucus production in the respiratory tract is increased to facilitate viral clearance during infections. however, excessive mucus production can lead to airway obstruction by impeding mucociliary clearance ( ) . second, viral infections can also reduce ciliary beat frequency and the number of ciliated cells, disrupt the coordinated movement of cilia, and impede the repair of respiratory epithelial cells, further leading to reduced mucociliary clearance ( , ) . third, respiratory viral infections impair innate immune responses against bacteria ( ) ( ) ( ) . innate immune cells including macrophages and neutrophils are recruited to the lung by cytokines and chemokines for phagocytosis and bactericidal activity. prior viral infections dysregulate both alveolar macrophages ( , ( ) ( ) ( ) ( ) ( ) ( ) and neutrophils ( , , ) , thereby inhibiting bactericidal activity. thus, with multiple aspects of pulmonary host defense impaired, it would not be entirely surprising if a subset of influenza infected patients developed secondary bacterial pneumonia as a result of being unable to clear aspirated pathobionts from the urt. in addition to enabling us to determine what is present during states of health, large-scale sequencing-based microbiome analyses have also revealed who is not present during disease. it has long been appreciated that mechanisms have evolved in bacteria that confer competitive advantages, permitting them to survive in an otherwise inhospitable host environment. however, interspecies competition also maintains homeostasis of the microbial community, either through their abilities to capture scare resources (e.g., iron), or targeted killing of other bacteria (e.g., bacteriocins), preventing one microbe from dominating the community. thus, it is possible that the immune response incited by acute viral infections, changes in the host epithelial surface caused by the virus, or the virus itself might lead to elimination of a host commensal that is responsible for keeping pathobionts in check. for example, s. epidermidis and propionibacterium acnes abundance in the nares has been shown to be negatively associated with s. aureus carriage ( ) . understanding these interactions may create new avenues for therapeutic interventions aimed at reducing colonization by pathogenic bacteria during influenza epidemics or pandemics. one group of commensals that has been examined for its role in inhibiting nasal carriage by s. aureus and s. pneumoniae is corynebacterium spp. an early study in japan reported on the effects of introducing a corynebacterium strain into the nares of healthy adult hospital workers who were persistent carriers of s. aureus, with successful eradication in % of subjects ( ). the mechanism appeared to be bacteriocin-independent. in comparison, s. epidermidis implantation did not have an effect. whether the s. epidermidis strain used expressed the serine protease esp, which inhibits biofilm formation by s. aureus and nasal colonization ( ) , is unknown. subsequent studies by another group reported that c. pseudodiphtheriticum inhibited s. aureus growth, whereas c. accolens and s. aureus appeared to support each other's growth ( ) . conversely, other investigators observed that corynebacterium spp. were enriched in children who were not nasally colonized with pneumococcus, and demonstrated that c. accolens inhibit s. pneumoniae growth in vitro by expressing a lipase that releases free fatty acids from skin surface triacylglycerols, which inhibit pneumococcal growth. thus, painstaking identification and mechanistic interrogation of interspecies competition between commensals might lead to novel insights as to how viral infections might confer competitive advantage to pathobionts, and how to exploit natural strategies employed by commensals to restore homeostasis to the host microbial niche. interestingly, a recent preclinical study using a murine model of rsv and s. pneumoniae superinfection employed nasal priming by a c. pseudodiphtheriticum strain to augment host defense against the viral infection, which enhanced clearance of secondary bacterial challenge and reduced lung injury measures ( ) . finally, direct effects of the infecting virus on bacteria that comprise the microbiome may facilitate the transition from pathobiont to pathogen. a metagenomic analysis showed that ph n -associated airway microbiotas were enriched in genes associated with cell motility, transcriptional regulation, metabolism, and response to chemotaxis compared to the same bacteria in non-infected patients ( ) . these data imply that influenza infection perturbs the respiratory microbiome, leading to the production of secondary metabolites including immune-modulating molecules. viruses have also been found to impair bacterial biofilm formation and disrupt existing biofilm ( ) ( ) ( ) ( ) . influenza has been shown to affect the s. pneumoniae transcriptome in terms of downregulating expression of genes associated with the colonizer state and upregulations of bacteriocins ( ) . thus, direct effects of viruses on bacterial transcriptional patterns might be a mechanism by which colonizing bacteria acquire invasive potential, thereby leading to bacterial superinfections. there are several areas that must be addressed by future respiratory microbiome research. first, it is necessary to standardize protocols used to analyze the respiratory microbiome, including sampling, processing, and bioinformatics methodologies. for example, sputum may be an appropriate material for investigations of respiratory diseases since it contains components of the lrt and can be obtained easily. however, more reliable information on the lrt requires invasive samples such as bal or protected specimen brush frontiers in immunology | www.frontiersin.org or bronchial/lung biopsies. second, most studies are limited to experiments conducted in animal models. even in human studies, most analyses have been performed in a small number of patients and have described bacterial communities in the urt. the role of microbial communities outside of the lungs including gut, sinus, and skin should be considered in the context of airway diseases. third, most studies on the microbiome have focused on the bacterial component, and have largely omitted fungi and viruses. the role of viruses-including the vast number of phages that infect bacteria-and fungi in respiratory diseases cannot be examined through s rrna gene analyses, and there are no studies describing the composition and role of the respiratory virome due to the difficulty of comprehensive analyses for viruses. fourth, it is not sufficient to study microbial communities based on species composition; a functional characterization through transcriptome and proteasome analyses is necessary to understand mechanistic role of microbiome on outcomes of infection. finally, mucosal microbiome manipulations by vaccines, antibiotics, and probiotics in the gastrointestinal and respiratory tract niches represent novel approaches for the prevention, treatment, and management of acute and chronic lung diseases. however, given that antibiotic therapy could affect commensal bacteria and hasten the emergence of drug-resistant bacteria, more research is needed on the long-term effects of this therapy. animal models should be developed to study the influence of the urt and lrt microbiomes on immune responses to respiratory viral infections; only then will it be possible to consider the clinical application of microbiome modulation strategies. respiratory viral infections can initiate a cascade of host immune responses that alter microbial growth conditions in the urt, lrt, and the gut (supplemental table ). activation of influenzainduced antiviral interferon pathways can lead to inadequate innate immune cell responses during host defense against secondary bacterial infections, resulting in the proliferation of potentially pathogenic bacterial species. concomitant changes in the gut microbiome caused by the initial viral infection may also alter immune cell priming against secondary bacterial challenge, although this has not been examined to date. although the picture is incomplete, recent microbiome literature provides additional insights into the pathogenesis of dysregulated immune responses following acute viral infections, that may promote the development of secondary bacterial pneumonias (figure ) . clarifying the differences and dynamics of respiratory microbiota in healthy subjects and chronic lung diseases during acute respiratory viral infections can elucidate pathogenesis of viralbacterial interactions and provide a basis for developing novel approaches for the prevention, treatment, or management of acute respiratory infection and exacerbation of chronic lung diseases. sh and jd co-wrote the manuscript. sh, jd, and mp designed the figures and table. kc and mp edited and provided critical revisions of the manuscript. all authors approve the final version and agree to be accountable for the content of the manuscript. jd is supported by a research grant from the national institutes of health (grant no. r hl ). the views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the department of veterans affairs or the us government. we thank cat meyer for her assistance with the figures. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness bacterial pathogens and death during the influenza pandemic updating the accounts: global mortality of the - "spanish" influenza pandemic insights into the interaction between influenza virus and pneumococcus the influenza pandemic: insights for the st century predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness the origin and virulence of the "spanish" influenza virus pneumonia and 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changes induced by influenza a virus lead to staphylococcus aureus biofilm dispersion and transition from asymptomatic colonization to invasive disease dynamic changes in the streptococcus pneumoniae transcriptome during transition from biofilm formation to invasive disease upon influenza a virus infection interkingdom signaling induces streptococcus pneumoniae biofilm dispersion and transition from asymptomatic colonization to disease streptococcus pneumoniae modulates staphylococcus aureus biofilm dispersion and the transition from colonization to invasive disease key: cord- -qcx ehnh authors: calistri, arianna; munegato, denis; carli, ilaria; parolin, cristina; palù, giorgio title: the ubiquitin-conjugating system: multiple roles in viral replication and infection date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: qcx ehnh through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. in this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells. ubiquitin (ub) is a highly conserved protein of aminoacids that can be covalently linked to target proteins through a multistep process known as ubiquitination. protein ubiquitination represents open access one of the best characterized post-translational modifications that controls the fate/function of proteins [ ] . protein ubiquitination involves a series of cellular enzymes in an enzymatic cascade, starting with the ub-activating enzyme e , followed by the ubiquitin-conjugating enzyme e and by the ub ligase e , which form an isopeptide bond between the carboxyl terminus of ub and the ε-amino group of a lysine residue on the target protein [ ] . the e ligase usually determines the substrate specificity, although the e -conjugating enzyme can also play a role in the substrate selection. accordingly, while there are few known e enzymes in mammals and roughly thirty five e s in humans, hundreds of e /ub ligases have been identified so far. e enzymes are currently classified into three main classes with different structural and functional characteristics: the hect domain family of ub ligases, the cullin-ring family of ub ligases, and the u-box containing ub ligases [ ] [ ] [ ] . the final outcome of the first round of the ubiquitination cascade is the mono-ubiquitination of the target protein. after mono-ubiquitination, a specific lysine of the first ub can be used by the same set of proteins to mediate the consecutive attachment of additional ubs, resulting in the formation of poly-ub chains ( figure ). in the most studied event, the ubiquitinated misfolded or damaged cytoplasmic and nuclear proteins are delivered to the proteasome for degradation as final event of the well-known ub-proteasome system (ups) [ ] . on the other hand, ubiquitination of the cytoplasmic domains of transmembrane proteins results in their sorting to lysosomes via the multivesicular body (mvb) pathway [ ] . ub-mediated degradation is important not only for the regulation of protein turn-over, but it also plays a role in dna damage repair, cell-cycle regulation, cellular growth, as well as in the immune system functions [ ] . moreover, it has been demonstrated that ub can also function act independently from its proteolytic activity, by regulating protein function and protein/protein interaction [ , ] . an important role in this context is played by specific ub hydrolases (deubiquitinating enzymes or dubs) that catalyze the removal of ub from the target proteins. similar to the function of kinases and phosphatases during the phosphorylation process, ub ligases and dubs can affect substrate function by transient ubiquitination. thus, protein ubiquitination represents a highly versatile and reversible event that may influence different features of a protein and not only its stability. part of this versatility is clearly linked to the fact that ub contains at least seven lysines (k) and additional residues that can be employed by the ub ligases to generate different types of ub chains on the target proteins, which, in turn, will interact with different downstream factors ( figure ) [ ] . for instance, it is well established that k- -based linkages lead mainly to the proteasome-mediated degradation of the ubiquitinated protein, while k- -based ub chains control primarily protein endocytosis, as well as trafficking and enzyme activity ( figure ) [ , ] . in addition to ub, a number of ub-like (ubl) proteins can also be conjugated to target substrates by specific e , e , and e enzyme-like proteins. among them are sumo (small ub modifier), isg (interferon-stimulated gene ), nedd (neural precursor cell expressed, developmentally downregulated ), fat (hla-f adjacent transcript ), atg (autophagy-related protein ) and lc (microtubule-associated protein light chain ) which display different functions and roles in the cellular physiology [ ] . schematic representation of the ub molecule and of the enzymatic cascade leading to protein ubiquitination. the seven lysines (k) involved in the process, the ubiquitin-activating enzyme e , the ubiquitin-conjugating enzyme e and the ubiquitin ligase enzyme e are highlighted, along with the main fate of the target proteins. as obligate intracellular parasites with limited genome size, viruses must co-opt the host cellular machineries in almost every step of their life cycle, from entry into the host cell, replication and transcription of their genome (either rna or dna), synthesis of proteins, assembly of new particles, till the egress from the infected cell. in addition, in order to establish an infection, viruses must overcome different host immune defenses. thus, there is a constant interaction between the virus and its host, with the virus trying to either counteract or exploit different complex cellular mechanisms and pathways to efficiently produce infectious progenies, or to establish a long-term persistence in the host, depending on the virus taken into consideration. under this respect, given the role played in many cellular fundamental processes, it is to be expected that ub and ubl proteins are involved in almost every aspect of the viral life cycle and pathogenesis [ ] . in this review, we will present different examples of how viruses interact with ub/ubl-mediated cellular processes in order to optimize their chance of survival, with a special focus on the mechanisms evolved, in this context, by one of the most important human pathogens, the human immunodeficiency virus type (hiv- ). the first evidence supporting the ability of viruses to utilize the ups to their own advantage came from the study of small dna tumor viruses and of their capability to interfere with the regulation of the cell cycle [ ] . since then, it has become clear that members of almost all viral families subvert or exploit both the cellular ub-conjugating and -deconjugating machineries in different phases of their replication cycle [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . under this respect, studies based on the treatment of infected cells with proteasome inhibitors have been instrumental, as such a treatment not only blocks the ups, but also depletes the cellular pool of free ub, affecting de facto all the cellular pathways involving this protein. proteasome inhibitors have been shown to interfere with the replication of major human pathogens such as herpesviruses [ , ] , poxviruses [ , ] , hepadnaviruses [ ] , adenoviruses [ ] , influenzaviruses [ ] , retroviruses [ ] [ ] [ ] , coronaviruses [ ] , paramyxoviruses [ ] , picornaviruses [ ] and rotaviruses [ ] . ub already plays a role in the first steps of viral replication. as an example, proteasome inhibitors have been reported to inhibit herpes simplex virus (hsv) entry at an early step, immediately after the penetration of the viral capsid into the target cell [ , ] . the kaposi sarcoma associated herpesvirus (kshv) [ ] , the influenza virus [ ] and adenoviruses [ ] are additional examples of viruses which rely upon the ups for their entry into target cells. for instance, it has been demonstrated that the ups is linked to the ability of the human pathogen kshv to penetrate into endothelial cells and to traffic to the nuclei. not only entry and early post-entry events, but also other steps of the viral life cycle can be affected by impairment of the proteasome activity. indeed, ub-mediated mechanisms regulate gene expression in epstein-barr virus (ebv) [ ] , hiv- [ ] , and human t lymphotropic virus (htlv) [ ] . in all these cases, specific viral proteins which function as transcriptional transactivators are able to interact with the ub/ubl-conjugating machinery, leading to an increase in viral protein activity. ups is also involved in the ability of herpesviruses to establish lifelong infections in their hosts, a phenomenon known as latency, as reported for kshv [ , ] and ebv [ , ] . specifically, in the case of ebv, the viral latent membrane protein a (lamp a) [ ] and lmp [ ] regulate viral lytic/latent replication through the interaction with specific ubligases and dubs. finally, ub plays a role in viral release from infected cells [ ] , as it will be discussed in more details later on. innate immunity represents a first line of defense employed by the cells against microorganisms. microorganisms are recognized by specific molecules named pattern recognition receptors (prr) that bind to pathogen-associated molecular patterns (pamps). this binding leads to the activation of different signaling cascades, with the involvement of the pro-inflammatory transcription factors ap- , nf-kb and/or one or more members of the interferon-regulatory factor (irf) family, with the final production of pro-inflammatory cytokines and interferon (ifn). not only regulation of innate immune signaling relies on post-translational modifications such as conjugation of ub/ubls to keys cellular proteins [ ] , but the ub/ubl conjugating system itself is adopted by many viruses to counteract this host defense response [ ] . first of all, viruses prevent the induction of nf-kb and/or ifn. to this end, the use of the cellular ub conjugating machinery represents one of the most exploited strategy. nf-kb is a complex of dimeric transcription factors, which in mammals comprises rela (p ), relb, c-rel, nf-kb (p ) and nf-kb (p ) [ ] . in normal conditions, nf-kb dimers are bound to nf-kb inhibitory proteins (ikbs) and retained in the cytoplasm. upon activativation, a multiprotein complex constituted by the enzyme transforming growth factor beta activated kinase- (tak ), the nf-kb essential modifier (nemo), and the ikb kinase (ikk) is formed. this complex phosphorylates the nfkb inhibitor (ikb), leading to its ubiquitination and degradation by the proteasome. once released by ikb, nf-kb can translocate into the nucleus where, along with specific irfs and other co-factors, is able to stimulate ifn transcription. among other mechanisms, it has been described that viruses can directly influence nf-kb stability. for instance, the case of the murid herpesvirus- (muhv- ) latency associated protein orf leads to p /rela degradation. [ ] . viruses can also use ups to impair nf-kb translocation to the nucleus by blocking ikb degradation. for instance, the rotavirus nsp protein mediates the ubiquitination and degradation of the β-transducin repeat containing protein (β-trcp) that, otherwise, would bind to and degrade ikb [ ] . viruses can also affect the stability of irfs and in particular of irf . one example is given by the varicella zoster virus (vzv) orf , a protein displaying a ring finger e ubiquitin ligase activity. orf specifically interacts with the phosphorylated/activated form of irf leading to its ubiquitination and proteasome mediated degradation [ ] . in addition, viruses can act downstream the ifn induction, by inhibiting the signal cascade activated by ifn binding to its receptor [ ] . as an example of ups-mediated viral interference with proteins belonging to this cascade, viruses in the rubulavirus genus of the paramyxoviridae family lead stat proteins to proteasome-mediated degradation, by assembling stat-specific ubiquitin ligase complexes from cellular components [ ] . finally, viruses can directly target the antiviral ifn-induced proteins (isgs) [ ] . some of these proteins, such as the protein kinase r (pkr), the ', '-oligoadenylate-synthetase (oas), the ribonuclease l(rnasel), and the mx gtp-ase, along with the respective viral counteracting mechanisms have been extensively studied [ ] [ ] [ ] . recently, different reports have been focused on the isgs belonging to the tripartite motif (trim) containing family, especially after the discovery of the role played by trim α in the establishment of a cross-species barrier against hiv- infection [ ] . interestingly, trim α and many other members of this family of isgs function by ubiquitinating, sumoylating or isgylating host/viral proteins with different outcomes on the antiviral response [ , ] . not only, the isg ub-like protein is itself an isg, and one of the most potently induced upon viral infection. it has been demonstrated that isg displays a broad antiviral activity [ ] , even though the mechanism/s accounting for this effect is/are still under investigation. what is known is that isgylation can specifically inhibit the functions of an ub ligase, nedd , which plays a role in the replication cycle of different rna viruses, such as ebola virus and oncoretroviruses [ ] . moreover, it has been reported that viruses have evolved the ability to remove isg from target proteins. for instance, the coronavirus papain-like protease (plp) protein acts as a de-ubiquitinating and de-isgylating enzyme [ ] . another interesting isg is represented by a peculiar cellular protein, tetherin, that being one of the most recently characterized targets of the hiv- accessory protein vpu, will be described in more details later. this rapid overview on the involvement of ups in crucial steps of the viral life cycle suggests immediately that viruses are connected to ub in different ways, either by usurping the host's ub-conjugating system or by evolving their own one. first of all, viral proteins have been described that can modify the substrate specificity of cellular ub ligases. as a consequence, specific cellular proteins are targeted for degradation. such a strategy, is exploited by viruses in several of the examples described in the previous paragraphs. for instance, the muhv- orf represents one of the viral proteins that can subvert the substrate recognition of a cellular e enzyme. indeed, by binding to orf , the elonginc/cullin /socs ub-ligase complex is able to poly-ubiquitinate p /rela with its subsequent proteasomal degradation [ ] . the rotavirus nsp protein leads to β-trcp ubiquitination and degradation through the recruitment of the ubiquitin-ligase complex skp- /cul /f-box (scf) [ ] . furthermore, small dna viruses with known oncogenic activity, such as the human papillomavirus (hpv), adenoviruses and polyomaviruses, take control of the cell cycle by usurping specific cellular ub ligase complexes to target crucial cell cycle regulators such as p and the protein of the retinoblastoma (prb) for degradation [ ] . in this way, two of the best studied tumor suppressor cellular pathways are inactivated. indeed, different types of hpv (high-risk) are strongly linked to the onset of cervical cancers, as well as to other type of cancer such as those affecting vagina, vulva, penis, and the head and neck [ ] , while many others, classified as low-risk, are only very rarely associated with cancer. although the high-risk and low-risk hpvs share several biological features, the two groups display significant structural/functional differences at the level of the two main viral encoded oncogenes: e and e . indeed, high-and low-risk e and e proteins have a different ability in affecting p /prb stability and in modulating the activity of additional cellular proteins with an effect on cell cycle control and cellular proliferation [ ] . interestingly, the studies of viral interactions with the host cell cycle have been instrumental in the identification and characterization of p and prb [ ] . in addition to p and prb, another protein complex involved in cell cycle control, the anaphase-promoting complex (apc) is emerging as a key target for viral proteins. apc is a cullin-ring e ub ligase that leads to the proteasome degradation of multiple cell cycle regulators and, as a consequence, to the correct progression of the cell cycle itself [ ] . different viruses have been reported to affect the apc function. in particular, the human cytomegalovirus (hcmv), a known human pathogen, encodes a protein, pul a, that is able to induce proteasome-dependent degradation of apc subunits during viral infection [ ] . additional viruses, and among them important human pathogens with known oncogenic activity, such as the htlv- , hpv, hepatitis b virus (hbv) have been reported to interfere with apc [ ] . viral proteins themselves can be directly modified by ub or ub-like proteins and, as a consequence, they can be recognized by cellular pathways that can be then exploited by the virus to perform specific tasks. examples of these processes are found in the mechanisms evolved by several enveloped viruses to egress from infected cells, as it will be explained later. viruses can also alter the activity of cellular de-ubiquitinating enzymes, by encoding, for instance, proteins which are able to interact with cellular dubs. under this respect, the ebv ebna protein, which is involved in several crucial aspects of viral replication and pathogenesis such as maintenance of the viral genome, transcription and translation of the viral dna, viral persistence, and cellular transformation, interacts with usp or herpesvirus-associated ub-specific protease (hausp) [ ] [ ] [ ] . this cellular dub is able to remove ub from p and from ebna itself, thus preventing their degradation [ ] . during ebv infection, not only usp but several additional cellular dubs are known to increase their activity and one of the effects appears to be the stabilization of β-catenin, a key factor of the wnt signaling pathway. the disregulation of this signaling pathway has been implicated in tumor development [ ] . since ebv is associated, as well, with different types of cancers, such as burkitt's and hodgkin's and the nasopharyngeal carcinoma, the study of ebv interference with the wnt pathway and the role played by the cellular dubs in this context are relevant. some viruses, especially the large dna viruses such as herpeviruses and poxviruses, encode their own ubiquitinating enzymes (table ) . vzv orf mentioned above is an example of such viral encoded ub ligases. additional examples are found in the processes evolved by different viruses to overcome the host immune defenses. under this respect, kshv encodes two e ub ligases, k and k , able to ubiquitinate the class i major histocompatibility complex (mhc), leading to its downregulation from the cell surface or from the er and thus interfering with cell antigen presentation [ ] . k also affects other surface proteins important for the stimulation of t cells, such as icam- and b - [ ] . interestingly, several other members of the herpeviridae family have been described to down-regulate mhci and t-cell activation markers from the cell surface by employing different mechanisms [ ] , some relying again on ub, as in the case of hcmv [ ] . another interesting example is given by the hsv- icp protein, a multifunctional factor which displays, among other features, a ring domain e ub ligase activity [ ] . thanks to this activity, icp is able to disassemble cellular protein aggregates, known as promyelocytic leukemia nuclear bodies (pml nbs), that are present in the nucleus of infected cells where they are involved in different processes such as the interferon (ifn) response to viral infection [ ] . both herpesviruses and adenoviruses have been described to interfere with pml nbs [ ] and ups is at the basis of some of the mechanisms employed by these viruses to this end. hsv- icp , in particular, mediates the proteasomal degradation of one of the protein complexes that constitute these bodies [ ] . moreover, viral dubs have been described (table ). among them, the large tegument protein of herpesviruses belonging to all the three known families of these pathogens (α, β and γ) not only is an essential component of the viral particle, but displays a conserved and unique deubiquitinating activity [ ] . taking into account the functions played by the large tegument proteins in the herpesviral replication cycle, a role for these viral dubs during both the entry and the egress of the virus from infected cells is very likely. for instance, the ebv dub, bplf , is known to deubiquitinate and downregulate the viral ribonucleotide reductase [ ] , the cellular processivity factor pcna [ ] and the e ubiquitin ligase rad with a positive effect on the production of infectious particles [ ] . it is interesting to note that the herpes simplex virus ub-specific protease, ul , has been recently reported to inhibit β-interferon production by deubiquitinating the tnf receptor associated factor (traf ) [ ] . this finding would indicate a role for viral dubs in overcoming the cellular immune response, as in the case of several viral ub ligases. the impact played by the ub/ubl system on viral replication and on the establishment of a successful infection is particularly clear when the life cycle and the pathogenetic mechanisms evolved by one of the most studied human pathogens, the hiv- , is analyzed. hiv- is a lentivirus and, like the other members of the retroviridae family, is characterized by a non-icosahedral particle enwrapped in a lipidic envelope. its genome comprises two copies of single-stranded rna with a short dimerized region. hiv- is the etiological agent of the acquired immunodeficiency syndrome, aids, a condition that after years from its discovery and the introduction of the highly active antiretroviral therapy (haart), still represents one of the major public health problem world-wide [ ] . the hiv- life cycle is a typical retroviral replication cycle starting with viral entry into specific target cells, reverse transcription of the viral rna into double stranded proviral dna, integration of this proviral genome into the host chromosomal dna, transcription and translation of viral proteins, assembly of viral particles, followed by their budding from the cell surface with the acquisition of an envelope. all retroviral genomes consist of at least genes, gag, pol and env. in addition to these three main genes, complex retroviruses such as hiv- encode accessory proteins that enhance their replication and infectivity ( figure ). in particular, hiv- is characterized by six auxiliary genes (tat, rev, nef, vpr, vpu and vif), of which only two, tat and rev, are essential for viral replication in vivo and in vitro [ ] . on the other hand, nef, vpu, vif, vpr genes encode factors that are known as accessory proteins, as they appear to be dispensable for viral replication in several in vitro experimental settings. however, the high degree of conservation of these proteins suggest crucial functions in vivo. the relevance of hiv- as human pathogen and the consequent development and availability of different tools and techniques to manipulate its genome, has allowed to deeply dissect several aspects of hiv biology. even though different questions still need to be answered, the reliance of hiv- on numerous cellular pathways and factors for nearly every step of its replication is well appreciated [ ] [ ] [ ] . moreover, it is becoming clear that hiv- proteins, and especially the accessory proteins, have the ability to antagonize host molecules that represent first lines of defense against retroviral infections. these cellular proteins are known as intrinsic immunity factors or restriction factors [ ] . recent studies have highlighted how the hiv- accessory proteins nef, vif, vpu, and vpr have evolved in order to enable the virus to evade the host immune system [ ] . interestingly, a common mechanism of action of these proteins is the use of the ups to interfere with cellular proteins that would affect hiv- replication. in particular, vif, vpu, and vpr exploit and subvert the physiological activity of specific cullin-ring finger ub ligases (crls) to induce the polyubiquitination and proteasomal degradation of specific cellular targets [ , ] (table ) . crls are the largest family of ub ligases and are responsible for ubiquitination of almost % of cellular proteins degraded through the ups. the choice as target of members of this particular family of e enzymes operated by these hiv- proteins is not accidental. indeed, crls are multisubunit complexes composed of a cullin (at least seven cullins are known in vertebrates), an rbx/roc ring finger protein, a variable substrate-recognition subunit (srs), and in most cases, an adaptor that links the srs to the complex [ , , ] . thus, crls are extremely versatile comprising hundreds of distinct complexes with the potential to recruit several protein. this feature has been exploited by vif, vpu and vpr to optimize the chances of hiv- to overcome different restriction factors evolved by the cells to inhibit viral replication and spreading. vif is a kda protein that is required for production of infectious virus in a cell type-specific manner [ ] . experimental evidence indicated that cells non permissive to vif-defective hiv- express a host factor inhibiting viral replication [ ] , next identified in the cytidine deaminase apobec g [ ] . apobec g is clearly expressed in vif-defective hiv- nonpermissive cells where it acts as an intrinsic restriction factor. in the absence of vif, apobec g is incorporated into budding virions, and, in the newly infected cells, it determines cytidine to uracil mutations in the single stranded dna of hiv- , during the process of reverse transcription. this hypermutation results in viral replication impairment. when expressed, vif recruits a multi-subunit e ub ligase complex composed of a scaffold protein, cullin , ring-box protein, a socs box binding protein complex, elongins b/c, as well as the core binding factor beta (cbf-β) [ ] . vif directly binds cullin [ ] . the vif-cbf-β-elonginb-elonginc-cullin -rbx e complex is then able to polyubiquitinate apobec g leading to its protesomal degradation [ ] . as a consequence, apobec g is not incorporated into viral particle and hiv- can replicate in de novo infected cells. it has to be mentioned that additional apobec molecules (i.e. apobec de/ f) have been characterized for their ability to restrict vif-defective hiv- . in vif expressing cells, all these restriction factors are substrates of the same e complex described in the case of abobec g and are as well subjected to proteasomal degradation [ ] . interestingly the crystal structure of the vif/crl complex has been recently resolved [ ] . this finding will help to further clarify the molecular basis of vif function. vpu is an amino acid dimeric integral membrane protein. one of the first characterized functions of this hiv- accessory protein was the ability of recruiting a crl (skp -cullin e ligase complex) to the cytoplasmic tail of cd , inducing its downregulation at the level of the er [ ] . in particular, vpu acts as an adaptor protein, directly interacting with cd in the er and with β-trcp, a component of the skp -cullin-f box (scf) ubiquitin ligase complex, leading to the cd polyubiquitination, dislocation from the er to the cytosol and proteasomal degradation [ ] . in addition to this important function, which is likely required for proper trafficking and maturation of the viral envelope glycoproteins, vpu has been more recently characterized for yet another crucial role, connected with the ability of the virus to evade a specific ifn- induced antiviral factor: the b cell stromal factor (bst- ) or tetherin. the latter name perfectly reflects the activity of bst- that, in the absence of vpu, physically retains (tethers) fully assembled hiv- particles to the surface of the infected cells [ ] . indeed, bst- is a type ii transmembrane protein with an unusual topology consisting of an n-terminal cytoplasmic tail, a transmembrane domain, a coiled-coil extracellular domain, and a glycosylphosphatidylinositol anchor at the carboxy-terminus. it has been demonstrated that this peculiar conformation, rather than the primary sequence, confers to tetherin the ability to physically retain budding virions on the cellular membrane [ ] . hiv- is not the only virus sensitive to bst- [ ] and human cells are not the only one encoding for such a restriction factor [ ] [ ] [ ] [ ] . indeed, tetherin represents also one of the major cross-species barrier factor especially in the context of lentiviral infection [ , , [ ] [ ] [ ] . there is still some uncertainty as to how vpu antagonism is accomplished, as different mechanisms have been reported. indeed, it has been described that vpu directly interacts with tetherin and can mediate its down-regulation from the cell surface with an increase in viral release [ ] . while some studies have indicated a vpu mediated β-trcp-dependent proteasomal degradation of tetherin [ , ] , there are also data supporting a role for the β-trcp-dependent endo-lysosomal pathway in bst- degradation [ , ] . in agreement with these latter studies rab a, a small gtpase essential for the maturation of late endosomes and lysosomal fusion, appears to be required for tetherin degradation [ ] , as along with the endosomal sorting complex required for transport (escrt)- [ ] . interestingly, β-trcp -dependent ubiquitination and subsequent degradation of tetherin do not seem to require lysine residues in the cytoplasmic domain of tetherin [ ] . it has to be mentioned that other studies have revealed a β-trcp -independent mechanism of tetherin antagonism by vpu, with the delocalization and retention of tetherin in a perinuclear compartment in the absence of degradation [ , ] . even though degradation does not appear to be the primary system by which vpu counteracts bst- , sequestration and degradation might be parallel existing and not mutually exclusive mechanisms by which vpu optimizes the chances to counteract the antiviral effect of tetherin. in agreement with the concept of multiple mechanisms, other viral proteins are able to counteract tetherin function, both by inducing its ub-dependent endosomal degradation, as in the case of the khsv k protein [ ] , or by sequestration in a perinuclear compartment, as in the case of the hiv- env [ ] . vpr is a amino acid protein whose function has been difficult to elucidate. one of the clearest activities of vpr is its ability to delay or arrest cells in the g phase of the cell cycle. in addition, in this case, the recruitment of a specific crl appears to be essential. indeed, vpr interacts with the cullin a-ddb -dcaf ub ligase complex [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , a binding that is required for the induction of the g arrest [ ] . however, the exact target/s of vpr-cullin a-ddb -dcaf activity that are linked to the g cell-cycle arrest is/are still unknown [ ] . in particular, the two substrates of vpr-mediated degradation, that have been better characterized so far, the uracil-dna glycosylase (ung) and the single-strand selective monofunctional uracil-dna glycosylase (smug ) [ ] do not seem to account for the above described vpr effect. recently, it has been demonstrated that also the endoribonuclease dicer is subjected to proteosomal degradation via vpr-recruited cullin a-ddb -dcaf ub ligase, with enhanced hiv- replication in monocyte-derived macrophages [ ] . it is well known that dicer is involved in the generation of mirna, a major component of the rna silencing machinery interfering with viral replication. thus, vpr would also act as a suppressor of silencing (srs) and vpr-mediated degradation of dicer would represent another example of a cellular restriction mechanism to hiv- infection bypassed by a viral "usurped" crl complex. while the vpr accessory protein is encoded by all primate lentiviruses, including hiv- and hiv- , its paralog vpx is expressed only by hiv- and by certain simian lentiviruses. myeloid cell types are known to be less permissive to hiv- infection with respect to cd -positive t lymphocytes [ , ] . this difference in susceptibility to hiv- infection has been linked to the expression in myeloid cell types of the vpx-interacting protein samhd [ ] . samhd is a nucleotide triphosphohydrolase that can inhibit lentiviral reverse transcription by depleting the intracellular pool of available dntps [ , ] . the hiv- and siv vpx proteins counteract this restriction by inducing samhd degradation following its ubiquitination. once again, as just described in the case of vpr, the crl involved in such a process is the vpx recruited cullin a-ddb -dcaf [ , ] . it has been recently reported that hiv vpr and vpx exploit not only cullin a but also cullin b to mediate ubiquitination of target proteins. interestingly, in primary macrophages vpx appears to need both cullin a and cullin b to obtain maximal samhd degradation [ ] . since hiv- does not have a vpx protein and hiv- vpr is not capable of interacting with samhd , myeloid cell types display resistance to hiv- infection. thus, samhd represents an additional restriction factor that interferes with hiv- infection, at least in a specific cell type, and that can be overcome by the virally recruited cullin a-ddb -dcaf ub ligase. viruses have developed sophisticated mechanisms for exiting from infected cells. enveloped viruses leave the cells through a complex process, known as budding, that requires two main steps, (i) the cell membrane deformation around the assembling virions; and (ii) a fission, resulting in the detachment of the viral particles from the cellular surface [ , ] . studies on retroviruses, and in particular on hiv- , have been instrumental in dissecting the complex viral/cellular interplay which ensures a successful egress of enveloped viruses. indeed, the gag polyprotein is the only retroviral protein necessary at this level. this feature allowed the development of viral mutants and tools to identify the molecular mechanisms involved in budding. in , göttlinger and co-workers, along with other groups, identified in the c-terminal p domain of hiv- gag a highly conserved motif (pt/sap) as crucial player in the detachment of budded virions from the cell surface [ , ] . starting from these initial findings, short proline-rich sequences, named late assembly or l-domains, functionally equivalent to the hiv- pt/sap motif, have been identified in the gag of different retroviruses [ ] . to date, in addition to the pt/sap motif, typical of most lentiviruses, two further l-domains have been well characterized: the ppxy-type l-domain present in the gag proteins of oncoretroviruses and the ypx n l-type motif, identified in the gag protein of the equine infectious anemia virus (eiav) [ ] . besides retroviruses, l-domains have been also found in the structural proteins of most rna enveloped viruses such as rhabdoviruses, filoviruses, arenaviruses, and paramyxoviruses [ ] , and in some dna enveloped viruses [ ] [ ] [ ] [ ] [ ] . several data have indicated a connection between ub, l-domains and retroviral egress from infected cells. firstly, a functional l-domain leads to gag ubiquitination [ ] and when directly fused to different retroviral gags, ub can functionally replace the l-domains [ , ] . furthermore, ub can be found in retroviral mature particles [ , ] and its depletion inhibits virus budding [ , ] , while the recruitment of hect ub ligases, belonging to the nedd -like family, is clearly involved in retroviral particle release [ , [ ] [ ] [ ] [ ] [ ] . interestingly, the ppxy type of l-domain interacts with the members of the nedd -like family of ubiquitin ligases by directly binding the ww domain characteristic of these cellular proteins [ ] . finally, the l-domains act independently from their position in the viral protein, frequently occur in combination and can be exchanged between unrelated viruses without losing their ability to mediate budding [ ] [ ] [ ] [ ] [ ] . overall, these features are suggestive of a role for the l-domains as docking sites for cellular factors belonging to a specific pathway involving ub and exploited by retroviruses to efficiently execute budding. the role played by ub in the endocytosis of transmembrane proteins suggested initially that the endocytic pathway could represent this cellular pathway [ ] . furthermore, some transmembrane proteins, such as the epithelial na+ channel, were known to contain sequences perfectly overlapping retroviral l-domains which are involved in the process of endocytosis [ ] [ ] [ ] . moreover, it was demonstrated that the ub residues involved in retroviral budding were indeed the residues involved in protein endocytosis [ ] . however, the vesiculation process taking place during endocytosis is topologically opposite to the one occurring during viral budding. indeed, while the budding of a virus happens from the cytosol toward the extracellular space and the factors that catalyze membrane fission must work from within the bud neck, during endocytosis the vesicles bud into the cytoplasm and membrane fission is driven by dynamin from outside of the bud neck. thus, the cellular machinery involved in the formation of endocytic vesicles could not be the one exploited by viruses for their egress. this apparent discrepancy between experimental data started to find a solution thanks to the seminal discoveries of the carter's laboratory [ ] and of the sundquist's group [ ] , that identified in the cellular protein tumor suppressor gene (tsg ) the binding partner of the hiv - pt/sap motif, an interaction which is crucial for hiv budding. these initial findings, along with the work done beforehand and in parallel by cellular biologists, allowed to establish the connection between a cellular pathway, to which tsg belongs, and the hiv- egress from infected cells: the biogenesis pathway of an organelle of the endocytic pathway, the multivesicular bodies (mvb) [ ] . since then, more than years of research have clarified that retroviruses, and in general most enveloped rna and some dna enveloped viruses, exploit mvbs during the latest steps of their replication cycle [ ] . these organelles give reason of the connection between viral budding, ubiquitin, l-domains and the endocytosis of transmembrane proteins. indeed, mvbs represent the organelles that eukaryotic cells have evolved to make the degradation of transmembrane proteins possible [ ] . when such a protein needs to be removed from the plasma membrane, it is ubiquitinated and endocytosed on the surface of endosome. then, a budding of vesicles from the membrane into the lumen of the endosome takes place, which allows the delivery of the trans-membrane protein into the lumen of the endosomes. this vesiculation event leads to the biogenesis of the mvb, which will eventually fuse with a lysosome resulting in the degradation of its cargo ( figure ) [ , ] . it is clear that, if the transmembrane protein would not get access to the lumen of the endosome, thanks to the formation of the mvb, its degradation through the lysosome could not occur. thus, the budding of vesicles from the endosomal membrane into the interior of the organelle (the biogenesis of the mvb) is the crucial step in this process. this vesiculation process, which takes place from the cytosol towards an environment that is equivalent to the extracellular space (the endosomal lumen), is now an event topologically identical to the budding of viruses from the plasma membrane. as a consequence, the connection between viral budding and mvbs biogenesis is functionally and physiologically sustainable. this vesicle budding step requires the sequential recruitment from the cytosol to the endosomal membrane of a highly conserved set of proteins, the escrt machinery [ ] and different members of such a machinery, as tsg and aip /alix are recruited by the viral l-domains to allow viral budding [ , ] . what is still apparently missing is the link between ub, mvb and viral budding. instead, several links do exist, with the most interesting one represented by the evidence that ub plays a central role in the regulation of the mvb biogenesis pathway. indeed, ubiquitination is necessary and sufficient to trigger the escrt-dependent endosomal sorting of membrane proteins and their degradation through the mvb/lysosomal pathway [ ] . moreover, precise and finely controlled cycles of ubiquitination and deubiquitination of target proteins and of specific components of the escrt machinery are essential for the function of the entire pathway of mvb biogenesis, till the vesicle budding [ ] [ ] [ ] . indeed, different upstream components of the escrt machinery with an essential role in viral budding, such as tsg and aip /alix, are ubiquitinated and are able to bind ub [ ] [ ] [ ] [ ] . the sequential recruitment of the escrt complexes to the mvb membrane is described along with the additional factors involved in the cargo protein delivery into the organelle lumen. the extracellular environment and its equivalents are colored in light blue, while the cytoplasmic environment is colored in yellow. details on the escrt proteins and on the other mvb key factors can be found in several comprehensive reviews [ , , , , ] . overall, the aforementioned crucial role played by ub in the control of the mvb biogenesis pathway by itself supports the strong evidence of an involvement of this cellular protein in viral budding. however, in addition or in parallel to this regulatory function, there is evidence that also direct ubiquitination of specific viral/cellular proteins might be important for the escrt-dependent viral budding. in particular, the importance of a direct gag ubiquitination is supported by several studies, as reviewed by votteler and sundquist [ ] . the working model foresees that ubiquitination of gag would facilitate its interaction with the escrt machinery. in other words, ubiquitination of gag would mimic ubiquitination of transmembrane proteins along the endocytic/mvb pathway, thus mediating the engagement of the escrt components. under this respect, it has been shown that the binding of tsg to gag has an increased affinity when gag is linked to ub [ , ] . furthermore, the bouamr's group, by fusing the dub domain of the hsv- ul to gag and to specific escrt proteins, has recently reported evidence supporting a crucial role for gag ubiquitination in hiv budding [ ] . however, along with these data, other studies indicate a more complex scenario. for instance, it has been demonstrated that ubiquitination of gag proteins can increase also in the absence of a functional l-domain and when budding is inhibited [ ] . moreover, the artificial targeting of ub ligases to gag leads to its ubiquitination, but not always enhances budding [ ] . finally, zhadina and coworkers have nicely shown that ub-dependent viral budding can take place without viral protein ubiquitination [ ] . thus, the question whether gag ubiquitination is the result of a bystander effect or has a functional relevance is still open. interestingly, it has been reported that the identity of the protein to which ub is bound might not be the key element for viral budding [ ] . what appears to be crucial is, instead, the presence of ub at the site of budding, thus emphasizing its crucial role in the process. as very simple intracellular parasites, viruses rely on host cell factors and pathways to perform their life cycle and to establish a successful infection. the extensive use of ub and ubl-mediated pathways by a number of unrelated viruses in different steps of their life cycle emphasizes the central importance of these cellular machinery in the cell physiology and, as a consequence, for viral replication and spreading. in this review, we have tried to give an overview of the complexity of the interplay between viruses and ub/ubl-conjugating systems. while it appears evident that different aspects have been deeply analyzed and better understood, such as the role played by ups in the context of the mechanisms evolved by viruses to control the cell cycle and to counteract innate immunity responses, more work needs to be done in order to clarify the functional relevance of ub involvement in specific steps of viral replication, such as viral budding. moreover, different emerging findings require further clarification both from the cellular and from the viral point of view, such as the role and the substrates of the newly characterized viral and cellular ubl proteins. under this respect, the study of the viral connection with the ub/ubl-conjugating machinery still represents one of the better examples of how viruses can serve as potent tools to dissect complex cellular processes and pathways. ubiquitin linkages make a difference mechanisms underlying ubiquitination the hect family of e ubiquitin ligases: multiple players in cancer development cullin-ring ubiquitin ligases: global regulation and activation cycles u-box proteins as a new family of ubiquitin ligases ubiquitin enters the new millennium multivesicular body morphogenesis ubiquitin and ubiquitin-like proteins as multifunctional signals back to the future with ubiquitin ubiquitin in chains polyubiquitin chains: polymeric protein signals ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets viral avoidance and exploitation of the ubiquitin system the e oncoprotein encoded by human papillomavirus types and promotes the degradation of p cellular proteasome activity facilitates herpes simplex virus entry at a postpenetration step proteasome subunits relocalize during human cytomegalovirus infection, and proteasome activity is necessary for efficient viral gene transcription inhibition of the ubiquitin-proteasome system prevents vaccinia virus dna replication and expression of intermediate and late genes orthopoxviruses require a functional ubiquitin-proteasome system for productive replication bortezomib inhibits hepatitis b virus replication in transgenic mice tip degradation by adenovirus relieves transcriptional repression of viral transcriptional activator eia inhibition of the ubiquitin-proteasome system affects influenza a virus infection at a postfusion step proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv- and hiv- a role for ubiquitin ligase recruitment in retrovirus release retroviruses have differing requirements for proteasome function in the budding process the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry decreased replication of human respiratory syncytial virus treated with the proteasome inhibitor mg- ubiquitination is required for effective replication of coxsackievirus b replication of the rotavirus genome requires an active ubiquitin-proteasome system a pre-immediate-early role for tegument icp in the proteasomedependent entry of herpes simplex virus the ubiquitin/proteasome system mediates entry and endosomal trafficking of kaposi's sarcoma-associated herpesvirus in endothelial cells epsin is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus a capsid-encoded ppxy-motif facilitates adenovirus entry ebna -mediated recruitment of a histone h b deubiquitylating complex to the epstein-barr virus latent origin of dna replication a non-proteolytic role for ubiquitin in tat-mediated transactivation of the hiv- promoter ubiquitination of human t-cell leukemia virus type tax modulates its activity kaposi's sarcoma-associated herpesvirus transactivator rta promotes degradation of the repressors to regulate viral lytic replication kaposi's sarcoma-associated herpesvirus rta promotes degradation of the hey repressor protein through the ubiquitin proteasome pathway the epstein-barr virus latent membrane protein a py motif recruits ww domain-containing ubiquitin-protein ligases the a deubiquitinase activity negatively regulates lmp activation of irf wrapping up the bad news: hiv assembly and release regulation of the innate immune system by ubiquitin and ubiquitin-like modifiers ubiquitylation in innate and adaptive immunity shared principles in nf-kappab signaling termination of nf-kappab activity through a gammaherpesvirus protein that assembles an ec s ubiquitin-ligase rotavirus nsp inhibits nfkappab activation by inducing proteasome-dependent degradation of beta-trcp: a novel mechanism of ifn antagonism varicella-zoster virus immediate-early protein orf abrogates the irf -mediated innate immune response through degradation of activated irf viral evasion of the interferon system paramyxoviruses sv and hpiv assemble stat protein ubiquitin ligase complexes from cellular components interferon-inducible antiviral effectors the tiers and dimensions of evasion of the type i interferon response by human cytomegalovirus interferon-stimulated genes: a complex web of host defenses innate immune interferon responses to human immunodeficiency virus- infection trim family proteins and their emerging roles in innate immunity the roles of the trim e -ubiquitin ligase family in innate antiviral immunity ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses role of multivesicular bodies and their components in the egress of enveloped rna viruses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases e ubiquitin ligases and human papillomavirus-induced carcinogenesis review of the current knowledge on the epidemiology, pathogenesis, and prevention of human papillomavirus infection how the rb tumor suppressor structure and function was revealed by the study of adenovirus and sv control the host cell cycle: viral regulation of the anaphase-promoting complex proteasome-dependent disruption of the e ubiquitin ligase anaphase-promoting complex by hcmv protein pul a protein interaction domains of the ubiquitin-specific protease, usp /huasp protein profiling with epstein-barr nuclear antigen- reveals an interaction with the herpesvirus-associated ubiquitin-specific protease huasp/usp huasp/usp as an epstein-barr virus target contributions of epstein-barr nuclear antigen (ebna ) to cell immortalization and survival wnt signaling in stem cells and cancer what has the study of the k and k viral ubiquitin e ligases taught us about ubiquitin-mediated receptor regulation? viruses a viral protein that selectively downregulates icam- and b - and modulates t cell costimulation herpesviruses and immunity: the art of evasion human cytomegalovirus immunity and immune evasion herpes simplex virus type immediate-early protein icp and is isolated ring finger domain act as ubiquitin e ligases in vitro pml and pml nuclear bodies: implications in antiviral defence the two functions of herpes simplex virus icp , inhibition of silencing by the corest/rest/hdac complex and degradation of pml, are executed in tandem a proteomic perspective of inbuilt viral protein regulation: pul tegument protein is targeted for degradation by icp during herpes simplex virus type infection herpes simplex virus e ubiquitin ligase icp protein inhibits tumor necrosis factor alpha-induced nf-κb activation by interacting with p /rela and p /nf-κb centromere architecture breakdown induced by the viral e ubiquitin ligase icp protein of herpes simplex virus type icp dismantles microtubule networks in herpes simplex virus-infected cells a viral e ligase targets rnf and rnf to control histone ubiquitination and dna damage responses ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection haploid genetic screens identify an essential role for plp in the downregulation of novel plasma membrane targets by viral e ubiquitin ligases the ring-ch ligase k antagonizes restriction of kshv and hiv- particle release by mediating ubiquitin-dependent endosomal degradation of tetherin kaposi's sarcoma-associated herpesvirus k and k proteins down regulate both dc-sign and dc-signr the march family e ubiquitin ligase k alters monocyte metabolism and proliferation through receptor tyrosine kinase modulation the ring finger domain of varicella-zoster virus orf p has e ubiquitin ligase activity that is essential for efficient autoubiquitination and dispersion of sp -containing nuclear bodies adenovirus ubiquitin-protein ligase stimulates viral late mrna nuclear export murine gammaherpesvirus orf c contains ubiquitin e ligase activity and requires pml sumoylation but not other known cellular pml regulators, ck and e ap, to mediate pml degradation newly discovered viral e ligase pk induces endoplasmic reticulum-associated degradation of class i major histocompatibility proteins and their membrane-bound chaperones the poxvirus p virulence factor is an e ubiquitin ligase viral ubiquitin ligase wssv is required for efficient white spot syndrome virus replication in shrimp arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling plp of mouse hepatitis virus a (mhv-a ) targets tbk to negatively regulate cellular type i interferon signaling pathway plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase mechanism of inhibiting type i interferon induction by hepatitis b virus x protein structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes the epstein-barr virus (ebv) deubiquitinating enzyme bplf reduces ebv ribonucleotide reductase activity epstein-barr virus bplf deubiquitinates pcna and attenuates polymerase η recruitment to dna damage sites the rad / ubiquitin complex interacts with the epstein-barr virus deubiquitinating enzyme, bplf , and contributes to virus infectivity herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf autocatalytic activity of the ubiquitinspecific protease domain of herpes simplex virus vp - cleavage specificity of the ul deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pul of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf molecular basis for ubiquitin and isg cross-reactivity in viral ovarian tumor domains mers-cov papain-like protease has deisgylating and deubiquitinating activities a compact viral processing proteinase/ubiquitin hydrolase from the otu family a viral deubiquitylating enzyme targets viral rna-dependent rna polymerase and affects viral infectivity the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions deubiquitinating function of adenovirus proteinase past, present and future: years of hiv research regulation of hiv- gene expression identification of host proteins required for hiv infection through a functional genomic screen host cell factors in hiv replication: meta-analysis of genome-wide studies host factors mediating hiv- replication host restriction factors in retroviral infection: promises in virus-host interaction hiv accessory proteins versus host restriction factors viral modulators of cullin ring ubiquitin ligases: culling the host defense hiv: ringside views selective protein degradation: a rheostat to modulate cell-cycle phase transitions the emerging family of cullin -ring ubiquitin ligases (crl s): cellular functions and disease implications structural basis for hijacking cbf-β and cul e ligase complex by hiv- vif role of hiv- vpu protein for virus spread and pathogenesis hiv- antagonism of cd is species specific and involves vpu-mediated proteasomal degradation of the restriction factor hiv- vpu neutralizes the antiviral factor tetherin/bst- by binding it and directing its beta-trcp -dependent degradation vpu directs the degradation of the human immunodeficiency virus restriction factor bst- /tetherin via a {beta}trcp-dependent mechanism inhibition of β-trcp-dependent ubiquitination of p by hiv- vpu promotes p -mediated apoptosis in human t cells besnard-gué rin, c. involvement of the betatrcp in the ubiquitination and stability of the hiv- vpu protein hiv vpr arrests the cell cycle by recruiting dcaf /vprbp, a receptor of the cul -ddb ubiquitin ligase vpr expression abolishes the capacity of hiv- infected cells to repair uracilated dna the hiv- protein vpr targets the endoribonuclease dicer for proteasomal degradation to boost macrophage infection samhd host restriction factor: a link with innate immune sensing of retrovirus infection new insights into the role of vif in hiv- replication evidence for a newly discovered cellular anti-hiv- phenotype isolation of a human gene that inhibits hiv- infection and is suppressed by the viral vif protein hiv- vif n-terminal motif is required for recruitment of cul to suppress apobec hiv- vif, apobec, and intrinsic immunity interactions between hiv- vif and human elonginb-elonginc are important for cbf-β binding to vif tetherin inhibits retrovirus release and is antagonized by hiv- vpu tetherin inhibits hiv- release by directly tethering virions to cells the antiviral activities of tetherin tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv- strains tetherin antagonism by primate lentiviral nef proteins feline tetherin is characterized by a short n-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein ancient origin of a deletion in human bst /tetherin that confers protection against viral zoonoses reacquisition of nef-mediated tetherin antagonism in a single in vivo passage of hiv- through its original chimpanzee host vpu antagonizes bst- -mediated restriction of hiv- release via beta-trcp and endo-lysosomal trafficking rab a is required for efficient production of infectious hiv- berlioz-torrent, c. the escrt- component hrs is required for hiv- vpu-mediated bst- /tetherin downregulation serine-threonine ubiquitination mediates downregulation of bst- /tetherin and relief of restricted virion release by hiv- vpu antagonism of tetherin restriction of hiv- release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment Β-trcp is dispensable for vpu's ability to overcome the cd /tetherin-imposed restriction to hiv- release hiv- vpu and hiv- env counteract bst- /tetherin by sequestration in a perinuclear compartment hiv- vpr-mediated g arrest involves the ddb -cul avprbp e ubiquitin ligase hiv- vpr activates the g checkpoint through manipulation of the ubiquitin proteasome system lentiviral vpr usurps cul -ddb [vprbp] e ubiquitin ligase to modulate cell cycle hiv- vpr function is mediated by interaction with the damage-specific dna-binding protein ddb ddb and cul a are required for human immunodeficiency virus type vpr-induced g arrest the hiv protein vpr acts to promote g cell cycle arrest by engaging a ddb and cullin a-containing ubiquitin ligase complex using vprbp/dcaf as an adaptor assembly with the cul a-ddb dcaf ubiquitin ligase protects hiv- vpr from proteasomal degradation human immunodeficiency virus type vif induces cell cycle delay via recruitment of the same e ubiquitin ligase complex that targets apobec proteins for degradation defining the interactions and role of dcaf /vprbp in the ddb -cullin a e ubiquitin ligase complex engaged by hiv- vpr to induce a g cell cycle arrest molecular mechanisms of hiv immune evasion of the innate immune response in myeloid cells intracellular nucleotide levels and the control of retroviral infections structural basis of lentiviral subversion of a cellular protein degradation pathway cullin a and cullin b are interchangeable for hiv vpr and vpx action through the crl ubiquitin ligase complex membrane budding and scission by the escrt machinery: it's all in the neck viral membrane scission effect of mutations affecting the p gag protein on human immunodeficiency virus particle release p gag is required for particle production from full-length human immunodeficiency virus type molecular clones expressing protease virus budding and the escrt pathway herpes simplex virus type cytoplasmic envelopment requires functional vps intracellular trafficking and maturation of herpes simplex virus type gb and virus egress require functional biogenesis of multivesicular bodies human cytomegalovirus exploits escrt machinery in the process of virion maturation cellular vps is required for efficient entry and egress of budded virions of autographa californica multiple nucleopolyhedrovirus functional characterization of the putative hepatitis b virus core protein late domain using retrovirus chimeras ubiquitin is part of the retrovirus budding machinery functional replacement of a retroviral late domain by ubiquitin fusion ubiquitin in avian leukosis virus particles ubiquitin is covalently attached to the p gag proteins of human immunodeficiency virus type and simian immunodeficiency virus and to the p gag protein of moloney murine leukemia virus hect ubiquitin ligases link viral and cellular ppxy motifs to the vacuolar protein-sorting pathway efficient and specific rescue of human immunodeficiency virus type budding defects by a nedd -like ubiquitin ligase sundquist, w.i. nedd l overexpression rescues the release and infectivity of human immunodeficiency virus type constructs lacking ptap and ypxl late domains role of the feline immunodeficiency virus l-domain in the presence or absence of gag processing: involvement of ubiquitin and nedd - s ligase in viral egress rescue of hiv- release by targeting widely divergent nedd -type ubiquitin ligases and isolated catalytic hect domains to gag positionally independent and exchangeable late budding functions of the rous sarcoma virus and human immunodeficiency virus gag proteins infectivity of moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses functional replacement and positional dependence of homologous and heterologous l domains in equine infectious anemia virus replication aip /alix is a binding partner for hiv- p and eiav p functioning in virus budding functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding ww domains of nedd bind to the proline-rich py motifs in the epithelial na+ channel deleted in liddle's syndrome regulation of the epithelial na+ channel by nedd and ubiquitination late assembly domain function can exhibit context dependence and involves ubiquitin residues implicated in endocytosis pr (gag) tsg and the vacuolar protein sorting pathway are essential for hiv- budding the role of ubiquitination in lysosomal trafficking of δ-opioid receptors ubiquitination in the first cytoplasmic loop of μ-opioid receptors reveals a hierarchical mechanism of lysosomal down-regulation dynamics of escrt proteins ubiquitin: same molecule, different degradation pathways how ubiquitin functions with escrts escrt ubiquitin-binding domains function cooperatively during mvb cargo sorting the circuitry of cargo flux in the escrt pathway tsg -specific e ubiquitin ligase, regulates receptor endocytosis and retrovirus budding the yeast alix homolog bro functions as a ubiquitin receptor for protein sorting into multivesicular endosomes structure and functional interactions of the tsg uev domain structure of the tsg uev domain in complex with the ptap motif of the hiv- p protein ubiquitin conjugation to gag is essential for escrt-mediated hiv- budding analysis of human immunodeficiency virus type gag ubiquitination ubiquitin-dependent virus particle budding without viral protein ubiquitination owing to space limitations and to the complexity of the topics we have predominantly cited recent publications and reviews, therefore we apologize to those whose original contributions have not been specifically referenced. the studies carried on in the authors' laboratory have been supported by the miur-prin- (# _ to cp), miur-prin- (# j rwm_ and m htt_ to cp and gp, respectively), aids grants from the istituto superiore di sanità (rome-aids projects # g. and h to cp, # g. and f. to gp), grants from the university of padova (ex- % to cp, gp, ac and cpda to ac) and from regione veneto to gp. dm is a phd student in biomedicine, at the department of molecular medicine, university of padova. the authors declare no conflict of interest. key: cord- -kxfc npg authors: blachere, francoise m.; lindsley, william g.; slaven, james e.; green, brett j.; anderson, stacey e.; chen, bean t.; beezhold, don h. title: bioaerosol sampling for the detection of aerosolized influenza virus date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: kxfc npg background influenza virus was used to characterize the efficacy of a cyclone‐based, two‐stage personal bioaerosol sampler for the collection and size fractionation of aerosolized viral particles. methods a collison single‐jet nebulizer was used to aerosolize the attenuated flumist® vaccine into a calm‐air settling chamber. viral particles were captured with bioaerosol samplers that utilize microcentrifuge tubes to collect airborne particulates. the first tube (t ) collects particles greater than . μm in diameter, while the second tube (t ) collects particles between . and . μm, and the back‐up filter (f) collects submicron particles. following aerosolization, quantitative pcr was used to detect and quantify h n and h n influenza strains. results based on qpcr results, we demonstrate that aerosolized viral particles were efficiently collected and separated according to aerodynamic size using the two‐stage bioaerosol sampler. most viral particles were collected in t ( ‐ . μm) and on the back‐up filter (< μm) of the bioaerosol sampler. furthermore, we found that the detection of viral particles with the two‐stage sampler was directly proportional to the collection time. consequently, viral particle counts were significantly greater at minutes in comparison to , and minute aerosol collection points. conclusions due to a lack of empirical data, aerosol transmission of influenza is often questioned. using flumist®, we demonstrated that a newly developed bioaerosol sampler is able to recover and size fractionate aerosolized viral particles. this sampler should be an important tool for studying viral transmission in clinical settings and may significantly contribute towards understanding the modes of influenza virus transmission. influenza infections are a public health concern accounting for more than deaths and hospitalizations annually. the primary populations at risk for infection include young children, elderly adults, and immunocompromised subjects. during influenza outbreaks or pandemics, healthcare workers are at an elevated risk for acquiring influenza infection due to the prolonged periods of exposure to influenza viruses within healthcare settings. the mechanisms of influenza virus dissemination and transmission are poorly understood. experimental studies have shown that influenza viruses can be transmitted via contact with respiratory secretions, large droplets, and aerosolized small particles ⁄ droplet nuclei. mucous membrane contact of large droplets expelled from the respiratory tract are thought to be the predominant mode by which influenza infection is transmitted, but small particle aerosols and droplet nuclei are also of concern due to the potential for prolonged airborne suspension. , given the epidemic potential and public health concern of newly emerging diseases such as avian influenza (h n ) and severe acute respiratory syndrome (sars), , it is important to develop methods to study aerosolized viral particles. such measures would not only enable improved monitoring and detection of viruses, but more importantly, would help prevent widespread transmission. to date, several bioaerosol samplers are available that use impaction or impinger sampling methodologies for viral aerosol detection. , however, limitations exist within each of these methods such as poor collection efficiency, limited sampling time, and inability to separate particles based on size. further complicating the study of viral aerosols is that in many environments where it would be desirable to sample for viral aerosols (e.g., hospitals, airports), the level of other biologically relevant particles such as bacteria, fungi, and pollens can also be very high. based on a one-stage, cyclone-based bioaerosol sampler developed at the national institute for occupational safety and health (niosh), a two-stage, cyclone-based bioaerosol sampler was recently fabricated. the two-stage bioaerosol sampler is a lightweight device that can be used either as an area sampler (e.g., in a hospital room) or as a personal breathing zone air-sampling device worn on the lapel of the subject (e.g., a healthcare worker). this bioaerosol sampler contains two microcentrifuge tubes and a back-up filter, which separate airborne particulates based on their aerodynamic diameter. the first tube (t ) of the sampler collects particles that are greater than approximately . lm in diameter, while the second tube (t ) collects particles from . to . lm in diameter and the back-up filter (f) collects submicron particulates. because the sample is deposited directly in microcentrifuge tubes, the sampler facilitates the direct processing of samples for downstream diagnostic applications such as quantitative polymerase chain reaction (qpcr) and enzyme-linked immunosorbent assay. the bioaerosol sampler potentially eliminates several limitations associated with other air sampling devices such as sample loss, contamination, and degradation. in this proof of concept study, we characterized the utility of the two-stage bioaerosol sampler for fractionating viral particles and separating them from other particulates. the influenza virus vaccine flumist Ò - formula was purchased from medimmune vaccine, inc. (gaithersburg, md, usa). flumist Ò is a live, trivalent vaccine, composed of the a ⁄ new caledonia ⁄ ⁄ (h n ), a ⁄ california ⁄ ⁄ (h n ), and b ⁄ jiangsu ⁄ ⁄ (b ⁄ shanghai ⁄ ⁄ -like) strains. these strains are genetically altered to attenuated, cold-adapted, and temperature-sensitive phenotypes, which limits viral replication to the nasal pharynx. each . ml dose has been formulated to contain approximately tcid ( . - . median tissue culture infectious dose) per viral strain. viral aerosols (flumist Ò ) were collected using two-stage bioaerosol samplers and two vertical reference samplers placed at the bottom of a calm air aerosol settling chamber, as described previously. the bioaerosol samplers were connected to personal air sampling pumps (model -pcxr ; skc, eighty four, pa, for aerosolization experiments, one dose ( . ml) of flu-mist Ò was diluted with . ml of saline ( . % nacl). one milliliter of this solution was initially drawn for assay, and the remainder placed in a collison single-jet nebulizer (bgi incorporated, waltham, ma, usa). the solution was aerosolized at kpa ( lbs ⁄ in ) air pressure, passed through a diffusion drier (model ; tsi incorporated, shoreview, mn, usa), and mixed with l ⁄ minute dry filtered air. the dry aerosol then flowed into the top of the calm air chamber. to avoid possible degradation of the virus by ozone, an unavoidable by-product, the bipolar ionizer employed in earlier studies was not used. instead, all conducting lines and the settling chamber itself were metal and grounded. previous tests with the calm air chamber indicated that these precautions were sufficient to avoid electrostatic aerosol deposition. the concentration and size distribution of the diluted aerosol was recorded using an aerodynamic particle sizer (aps model ; tsi), which drew air from a vertical probe placed at the same height as the bioaerosol sampler inlets. at the start of each experiment, the nebulizer was operated for minutes to allow the aerosol concentration to reach equilibrium. after minutes, the sampling pumps and vacuum source were activated simultaneously for all samplers. for aerosol collection studies, sampler pumps were switched off after , , , and minutes, while reference samplers were switched off after and minutes. after aerosol collection, the exterior of each sampler was disinfected (conflikt; decon labs, king of prussia, pa, usa) and the collection tubes and filters removed for analysis. a ml aliquot of the solution remaining in the nebulizer was also removed for analysis. the particle size-segregation characteristics of the twostage sampler were reported previously for a range of particle sizes. using this information, the particle size and number data from the aps, an estimate was made of the relative amounts of viral material that should have collected in the first and second tubes and on the back-up filter. this estimate was compared to the actual proportions of viral material found in each stage of the sampler as measured by qpcr (below) to indicate if the two-stage sampler was separating the aerosol particles based on size as expected. viral rna was extracted using the magmax tm viral rna isolation kit (ambion, austin, tx, usa). briefly, the supplied lysis ⁄ binding solution supplemented with carrier rna was used in the extraction of viral rna from either the neat flumist Ò (qpcr standards) or experimental samples (t and t ). the back-up filters (f) from the bioaerosol samplers were aseptically removed, immersed in ml of the supplemented lysis ⁄ binding solution and vortexed at moderate speed for minutes. viral lysis, rna binding, magnetic capture, washing, and drying of the rna-bound beads were all carried out according to the manufacturer's instructions. cdna was generated by reverse transcription of the isolated viral rna using taqman tm reverse transcription reagents (applied biosystems, foster city, ca, usa). in brief, ll of viral rna was added to the rt-pcr mixture containing ll of x rt buffer, ll of mm mgcl , ll of mm dntp mix, and ll of mm random hexamers. rnase inhibitor ( . ll, u ⁄ ml) and . ll of multiscribe reverse transcriptase ( u ⁄ ml) were added finally, for a final volume of ll. samples were briefly centrifuged, placed in a thermo hybaid thermocycler (ashford, uk) and run under the following conditions: °c for minutes, °c for minutes, and °c for minutes. a control without template was run for each experiment. as a pilot study to simulate the more biologically complex aerosols that one might encounter in field situations, flu-mist Ò was mixed with fungal spores and then aerosolized and collected. aspergillus versicolor (atcc , american type culture collection) was grown on malt extract agar for days at . °c and the spores isolated as previously described. spore concentrations were determined by hemacytometer count. for co-aerosolization, one dose of flumist Ò was diluted with . ml of . % nacl containing a. versicolor spores and ml of rnasecure reagent (ambion) to inhibit viral rna degradation. the aerosol was generated using a collison single-jet nebulizer, passed through a diffusion drier and mixed with dry filtered air using the same experimental procedure as described above. the aerosol was collected with two-stage samplers and two reference samplers. two of the two-stage samplers and one reference sampler collected the aerosol for minutes, while the remaining samplers collected the aerosol for minutes. genomic dna was extracted from a. versicolor-containing samples as described by griffin et al. briefly, ll of ap buffer supplemented with ll rnase a (dneasy plant mini kit; qiagen, valencia, ca, usa) was added to spores (qpcr standard) or experimental samples. filters were eluted in ml of the supplemented ap buffer, vortexed for minutes, and transferred to a microcentrifuge tube. approximately mg of . mm zirconia ⁄ silica beads (biospec products, inc., bartlesville, ok, usa) was added to each sample. samples were bead-beaten at maximum speed in a mini-beadbeater- (biospec products, inc.) for minute and chilled on ice for minutes. the bead-beating ⁄ cooling steps were repeated twice. samples were centrifuged for minutes at g and protein precipitation, washing, and drying of fungal dna was carried out according to the manufacturer's instructions. primer ⁄ probe design and optimization real-time pcr primers and probes were specifically targeted against influenza surface glycoproteins and designed using primer express software (applied biosystems). sequence information for a. versicolor was obtained from the u.s. environmental protection agency (http://www.epa.gov/microbes/moldtech.htm). synthesis of all primers and probes was performed by applied biosystems. table lists the sequence and dye labels for primers and probes used in qpcr detection. to determine the optimal primer concentrations for qpcr analysis, an optimization qpcr matrix was performed for each primer ⁄ probe combination (singleplex). optimal primer and probe concentrations used in qpcr detection ranged from to nm. for detection of influenza or a. versicolor in aerosol samples, ll of the generated viral cdna or fungal genomic dna, respectively, was added to ll applied biosystem's taqman tm universal pcr master mix. the appropriate concentration of primers and tamra-labeled probes was added, and brought to the final reaction volume of ll with pcr-grade water. all reactions were run using the applied biosystem's real-time pcr system at the following conditions: °c for minutes, °c for minutes, cycles at °c for seconds, and °c for minute. in order to quantify the relative amount of viral particles or fungal spores collected at each stage of the bioaerosol sampler, qpcr was performed in parallel using either serial -fold dilutions of cdna generated from a single dose of non-aerosolized flumist Ò containing approximately tcid per influenza strain or genomic dna isolated from spores. a negative control without template was included in all qpcr reactions. statistical analysis was conducted on the experimental setup as repeated-measures anova, with main effects of stage and time. stage is a three-level categorical variable with factors tube , tube , and back-up filter. to determine the effect of time, measurements were taken at , , , and minutes. proc mixed in sas v . (sas institute, cary, nc, usa) was used to analyze the data for significance and to calculate regression parameters. proc mixed was also used to insure similarity of experiments by testing variability between replicates. results were considered significant if p < . . standard curves were generated for influenza strains h n and h n using serial -fold dilutions of cdna isolated from a single dose of flumist Ò ( tcid ⁄ viral strain). for qpcr detection of a. versicolor, a standard curve was generated using genomic dna isolated from spores and serially diluted -fold. the standard curves for h n , h n , and a. versicolor were linear over a -log range of . · - . · particles. we were unable to generate a reproducible signal for the b strain virus using the specific primers that were designed. standard curves were used to extrapolate relative viral or spore numbers in unknown samples using the titer reported by the manufacturer or hemacytometer counts, respectively. when flumist Ò was aerosolized the virus-laden aerosol in the calm air chamber typically contained about particles ⁄ cm with a median aerodynamic diameter of . lm and a geometric standard deviation of . . as part of methods development, the optimal back-up filter composition for virus collection was determined. four bioaerosol samplers were fitted with different types of filters -gelatin, glass fiber, ptfe, and polyvinyl chloride. following a minute collection, several gelatin filters were found to be fractured. during rna extraction, the glass fiber filters released a high number of particulates that presumably interfered with qpcr detection. based on an initial experimental run, the ptfe filter was found to be optimal for the recovery of viral particles ( figure ). all subsequent aerosol experiments were performed with the ptfe filters. to characterize the collection efficiency of the bioaerosol sampler, three identical experiments were performed. figure (a, b) illustrates the collective results for viral aerosols collected at , , , and minutes. for both h n and h n , collection of viral particles with the two-stage sampler was linear up to minutes. viral particle counts were significantly greater at minutes in comparison with the , , and minute collection times (p < . ). furthermore, as predicted by the aerodynamic particle size data, the greatest proportions of the viral particles were detected in t and the back-up filter f, regardless of the collection time. for strain h n at minutes, % of the viral particles were detected in stage t and f, while only % of particles were detected in t . likewise, with strain h n , % of the particles were detected in stages t and f with % of the particles detected in stage t . these results were consistent throughout all experiments and demonstrate the ability of the bioaerosol sampler to separate particles based on aerodynamic size. while a similar distribution of h n particles was recovered, for unknown reasons the absolute particle numbers were significantly lower. to examine the effectiveness of the bioaerosol sampler to separate a mix of aerosolized particles, we co-aerosolized flumist Ò with a. versicolor spores (aerodynamic diameter . lm). following minutes of aerosolization (figure ), the bioaerosol sampler was able to separate these particles of differing size. both qpcr and spore counts confirmed that stage t retained % of the a. versicolor spores while stage t , with % of the spores, was found to contain the greatest amount of viral particles ( %). an overall shift in the deposition of viral particles toward t was observed when co-aerosolized with a. versicolor spores ( %- %). significantly fewer viral particles were detected on the back-up filter f ( %) in comparison with the back-up filter f using flumist Ò alone ( %), suggesting some interaction or aggregation of the particles during aerosolization. the need for rapid and accurate methods for detecting airborne viruses has increased in recent years, particularly following the reported outbreaks of avian influenza (h n ) and sars. various aerobiological monitoring studies have shown a high degree of variability with capturing, recovering, and detecting aerosolized viral particles in environmental and clinical samples. , - using the niosh bioaerosol sampler, we were able to overcome some common viral particle sampling limitations and fractionate aerosolized influenza particles from an artificially generated aerosol. bioaerosols vary in size, concentration, composition, and settling times. , an aerodynamic particle sizer was used to monitor the concentration and size distribution of the flumist Ò aerosol within the settling chamber. using the bioaerosol sampler, it could be demonstrated that capture, recovery, and subsequent detection for each sampler stage (t , t , f) were consistent with the expected values based on particle size. as anticipated, qpcr results confirmed that a significant number of the aerosolized viral particles were localized within stages t and f. these results are in agreement with the sampler cutoff size of . and < lm for stages t and f, respectively. the small number of aerosolized particles detected in stage t can be attributed to the collection efficiency of the bioaerosol sampler. the engineering design of the bioaerosol sampler is critical in the size-fractionation of bioaerosols, but sampling time must also be taken into consideration. qpcr results of aerosolized viral samples collected at , , , and minutes confirms that a sampling time of minutes yields the highest quantity of viral particles; however, this does not necessarily represent an ideal environmental sampling time as the quantities of aerosolized influenza virus expelled from patients have not yet been evaluated. in aerobiological studies using different viral strains, , it was found that prolonged sampling periods resulted in decreased viral recovery. likewise, because influenza viruses are stress sensitive, the possibility exists that lengthier sampling times may result in the desiccation of viral-laden aerosol and consequently compromise stability. future studies will aim at addressing the effects of prolonged sampling time on viral recovery and stability. using the bioaerosol sampler, the collection of aerosols within disposable microcentrifuge tubes limits sample loss and contamination. moreover, samples are directly processed within the respective tubes and further analyzed by different diagnostic methods including immunoassays or molecular detection techniques such as qpcr. currently qpcr is the preferred methodology for the rapid and sensitive detection of viruses; however, several limitations still exist. the sensitivity and specificity of qpcr detection is limited to the targeted template. in this study, primers and probes were designed to selectively amplify the ha and na genes from strains h n and h n , respectively. interestingly, the results showed a considerable difference in the number of viral particles that were detected for strains h n and h n . the flumist Ò vaccine is formulated so as to contain an equivalent concentration of each viral strain using the median culture infectious dose (tcid ) assay but does not account for non-viable viral particles. qpcr can detect viral cdna from both viable and non-viable viral particles, and perhaps explain this discrepancy. as for the qpcr detection of strain b, erratic results were obtained. as qpcr detection was successful for both strains h n and h n , this may not be due to the presence of inhibitors in the reaction but instead is more likely due to poor primer or probe design or the presence of secondary ( ) and flumist Ò were co-aerosolized together into the calm air chamber. samples were collected at minutes and analyzed for influenza viruses using qpcr and a. versicolor using qpcr and hemacytometer counts. data for each stage is presented as the percentage of total number of particles collected in the three stages. spore counts were the average of eight replicate hemacytometer counts. values for the flumist Ò with no fungal spores were taken from the previous experiment presented in table and represent the combined average values for h n and h n strains. structures in the influenza b rna, resulting in poor reverse transcription and insufficient target template. environmental bioaerosols vary considerably and may consist of a number of different microorganisms including viruses, bacteria, and fungi. recent findings by lindsley et al demonstrated that the two-stage bioaerosol sampler was effective at collecting and separating aerosolized fungal spores and fragments. results of the current study provide supporting evidence that the bioaerosol sampler is able to successfully recover and size-fractionate viral-laden aerosols. the culmination of these results suggests that the bioaerosol sampler would be an ideal air-sampling device for the aerobiological monitoring of various microorganisms within occupational environments. however, preliminary findings from a co-aerosolization study suggest fractionation of environmental samples may be more complex. namely, an overall shift in the viral particle deposition in the presence of a. versicolor spores was observed. the shift in viral particle deposition may be attributed to the attachment of viral particles to a. versicolor spores. aps data (not shown) further suggest that particles adhere to one another. given that the viral and spore-laden solution is pre-mixed prior to aerosolizing, it remains unclear as to whether the binding may occur before, during or after aerosolization within the calm-air settling chamber. future studies will further elaborate on the use of the bioaerosol sampler to capture and effectively size-fractionate co-aerosolized particles of different species. currently, there are conflicting views as to whether influenza viruses are spread by direct contact with secretions, large droplet transmission, or aerosol transmission. due to the lack of virtually any environmental data, aerosol transmission of influenza viruses is often overlooked as a possible mode of transmission. in this study, by aerosolizing flumist Ò , we demonstrate the recovery of aerosolized viral particles using the bioaerosol sampler and detection of influenza by qpcr. whether aerosolized influenza particles are a significant contributor to influenza transmission in work environments and the community remains to be determined. future experiments will focus on studying the dissemination of viral-laden aerosols utilizing an artificial cough generator to simulate cough dispersal of influenza viruses within a room-sized aerosol chamber. these findings would significantly contribute toward understanding the transmission of aerosolized influenza viral particles. furthermore, we will test the bioaerosol sampler in a healthcare setting to monitor the prevalence of airborne influenza viral particles and to study the transmission of influenza via the inhalation of aerosolized viral particles. such studies will help to further elucidate the routes of transmission of influenza and would ultimately contribute to better patient management as well as improve infection control guidelines and decrease worker health risk. avian flu to human influenza transmission of influenza: implications for control in health care settings toward understanding the risk of secondary airborne infection: emission of respirable pathogens review of aerosol transmission of influenza a virus the origins of pandemic influenza-lessons from the virus the severe acute respiratory syndrome sampling methodologies and dosage assessment techniques for submicronmetre and ultrafine virus aerosol particles new personal sampler for viable airborne viruses: feasibility study development of a personal sampler for collecting fungal spores a two-stage cyclone using microcentrifuge tubes for personal aerosol sampling design and use of a settling chamber for sampler evaluation under calm-air conditions a rapid and efficient assay for extracting dna from fungi the value of a database in surveillance and vaccine selection long-term sampling of viable airborne viruses collection efficiencies of aerosol samplers for viruscontaining aerosols optimization of a sampling system for recovery and detection of airborne porcine reproductive and respiratory syndrome virus and swine influenza virus on-line monitoring and identification of bioaersosols air sampling for the detection of exotic newcastle disease virus in poultry houses spincon based air sampler foot and mouth disease virus test report. cambridge: lares corporation molecular diagnosis of influenza a simple method of estimating fifty percent end points this work was supported by internal funds from the health effects laboratory division. the findings and conclusions in this report have not been formally disseminated by the niosh and should not be construed to represent any agency determination or policy. key: cord- -s rxzm t authors: burnouf, thierry title: modern plasma fractionation date: - - journal: transfus med rev doi: . /j.tmrv. . . sha: doc_id: cord_uid: s rxzm t protein products fractionated from human plasma are an essential class of therapeutics used, often as the only available option, in the prevention, management, and treatment of life-threatening conditions resulting from trauma, congenital deficiencies, immunologic disorders, or infections. modern plasma product production technology remains largely based on the ethanol fractionation process, but much has evolved in the last few years to improve product purity, to enhance the recovery of immunoglobulin g, and to isolate new plasma proteins, such as α -protease inhibitor, von willebrand factor, and protein c. because of the human origin of the starting material and the pooling of to donations required for industrial processing, the major risk associated to plasma products is the transmission of blood-borne infectious agents. a complete set of measures—and, most particularly, the use of dedicated viral inactivation and removal treatments—has been implemented throughout the production chain of fractionated plasma products over the last years to ensure optimal safety, in particular, and not exclusively, against hiv, hepatitis b virus, and hepatitis c virus. in this review, we summarize the practices of the modern plasma fractionation industry from the collection of the raw plasma material to the industrial manufacture of fractionated products. we describe the quality requirements of plasma for fractionation and the various treatments applied for the inactivation and removal of blood-borne infectious agents and provide examples of methods used for the purification of the various classes of plasma protein therapies. we also highlight aspects of the good manufacturing practices and the regulatory environment that govern the whole chain of production. in a regulated and professional environment, fractionated plasma products manufactured by modern processes are certainly among the lowest-risk therapeutic biological products in use today. protein products fractionated from human plasma are an essential class of therapeutics used, often as the only available option, in the prevention, management, and treatment of life-threatening conditions resulting from trauma, congenital deficiencies, immunologic disorders, or infections. modern plasma product production technology remains largely based on the ethanol fractionation process, but much has evolved in the last few years to improve product purity, to enhance the recovery of immunoglobulin g, and to isolate new plasma proteins, such as a protease inhibitor, von willebrand factor, and protein c. because of the human origin of the starting material and the pooling of to donations required for industrial processing, the major risk associated to plasma products is the transmission of blood-borne infectious agents. a complete set of measures-and, most particularly, the use of dedicated viral inactivation and removal treatments-has been implemented throughout the production chain of fractionated plasma products over the last years to ensure optimal safety, in particular, and not exclusively, against hiv, hepatitis b virus, and hepatitis c virus. in this review, we summarize the practices of the modern plasma fractionation industry from the collection of the raw plasma material to the industrial manufacture of fractionated products. we describe the quality requirements of plasma for fractionation and the various treatments applied for the inactivation and removal of blood-borne infectious agents and provide examples of methods used for the purification of the various classes of plasma protein therapies. we also highlight aspects of the good manufacturing practices and the regulatory environment that govern the whole chain of production. in a regulated and professional environment, fractionated plasma products manufactured by modern processes are certainly among the lowest-risk therapeutic biological products in use today. a elsevier inc. all rights reserved. c ollected human plasma may be used as a therapeutic product (known as bclinical plasmaq or bfresh frozen plasmaq) or as source material for the production of pharmaceutical fractionated products (also called bplasma productsq or bplasma derivativesq). this complex biologic material contains hundreds of proteins covering a myriad of physiological functions. many components still have undiscovered roles. the most abundant proteins, albumin and immunoglobulin (ig) g, are present at about and g/l, respectively, representing about % of all plasma proteins. less abundant proteins include the protease inhibitors, like a -antitrypsin (aat) ( . g/l) and antithrombin (at) ( mg/l), and the coagulation factors such as factor viii (fviii) (a few ng/l), which exhibit potent physiologic activity. currently, about different plasma protein therapeutics are used for treating life-threatening diseases or injuries associated to bleeding and thrombotic disorders, immunological diseases, infectious conditions, as well as tissue degenerating diseases, thus addressing the clinical needs of countless patients. an updated list of the major therapeutic applications of plasma protein products can be found elsewhere. this industrial process used to isolate therapeutic plasma proteins is known as bfractionation.q over to million liters of human plasma are fractionated each year in the world, in batches of several thousand liters, in about factories. modern plasma fractionation combines manufacturing steps to isolate, in a sequential and integrated manner, the crude fractions that are further purified into individual therapeutic products. validated dedicated steps inactivate and/or remove infectious agents potentially present in the starting plasma pool. this sophisticated industrial process is performed under highly hygienic conditions in licensed facilities (plasma fractionation plants) that are operated in compliance with good manufacturing practices and following quality assurance principles. over the years, plasma fractionation has evolved from a medical service activity mostly oriented toward the needs of local communities into a global manufacturing industry conforming to high regulatory standards. these strict requirements start from the collection of plasma for fractionation and include product manufacture and distribution steps. in this article, we review the most current practices encompassing the collection of plasma for fractionation, the core industrial plasma fractionation process, and the purification and pathogen reduction technologies of individual plasma products. the practices used for the collection of plasma for fractionation have direct influence on the safety profile of protein products since individual donations contribute to large plasma pools used to manufacture therapeutic preparations intended for hundreds or even thousands of patients. it is therefore logical that the production of plasma is regarded as an integral part of the manufacture of modern fractionated products. collection requirements of plasma for fractionation may differ from those relevant to fresh frozen plasma. in a regulated environment, plasma for fractionation is collected by licensed/registered blood establishments (blood centers and apheresis collection centers) that are inspected by the relevant national regulatory authorities (nras). compelled by the same safety concerns, the plasma fractionators conduct audits to verify that the contractual plasma collection and quality and safety measures, agreed upon with the plasma supplier, are met. areas of specific relevance include (a) procedures for donor screening and donation testing; (b) labeling, documentation, and traceability requirements; and (c) the handling of blood and plasma. such information is part of the marketing license of plasma products and, in europe, is assembled into a document called the bplasma master file.q various requirements for the collection of plasma for fractionation have been described in various guides, eg, from the pharmaceutical inspection convention and pharmaceutical inspection cooperation scheme (jointly referred to as pic/s), us food and drug administration (fda) and in recent world health organization recommendations. they are summarized below. candidate donors are provided with educational materials and undergo a medical interview to establish the absence of risks or signs of infections and to prove compliance for a donation of plasma for fractionation (table ) . potential donors presenting a health hazard are asked to exclude themselves. medical information of donors is acquired and archived. continuous epidemiologic surveillance of the donor population is being required in some jurisdictions. it helps to establish the background level (prevalence and incidence) and trends of known infectious markers (eg, hiv and antibodies, hepatitis c virus [hcv] antibodies, and hepatitis b surface antigen [hbsag]) in the population. this is also of interest for the early detection of emerging diseases, allowing early implementation of counter measures (such as more stringent donor screening processes or requirements for additional testing procedures). donors eligible to donate plasma for fractionation are individuals who meet donation criteria (such as age and donation frequency), do not present risk factors of blood-born infectious agents, and comply with requirements defined by the plasma fractionator and the nras of the country of plasma collection and of use of the products. in most situations, eligibility of whole blood donors and apheresis donors overlap, apart from donation frequency which is higher for plasmapheresis donors. eligibility criteria take into account scientific information about the risks of transmission of infectious agents by pooled plasma products (which may differ from those by blood components). special criteria may exist for the collection of hyperimmune plasma (used to make hyperimmune igg preparations), such as procedures for donors' immunization and minimal antibody titer. currently, about % of the plasma fractionated in the world is obtained by centrifugation of whole blood (brecoveredq plasma), and % is obtained by apheresis. blood/plasma collection, processing, and storage may affect plasma quality, as well as having an impact on the recovery of the most labile proteins such as fviii. in particular, risks of activation of the coagulation, complement, and fibrinolytic systems, which may lead to generation of plasma proteases, should be avoided. to better preserve the integrity of recovered plasma and limit risks of activation of the coagulation cascade and of cellular components, (a) good mixing of the blood with the anticoagulant solution (a sodium citrate based solution ) should be ensured from the initiation till the end of the collection process; (b) the duration of the collection should not exceed minutes; and (c) temperature variations of the blood should be avoided. a few hours after donation, whole blood is subjected to a centrifugation that separates the cellular elements (most specifically red cells) from plasma. the mean plasma volume obtained from one whole blood donation is about ml but varies depending upon the volume of collected whole blood (most often - ml) and donor's hematocrit. apheresis plasma (also called bsource plasmaq) is collected from donors through a process where blood is removed from the donor, anticoagulated (generally with a % sodium citrate solution), and immediately separated by physical means (centrifugation or filtration, or a combination of both) into components. at minimum, the red cells are returned to the donor while plasma is retained and collected in a container (bag or plastic bottle). the duration of a typical plasmapheresis procedure depends on the number of cycles (and, hence, the volume of plasma collected) and lasts generally from to minutes. apheresis plasma volume may range from to ml, depending upon the country's regulations and collection protocol. apheresis plasma can also be prepared as a byproduct of plateletpheresis (bconcurrent plasmaq), a procedure used primarily for the collection of platelets. both recovered and apheresis plasmas are suitable for the manufacture of the whole range of fractionated plasma products. the mean content in coagulation factors, more particularly fviii, is lower in recovered than in apheresis plasma because of (a) longer processing time before freezing (whole blood must be further processed to separate cellular components and plasma), (b) higher ratio of anticoagulant and, possibly, (c) the higher level of cellular contamination that may release proteolytic enzymes affecting the stability of coagulation factors. apheresis plasma contains less igg when collected from frequent donors. protein content and quality of fractionated proteins is apparently not affected by the apheresis system used, although residual cell content differ based upon the type and configuration of the cell separation device. plasma from membrane apheresis procedures, as does recovered plasma prepared from whole blood leukoreduced on positively charged filters, may performed by most fractionators. ymandatory in europe for hcv. zmay contribute to viral clearance but does not necessarily result in robust and consistent removal. §for small viruses, robust removal is achieved by narrow pore size membranes (v nm). texpected contribution based on experimental studies using spiked tse agents, in the absence of information of the biological nature of the tse-human plasma associated agent. contain more activated complement component and (c a, c a) anaphylatoxins, - but the impact on the quality or yield of fractionated products is unknown. various infectious agents have been identified as potential contaminants of human blood. bacteria, parasites, and intracellular viruses are not transmitted by plasma products because they are destroyed by freeze-thaw steps or removed by the . -to -lm filtration steps used during the processing of fractionated products. pathogenic plasma-borne viruses include hiv, hcv, hepatitis b virus (hbv), west nile virus (wnv), hepatitis a virus (hav) and parvovirus b (b ). the various complementary safety nets in place during the production chain of fractionated products, from donor selection to industrial product extraction, to optimize safety against these agents are summarized in table . the importance of viral testing on the safety of plasma products has been reviewed. , , the extent of viral testing of plasma for fractionation takes into account the ability of validated fractionation processes to eliminate viral risks. some testing is performed by blood establishments, other by plasma fractionators (table ) . individual plasma donations must be negative for anti-hiv and , anti-hcv, and hbsag. genomic assays of plasma minipools for nonenveloped hav and b may be performed. , relevance of testing for the absence of hiv p ag or wnv nucleic acid testing (nat), which may be justified for the safety of non-virally inactivated blood components, is arguable for plasma for fractionation subjected to robust viral reduction steps of enveloped viruses. the industrial manufacturing pool (usually the cryo-poor plasma that is the first homogeneous pooled plasma fraction) is also tested to confirm the absence of serologic and/or genomic viral markers of hiv, hbv, hcv, hav, and b . in spite of the most rigorous donor screening and donation testing, infectious viruses may still be present in plasma fractionation pools. therefore, the viral inactivation-removal steps that have been deliberately introduced during plasma products manufacture-and that are described below-play a most critical role in ensuring safety. altogether, these overlapping tests should ensure that the viral load of the manufacturing should is both minimal and significantly below the viral reduction capacity of the manufacturing processes used. preserving fviii during blood/plasma collection and preparation is important for most fractionators preparing coagulation factor concentrates. apheresis plasma can generally be frozen quickly, ensuring optimal fviii preservation. by contrast, whole blood has to be centrifuged to separate the various components. when blood is cooled to c after collection, plasma should be separated and frozen within to hours to preserve fviii, but when cooled rapidly at constant c using devices like butanediol plates, coagulation factors are stable for up to to hours. plasma frozen within hours is suitable for igg and albumin production. after separation from cellular elements, plasma for fractionation should be frozen rapidly below À c, À c, or À c, depending upon local regulations. plasma used to manufacture only albumin and igg may be frozen below À c within hours of collection. in the us code of federal regulations, apheresis plasma should be stored at À c or colder immediately after collection. rapid plasma freezing, to ensure rapid ice front velocity and core temperature of À c, preserves fviii and appears more important than the actual freezing temperature itself. plasma for fractionation is stored at less than À c, or colder, typically for several months or more. storage temperature should be as constant as possible, including the transportation to the fractionation facilities. cross-continent or intercontinent shipment of plasma for fractionation is frequent. production steps taking place at fractionation plants are summarized in figure , and typical plasma protein downstream methods are presented in table . physical compliance with shipping requirements is verified at delivery of the plasma frozen plasmas are expelled from the plastic containers and pooled for cryoprecipitation and further manufacturing steps, as described below. intermediate fractions generated during production may be stored for subsequent pooling and processing. purified sterile-filtered products are aseptically dispensed into final containers (glass vials or bottles). albumin bottles undergo terminal pasteurization. many products, but albumin and some igg preparations, are freeze-dried, typically for a duration of to days, depending upon physicochemical characteristics and filled volumes. batches are quarantined while quality controls and checks of production files take place. batches meeting specifications are labeled and packaged and subsequently boxed and shipped for distribution. in a few countries, product batches may be released by regulatory authorities. the production cycle of fractionated products takes a few weeks to several months. current core fractionation technology largely relies on a backbone process encompassing cryoprecipitation and cold ethanol precipitation steps, as developed in the s by cohn et al in the united states, or modified by kistler and nitschman in europe. this process involves successive processing steps at defined ethanol concentrations, associated with shifts in ph, temperature, and osmolality that result in selective precipitation of proteins, most notably igg and albumin. precipitates are separated by centrifugation or filtration. in the last few years, the complexity of the fractionation process has increased by (a) the introduction of chromatography to isolate new proteins from existing fractions such as cryoprecipitate, cryo-poor plasma, and cohn fractions; (b) the integration of chromatography to the ethanol fractionation process to increase igg recovery; and (c) the implementation of dedicated viral inactivation or removal steps. chromatography was introduced in the s; however, its application developed mostly in the mid/late s. anion-exchange chromatography and affinity chromatography are frequently used to capture proteins at physiological ph and ionic strength, therefore best preserving functional activity. , immobilized heparin and monoclonal antibodies are common affinity chromatography ligands. chromatography is used for specific goals: (a) improvement of products purity, (b) extraction of trace labile proteins, (c) optimization of protein recovery, and (d) removal of viral inactivation agents. figure illustrates a typical industrial fractionation scheme of standard plasma. plasma packs (typically for a batch of - l) are opened under hygienic conditions, and plasma is expelled from the containers and thawed at c to c. cryoprecipitate is isolated using refrigerated continuous centrifuges, recovered from the centrifugation bowls and frozen at À c or colder for storage until further pooling and processing. the cryo-poor plasma is immediately processed for primary chromatographic capture of labile coagulation factors (such as the factor ix [fix] complex and its components) and protease inhibitors (such as at and c esterase inhibitor [c -inh]). the prepurified intermediates may be stored frozen until further processing. the coagulation factors/ anticoagulant-depleted plasma undergoes sequential ethanol precipitation steps. this leads to successive precipitations of fibrinogen, igg and albumin fractions, and intermediates for extraction of other therapeutic proteins, such as aat (fraction iv- ), or igm (fraction iii). depth filtration is preferred to centrifugation to separate precipitates and improve protein recovery. the fractionation of hyperimmune plasma (eg, anti-rhesus) is usually performed on small plasma batch sizes, increas- ingly using full chromatographic processes to optimize the recovery of igg. no documented transmission of hiv, hbv, or hcv by products subjected to dedicated viral inactivation treatments has been recorded since the end of the s. viral reduction treatments include inactivation steps (where viruses are bkilledq) and removal steps (where viruses and proteins partition into distinct fractions). use of one, or preferably two, distinct dedicated viral reduction treatments is the current bgold standardq for all plasma products. the first treatment is performed primarily to inactivate the most pathogenic viruses (hiv, hbv, and hcv), whereas the second reduction step targets nonenveloped viruses but also contributes to added safety against all agents. most viral reduction treatments are integrated with the protein fractionation process (bin-processq treatments), but some currently based on heat inactivation procedures are applied on products filled in their final container (terminal treatment). fractionators are required by regulatory authorities to conduct down-scale experimental validation studies using relevant model viruses to establish the efficacy and robustness of viral reduction procedures. the robustness of the viral reduction procedures in place is exemplified by the absence of transmission of wnv, an emerging virus. similarly, the lipid-enveloped severe acute respiratory syndrome coronavirus has been shown to be inactivated by core viral inactivation treatments of plasma products. , it is also likely, but not proven by validation experiments yet, that avian flu and simian foamy viruses, which also have a lipid envelope, would be inactivated by current processes in place if present in plasma. details on viral validation procedures can be found elsewhere. , the major characteristics of current viral reduction treatments are summarized in table , and discussed briefly here. in-process viral inactivation treatments. the solvent-detergent (sd) treatment, developed in the mid s, remains the most frequent core viral inactivation procedure of plasma products. protein solutions are incubated for to hours at c to c in the presence of . % to % tri-n-butyl phosphate (tnbp) and % tween- or triton x- . typically, lipid enveloped viruses are inacti-vated in a matter of minutes, and the functional activity of even the most labile plasma proteinswith the possible exception of some serine prolease inhibitors-is well preserved, but nonenveloped viruses (less pathogenic in most individuals) are not inactivated. the sd agents are removed down to a level of a few parts per million usually by chromatographic adsorption or specific precipitation of proteins, or selective adsorption on hydrophobic chromatographic support. pasteurization, another common viral inactivation procedure, is a heat treatment of protein solutions for hours at c, a treatment that denatures viral proteins and inhibits virus replication. pasteurization can inactivate both enveloped and nonenveloped viruses, but stabilizers, needed to limit loss of protein functionality, may decrease the rate and extent of viral inactivation. stabilizers may be removed by ultrafiltration, protein precipitation, or chromatography. vapor heat has also been used by one company; extent of virus inactivation is influenced by the temperature, duration, and pressure during treatment. risk of neoantigen formation, which can enhance protein immunogenicity, should be considered when using heat-based inactivation processes. low ph incubation, usually at ph , at c to c for more than hours, was introduced in the early s to allow the intravenous infusion of igg. this form of treatment was subsequently found to inactivate most lipid-enveloped viruses. caprylic (octanoic) acid precipitation/incubation at ph below is a recently introduced treatment of human igg that can inactivate lipid-enveloped viruses. , terminal viral inactivation treatments. in modern plasma fractionation, heat treatment of lyophilized products (dry heat) is used, due to limitations, mostly as a secondary viral inactivation step rather than the core inactivation treatment. the treatment is applied to some coagulation factor concentrates. performed at c for hours or at c for minutes, generally in the presence of protein stabilizers, it provides added safety against havand other heat-sensitive viruses but may not be sufficient to exclude b transmission. terminal (liquid) pasteurization at c for hours is the bgold standardq treatment of albumin preparations. the fatty acids, caprylate, and tryptophanate, which protect albumin from heat denaturation, are added at doses compatible with therapeutic use and, therefore, are not removed before product infusion. viral removal treatment. nanofiltration is a specific viral filtration process applied to protein solutions using -to -nm multi-layers membranes, or equivalent systems, to remove viruses mostly by a sieving mechanism. , introduced in the early to mid s, it has reached wide acceptance as a robust viral removal step for essentially all products, apart from albumin. nanofiltration is used to complement the core viral inactivation treatment and to provide enhanced safety against nonenveloped viruses or other resistant infectious agents. virus removal can also incidentally take place during protein precipitation, chromatography, or filtration steps; these steps contribute to the lowering of virus load from the protein production stream; they are difficult to monitor; and therefore do not guarantee, as standalone procedures, sufficient safety margin. prion removal methods. variant creutzfeldt-jakob disease (vcjd) can be transmitted by red blood cell concentrates, but to date, transmission has not been identified from plasma or plasma products. because of its biological nature, the prion agent is thought to be resistant to current viral inactivation procedures used during plasma fractionation. the methods known to inactivate abnormal misfolded prion proteins associated with transmissible spongiform encephalopathies (prp tse ) (such as oxidation, treatment with strong base, chaotropic agents, and extreme heat) destroy plasma proteins and, therefore, cannot be used. still, modern processes of fractionated products would seem to ensure significant removal of prp tse , as suggested by experimental spiking studies. as scale-down and experimental transmissible spongiform encephalopathy (tse) spiking models are developed, knowledge on the capacity of manufacturing processes of plasma proteins to remove prions is growing rapidly, although much uncertainty remains because the unknown biological nature of the human plasma associated infectious agent. several processes for manufacturing fviii, fibrinogen, von willebrand factor (vwf), fix, igg, and albumin products have been shown to be capable of removing prions in a consistent and reproducible manner. [ ] [ ] [ ] generally, multistep fractionation processes are thought to be a contributing factor to prp tse elimination. two to more than log removals of spiked prions occur during standard protein purification steps such as precipitation with ethanol or polyethylene glycol (peg), anion-exchange chromatography, and depth filtration. , , mechanism of removal that may encompass an adsorption mechanism is still not fully understood and appears to be influenced by ph and the concentration of the precipitating agent. the partitioning process may reflect some prion aggregation because of prion hydrophobicity and insolubility. the removal capacity of nanofiltration membranes with pore sizes less than or nm has been extensively investigated, , and prion removal is likely due in part to molecular sieving. extent of removal is related to the pore size of the filters and, presumably, to the aggregation state of prp tse . removal is superior with membrane of pore sizes of nm, compared to nm. the nature and origin of the spiking agent used for prp tse clearance studies is of critical importance because various spikes differ in size and characteristics. brain homogenate or brain-derived microsomal fractions from infected animals have usually been used as the source of prp tse spiking material. the most reliable and quantitative detection method of prp tse is based on animal bioassays, which require many animals and a time frame generally longer than months. in vitro immunochemical methods, such as a western blot assay, are being used, at least as a first marker of the presence of prp tse and associated infectivity, by detecting the protease-resistant fragment. thus, the risk transmission of vcjd by human plasma products appears remote, but caution should prevail since the biochemical nature of the infectious agent in human blood is not known. technologies to extract coagulation factors, protease inhibitors, and igg have evolved considerably in the last years, leading to the development of products with improved safety and purity profiles. a description of major manufacturing techniques is given below. factor viii. several generations of fviii preparations have been developed since the mid- s, providing (a) safety from hiv and then hcv and hbv, (b) improved purity, and (c) enhanced safety from hav and b . current development efforts focus on establishing prion removal. all currently licensed plasma-derived fviii concentrates are purified from cryoprecipitate. in a typical process, cryoprecipitate is subjected to a combination of aluminium hydroxide adsorption and precipitation, or precipitation only (eg, using glycine), to reduce the level of trace vitamin k coagulation factors (as they may activate fviii during the downstream purification steps) or load proteins such as fibrinogen. the purified cryoprecipitate extract usually undergoes viral inactivation typically by sd or pasteurization. many processes include subsequent chromatography by anion exchange, monoclonal antibody affinity (using anti-fviii or anti-vwf murine antibodies), or immobilized heparin affinity to remove protein contaminants (such as fibrinogen or fibronectin), most or part of the vwf, and the sd agents. immunopurified fviii eluate is further purified by chromatography to remove murine igg ligands that may have leached. prior to formulation and sterile filtration, some fviii products are nanofiltered using membranes with a pore size of , , or even nm if a partial dissociation of fviii and high-molecular-weight vwf multimers is initiated. alternatively, some freeze-dried preparations are subjected to heat treatment at c or c to inactivate nonenveloped viruses like hav. recovery of fviii, as expressed per liter of plasma, is usually comprised between and iu ( iu is defined as the physiological activity present in ml of plasma). factors lowering fviii yield include cryoprecipitation (ca % loss), chromatographic purification (ca % to %), and viral heat inactivation ( %- %). current fviii concentrates have a specific activity between and iu/mg. some products are formulated with human plasma-derived albumin, whereas others contain copurified vwf that helps stabilize fviii. purity of fviii concentrates has not been convincingly demonstrated to enhance immunological safety. long-term clinical experience indicates that the alleged reduced immunosuppressive effects of immunopurified preparations compared to lowerpurity plasma-derived preparations, as claimed in the late s, were probably unfounded. processes used that, in part, influence residual vwf, may have an impact on the immunogenicity of plasma-derived fviii products. retrospective studies of previously untreated patients show that an sd-treated, nanofiltered, ion-exchange purified fviii product containing vwf appears to be times less likely to induce anti-fviii inhibitors in hemophilia a patients than full-length recombinant fviii concentrates. von willebrand factor. because fviii chromatographic purification removes all, or part, of vwf, fviii products effective in treating von willebrand disease (vwd) are generally low-purity (bintermediate purityq) preparations prepared from cryoprecipitate by precipitation steps coextracting vwf and fviii in a ratio of higher than . the low purity and the high protein content of these products technically restrict the choice of viral inactivation treatments to pasteurization or terminal dry heat at c for hours. one highly purified vwf concentrate, largely devoid of fviii and, therefore, specific for vwd treatment, is prepared from cryoprecipitate by a -step chromatographic procedure (integrated with fviii and fibrinogen purification processes) using anion exchangers and immobilized gelatin polishing (to remove fibronectin). viral reduction is by sd, -nm nanofiltration, and terminal dry heat at c for hours. fibrinogen. there are registered fibrinogen preparations available for treating a fibrinogenemia or hypofibrinogenemia. traditional preparations are obtained by multiple precipitation steps of plasma or cryoprecipitate using ethanol and glycine, whereas other modern products are purified by chromatography. viral reduction is achieved by sd treatment, often complemented by -nm nanofiltration or terminal dry heat treatment. single-step pasteurization at c for hours is used for product. fibrin sealants. fibrin sealants (fibrin glues) comprise fibrinogen-rich and purified thrombin concentrates. when mixing the components, a strong adhesive clot exhibiting hemostatic, sealing, and healing properties is formed almost instantaneously or within a few seconds, offering multiple topical surgical applications. fibrinogen is prepared by precipitation methods from cryoprecipitate, or from the cohn fraction i; the fraction may also contain fibronectin, vwf, or factor xiii (fxiii), which may confer other physiological functions. fibrinogen fractions are virally inactivated by sd, pasteurization, vapor-heat treatment, and/or nanofiltration. the fibrinogen concentration is typically above g/l and may be formulated in the presence of an antifibrinolytic agent. prothrombin complex. prothrombin complex concentrate (pcc) is a mixture of vitamin kdependent coagulation factors in which fix, factor ii, and factor x and proteins c and s have a low specific activity between . and iu/mg. a few products contain also factor vii (fvii), but usually at levels lower than that of fix. the manufacture is usually based on a s method that involves diethylaminoethyl (deae) sephadex or deae cellulose adsorption of cryo-poor plasma, but downstream ethanol fractions can also be used as starting materials. anion exchangers coextract proteins sharing the presence of gamma-carboxyglutamic acid residues, whereas bulk plasma proteins, such as albumin and igg or at and aat remain in the unbound fraction. precipitation with tricalcium phosphate is used for one product. viral reduction is most often achieved by sd (tnbp-tween ) treatment, complemented by -or -nm nanofiltration or by terminal dry heat. pasteurization and vapor heat are applied to products. viral inactivation treatment by sd requires one subsequent ionexchange chromatographic step for removal of the virus-inactivating agents. recovery is in the range of to iu of fix per liter of plasma. single fix. high-purity fix products were developed in late , leading to reduced risks of thromboembolism, compared to pcc, in hemophilia b patients. fix is isolated by chromatographic purification of the pcc using anion exchange combined with either immobilized heparin, metal chelate affinity, or monoclonal antibody. these processes yield fix concentrates with a mean specific activity in the range of to iu/mg and a yield between and iu/l of plasma. factor vii. three specific concentrates rich in fvii and with reduced amount of other vitamin kdependent clotting factors are currently licensed to control bleeding in deficient patients. the manufacturing process includes ion-exchange chromatography or aluminium hydroxide adsorption, following a downstream procedure similar to that of pcc and fix. viral inactivation is achieved by sd treatment, vapor heat, or dry heat. factor xi. two factor xi (fxi) concentrates are currently available for deficient patients. one, of low purity, is purified by chromatography of cryo-poor plasma on deae cellulose and immobilized heparin. after freeze-drying, the product is virally inactivated by dry heat. in the other product, fxi is captured by adsorptive filtration then highly purified by cation-exchange chromatography. this product undergoes dual viral reduction processing by sd and -nm nanofiltration. factor xiii. factor xiii is a transglutaminase that catalyzes the final step in the coagulation cascade, cross-linking the loose fibrin polymer into a highly organized structure. the early generation of fxiii concentrates for the treatment of fxiiideficient patients was extracted from placenta, but plasma-derived products have subsequently been developed. one is purified from a cold-ethanol fraction from cryoprecipitate supernatant, purified by precipitation with sodium citrate and removal of fibrinogen by heating. the product is pasteurized in sorbitol solution, ultrafiltered to remove sorbitol, adsorbed with bentonite, and freeze-dried. the other product is obtained by precipitation steps and is also pasteurized. factor xiii is also a component of some fibrin sealants. although still subject to discussion, the presence of fxiii is claimed to contribute to fibrin c-chain cross-linking and tensile strength, possibly improving the hemostatic effect. activated coagulation factors. human thrombin concentrates are available so far only as components of fibrin sealant. thrombin is prepared by activation of the pcc, usually in the presence of calcium chloride, followed by viral inactivation treatment by sd, purification by cation-exchange chromatography, and viral removal by -nm nanofiltration. final thrombin concentration is usually between and iu/ml, but preparations with lower potency are available when a slower speed for clot formation of the sealant is preferred for some surgical applications requiring longer time for tissue gluing. a procedure for large-scale production of a purified plasma-derived fviia has been developed in japan. fvii is purified by anion exchange and immunoaffinity chromatography and converted to fviia by autoactivation on an anion-exchange resin and incubation in the presence of ca + for hours at c. this preparation is virally reduced by nanofiltration and dry-heating and is intended for the treatment of hemophiliacs with antibodies against fviii or fix. antithrombin. antithrombin concentrates were the first plasma products extracted by affinity chromatography. production from cryo-poor plasma usually comprises ion exchange chromatography to remove the pcc components, followed by capture of at on immobilized heparin. viral inactivation is traditionally achieved by pasteurization in the presence of sodium citrate or a combination of sucrose and glycine, although sd treatment is used as well. because heat treatment may partially denature at, a second adsorption step on immobilized heparin can be used to remove altered molecules. recovery is between and u/l of plasma. fraction iv- is an alternative starting material, but yield is significantly lower. a -antitrypsin (a -protease inhibitor). there are now several licensed aat concentrates, including in the united states. a -antitrypsin augmentation therapy is indicated for the treatment of patients with lung emphysema secondary to congenital aat deficiency. because aat shares many physicochemical properties, in particular, molecular weight and isoelectric point, with albumin, it has been difficult to design production methods for aat not affecting the existing production process for albumin. most preparations are recovered from fraction iv [ ] [ ] [ ] [ ] . purification from this waste fraction is rather cumbersome and involves peg precipitation and ion-exchange chromatography, considerably compromising recovery (~ . g/l). the first preparations developed in the s were virally inactivated by pasteurization, but dual viral reduction steps using sd and nanofiltration are also now used. recent isoelectrofocusing studies have provided evidence indicating that new anodal aat variants are present in at least one of the fda-licensed product, suggesting alteration of some isoforms during purification. loss of a c-terminal positive charged lysine, secondary to carboxypeptidase activity, was proposed as an explanation for these isoelectrofocusing migration shifts. under circumstances when clinical demand for albumin decreases, extracting aat from upstream fractions, such as the supernatant ii+iii, appears a logical trend, offering the possibility of a more effective production scheme, characterized by a recovery of . to g/l. protein c. there are protein c concentrates manufactured in europe and in japan. in one process, the pcc undergoes cascade purification on ion exchangers, whereas in the other, immunoaffinity and affinity chromatographic processes are combined with ion exchange. viral reduction is achieved by sd treatment, which can be combined with -nm nanofiltration or vapor-heat treatment. c -esterase inhibitor. c -esterase inhibitor concentrates are used for the treatment of acute phases of angioedema, primarily in the oropharyngeal region and gastrointestinal tract in patients with congenital or acquired c -inh deficiency. there are products licensed in europe. products are generally purified by chromatography from the cryo-poor plasma after extraction of the pcc and, potentially, at. viral inactivation is achieved by pasteurization, vapor heat, or sd, possibly combined with nanofiltration. essentially all therapeutic albumin preparations are prepared by fractionation of cryo-poor (or pccpoor and/or at-poor and/or c -inh-poor) plasma by ethanol fractionation. a critical upstream process step (precipitation ii+iii) separates the igg fraction. to optimize recovery, the precipitates generated during the ethanol fractionation process are separated by depth filtration. albumin recovery of % to % ( - g/l) and purity of % to % are typically obtained. some processes combine ethanol fractionation with a polishing ion exchange chromatography, which generally improves product purity to ca %, whereas in one production method, albumin is purified mostly by anion exchange, cation-exchange, and size-exclusion chromatography. the adjustment of the concentration of the purified fraction, typically from % to %, is achieved by ultrafiltration. the standard viral inactivation method is pasteurization, which, according to most pharmacopeias, should be performed in the final container rather than on the albumin batch before aseptic filling. current mean albumin yield is to g/l of plasma. polyvalent igg preparations, either for intramuscular or intravenous uses, are traditionally prepared from the fraction ii that is obtained by stepwise fractionation of cryo-poor plasma using cold ethanol at concentrations up to %. increasingly, igg products are extracted from up-streamed ethanol precipitated fractions, such as supernatant iii or precipitate ii+iii, to optimize recovery. intermediate igg fractions are subjected to ion-exchange chromatography, caprylic acid, or peg precipitations to remove protein contaminants, proteolytic enzymes, and/or aggregates. most current viral inactivation procedures are low ph incubation, pasteurization, or sd; the caprylic acid treatment, recently introduced in the manufacture of human igg products, is also a robust viral inactivation process of igg. dedicated viral removal by -to -nm nanofiltration is commonly used to increase the safety against nonenveloped viruses, especially in a situation when the core viral inactivation treatments target only lipid-enveloped viruses. the igg recovery has long been in the . -to . -g/l range when combining traditional ethanol fractionation processes and centrifugation. depth-filtration and/or chromatographic purification from upstream fractions have improved the mean recovery to the . -to . -g/l range, or more. total chromatographic procedures are increasingly used for the production of hyperimmune igg products because such processes are amenable to the fractionation of smaller plasma volumes and can optimize recovery. the manufacturing process includes at least dedicated viral reduction treatments. other protein components have been fractionated at pilot-scale and subjected to experimental or human trials. inter-a-trypsin inhibitor (iti) is a kunitz-type serine proteinase inhibitor. its inhibitory capacity is carried by bikunin, a chondroitin -sulfate proteoglycan, which is covalently linked to its heavy chains h and h , but can be released by proteolytic cleavage. an iti concentrate has been obtained by fractionation of the prothrombin complex on an anion exchanger followed by immobilized heparin and viral inactivation by sd. iti, as a reservoir of bikunin, may be involved in control of inflammatory processes. in a porcine model of endotoxin shock, iti improved the hemodynamic, oxygenation, and coagulation parameters. administration of iti very early after the onset of sepsis or repeated injections at later time points ( and hours) maintains cardiovascular stability and significantly reduces mortality in a rat model. transferrin is the major iron binding plasma protein which may prevent cytotoxic effects or predisposition to septic infection due to accumulated free non-transferrin-bound iron when normal iron use is hampered and/or apotransferrin production is decreased. a liquid apotransferrin concentrate has been obtained from cohn fraction iv by ion exchange chromatographic steps and ultrafiltration. viral safety was ensured by sd treatment, nanofiltration, and peg precipitation. the product had intact iron binding capacity, and maintained the bacterial growth inhibitory effect in serum. in hematological stem cell transplant patients, the product prevented the appearance of non-transferrin-bound iron. apolipoprotein a-i is the principal protein component of the plasma high-density lipoproteins. it prevents the accumulation of cholesterol-loaded macrophages which deposit on the arterial wall as foam cells. apolipoprotein a-i inhibits hepatic lipase and lipoprotein lipase in vitro. a concentrate has been isolated by precipitation from cohn fraction iii. intravenous injections in men were well tolerated in an early clinical trial; clinical observations were consistent with combined inhibition of hepatic lipase and lipoprotein lipase activities. possible clinical applications include the treatment of hypercholesterolemic patients and atherosclerosis. recombination with lecithin forms a high-density lipoprotein complex that could help limit inflammation, endotoxin-induced activation of coagulation, and fibrinolysis in septic conditions. mannan-binding lectin (mbl) is a component of the innate (aspecific) immune system that can bind repetitive structures of mannan groups, such as those on the surface of micro-organisms, activating the complement system and leading to the destruction of a large variety of micro-organisms. the relatively frequent congenital deficiency of mbl is associated to recurrent infections, especially in infants when the specific immune system has not yet matured. an mbl concentrate has been produced from cohn fraction iii. plasmin is the major fibrinolytic enzyme in plasma. encouraging results as a new fibrinolytic agent have been obtained in animal models where plasmin was applied directly to the clot through a catheter to treat peripheral arterial occlusion. von willebrand factor cleaving protease (vwf-cp, adamts ) cleaves ultra-large multimers of vwf that enter the blood stream directly after biosynthesis by endothelial cells. if developed, a purified vwf-cp could be useful to treat, in place of plasma, patients with thrombotic thrombocytopenic purpura with congenital or acquired deficiency of vwf-cp. activated protein c can be of value for the treatment of sepsis, as demonstrated through the clinical use of a recombinant preparation. there is no therapeutic plasma-derived activated protein c products available yet for therapeutic use, although a process for a highly purified preparation, where cryo-poor plasma is purified by immunoaffinity and anion-exchange chromatographic steps, and sd viral inactivation has been described. there is also research needed to investigate alternative indications for currently available products. potential clinical use of c -inh, aimed at benefiting from its role as inhibitor or attenuator of the activation of complement and contact systems, include septicemia, myocardial infarction, capillary leak syndrome, pancreatitis, and organ transplantation. intravenous use of a fxiii concentrate was found to increase epidermal growth factor and transforming growth factor-b, suggesting that it may accelerate wound healing of anastomotic leaks and nonhealing fistulas. factor xiii was also found to have osteoinductive properties, suggesting use in bone tissue engineering. a review on plasma fractionation should also cover the role played by national and international regulatory authorities. over the last few years, assuring the safety of large-pool plasma products has posed formidable challenges to regulatory authorities and fractionators alike. the complexity of the field, encompassing the diversity in blood and plasma product types and manufacturing processes, made it difficult to enact balanced decisions toward ensuring both product safety and guarantee of supply. since the s, the agencies regulating the plasma fractionation industry have developed a comprehensive set of measures to ensure the viral safety of plasma products. multiple layers of regulatory oversight of the plasma industry have been established to ensure overlapping safeguards against the risks of the transmission of bloodborne infectious agents. several regulations, guidances, position statements have been issued by agencies like the us fda and the european medicine evaluation agency, which are updated as needed. those cover important safety aspects required at all stages of the manufacturing chain, from activities at blood establishment preparing plasma for fractionation, extending to the manufacturing and distribution of plasma products. regulatory oversight includes epidemiologic surveillance of the donor population; donor deferral policies and screening practices; mandatory donation testing; testing of manufacturing plasma pools; validation of viral reduction procedures and other production steps; as well as the assessment of product quality, safety, and efficacy for marketing authorization. most of relevant information on plasma collection is assembled in europe in the plasma master file, which allows establishing key levels of information regarding the quality and safety of the plasma raw material. post marketing, reports on adverse reactions associated with plasma products should be transmitted to nras and could prompt emergency response procedures and product recalls. some harmonization of the requirements for manufacture and supply of plasma products in the united states, europe, and japan is taking place under the auspices of the international conference on harmonisation, but much work remains to be done. such measures, nonetheless, provide the framework through which modern plasma products now exhibit a very high level of quality, safety, and efficacy. however, the rigidity of the regulatory system has been an impediment to more significant technological evolution of the plasma fractionation process because process changes are currently associated to major regulatory work. the cohn plasma fractionation method initially designed to obtain albumin has, over the years, developed rather successfully into a well-established industrial procedure isolating a wide range of clinically useful products. today, more than different protein products, and more if one considers the variety of hyperimmune igg preparations, can be extracted through large-scale fractionation of human plasma. the soundness and large-scale adaptability of the technology, on the one hand, and the rigidity of the current regulatory framework, on the other, explains why this technology remains the main core method in use at industrial scale, although it implies suboptimal yield for most proteins, apart from albumin. the technology has increased in complexity over the years, with the greatest progresses in purification being, without doubt, associated with the use of chromatographic methods that have made possible the development of new protein therapeutics and impressive improvements in product purity and quality. the fractionation scheme has also changed dramatically through the introduction of in-process viral reduction treatments, which have required the addition of downstream techniques such as chromatography and ultrafiltration. the change in protein drivers, with the prominent clinical role now played by igg, and the requirements to increase protein recovery and optimize the fractionation process may crystallize the incentive for most fractionators to abandon relative technical conservatism and introduce significant technological changes in processing technology. the production of igg from precipitate (i)+ ii+iii, already implemented by some manufacturers and, possibly, that of aat from supernatant (i)+ ii+iii, are signs of a gradual evolution in the direction of total chromatographic processing. with so much gained, over the last few years, in the understanding of the key parameters building plasma product quality, safety, and efficacy, one can hope that the regulatory paths, in particular, with regard to clinical studies, to license known protein therapeutics prepared by improved, more efficient technology should be simplified. this would, in turn, contribute to an improved supply. as the plasma fractionation industry has so far been targeting mostly proteins that were obvious candidates for replacement therapy, it is also hoped that it will invest more into developing new products because plasma remains a unique source of potential therapeutic proteins. proactive research and development work should be encouraged to isolate and evaluate new therapies among the well characterized plasma proteins with still unknown function. finally, one should not forget that plasma protein therapies are expensive and largely inac-cessible to the developing world. the new market drivers in rich countries are likely to diminish economical interest in the manufacture of fviii that remain a major protein therapy in need in the developing world. the belief that decreased use of plasma-derived fviii or fix in developed economies (as hemophiliacs are switching to recombinant therapies) would increase the supply of products to developing countries may be not economically viable. rather, this switch to recombinant products in rich countries can make poor countries unable to afford the increased cost of products no longer subsidized by the premium price paid in rich countries. it therefore remains important to develop affordable viral inactivation and processing technologies gradually allowing developing countries to make use of local plasma resources in a safe manner. guideline on epidemiological data on blood transmissible infections. for inclusion in the guideline on the scientific data requirements for a plasma master file current instrumentation for apheresis. apheresis: principles and practice assessment of complement activation during membrane-based plasmapheresis procedures the effect of leukocyte depletion on the quality of fresh-frozen plasma current safety of the blood supply in the united states farrugia a: guide for the assessment of clotting factor concentrates for the treatment of hemophilia. www.wfh.org. montreal, world federation of hemophilia preparation and properties of serum and plasma proteins. iv. a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids eight years experience with the alcohol fractionation procedure of nitschmann, kistler and lergier tabor e: the epidemiology of virus transmission by plasma derivatives: clinical studies verifying the lack of transmission of hepatitis b and c viruses and hiv type contribution and interpretation of studies validating the inactivation and removal of viruses (revised) inactivation of viruses in labile blood derivatives. i. disruption of lipidenveloped viruses by tri(n-butyl)phosphate detergent combinations pasteurization of antihemophilic factor and model virus inactivation studies enveloped virus inactivation by caprylate: a robust alternative to solventdetergent treatment in plasma derived intermediates evaluation de l'efficacité des procédés de purification des proteins plasmatiques à éliminer les agents transmissibles non conventionnels distribution of a bovine spongiform encephalopathy-derived agent over ionexchange chromatography used in the preparation of concentrates of fibrinogen and factor viii al: influence of the type of factor viii concentrate on the incidence of factor viii inhibitors in previously untreated patients with severe hemophilia a in vitro study of a triple-secured von willebrand factor concentrate fibrin sealant: scientific rationale, production methods, properties, and current clinical use properties of a highly purified human plasma factor ix:c therapeutic concentrate prepared by conventional chromatography large-scale production and properties of human plasma-derived activated factor vii concentrate effects of inter-alpha-inhibitor in experimental endotoxic shock and disseminated intravascular coagulation delayed administration of human inter-alpha inhibitor proteins reduces mortality in sepsis a minipool process for solvent-detergent treatment of cryoprecipitate at blood centres using a disposable bag system key: cord- -b dz lnn authors: domingo, esteban; perales, celia title: viral quasispecies date: - - journal: plos genet doi: . /journal.pgen. sha: doc_id: cord_uid: b dz lnn viral quasispecies refers to a population structure that consists of extremely large numbers of variant genomes, termed mutant spectra, mutant swarms or mutant clouds. fueled by high mutation rates, mutants arise continually, and they change in relative frequency as viral replication proceeds. the term quasispecies was adopted from a theory of the origin of life in which primitive replicons) consisted of mutant distributions, as found experimentally with present day rna viruses. the theory provided a new definition of wild type, and a conceptual framework for the interpretation of the adaptive potential of rna viruses that contrasted with classical studies based on consensus sequences. standard clonal analyses and deep sequencing methodologies have confirmed the presence of myriads of mutant genomes in viral populations, and their participation in adaptive processes. the quasispecies concept applies to any biological entity, but its impact is more evident when the genome size is limited and the mutation rate is high. this is the case of the rna viruses, ubiquitous in our biosphere, and that comprise many important pathogens. in virology, quasispecies are defined as complex distributions of closely related variant genomes subjected to genetic variation, competition and selection, and that may act as a unit of selection. despite being an integral part of their replication, high mutation rates have an upper limit compatible with inheritable information. crossing such a limit leads to rna virus extinction, a transition that is the basis of an antiviral design termed lethal mutagenesis. of its corollaries is the error threshold relationship, which marks the maximum mutation rate at which the master (or dominant) sequence can stabilize the mutant ensemble. violation of the error threshold results in loss of dominance of the master sequence and drift of the population in sequence space) [ ] [ ] [ ] [ ] . the core quasispecies concepts are described by two fundamental equations: replication with production of error copies, and the error threshold relationship (fig ) . they capture two major features of rna viruses at the population level: the presence of a mutant spectrum, and the adverse effect of an increase of mutation rate on virus survival, each with several derivations (fig ) . the existence of a mutant spectrum was experimentally evidenced first by clonal analyses of rna bacteriophage qβ populations whose replication had been initiated by a single virus particle. individual genomes differed from the consensus sequence in an average of one to two the equations are the mathematical expression of the major concepts implied by quasispecies theory. the first equation describes the change of concentration of molecule i as a function of replication parameters, and its production from other molecules of the same ensemble. the second equation is the error threshold relationship, indicating the maximum amount of information (ʋ max ) and the maximum average error rate p max (p = -q; q is the copying fidelity) for maintenance of genetic information. terms are defined in the box on the right. below, an evolving mutant spectrum (with mutations represented as symbols on the genomes), with an invariant consensus sequence. details in [ ] . mutations per individual genome [ ] . fitness of biological clones was inferior to that of the parental, uncloned population, a difference also documented for vesicular stomatitis virus (vsv) [ ] . the replicative capacity of a population ensemble need not coincide with that of its individual components. the finding that a viral population was essentially a pool of mutants came at a time when mutations in general genetics were considered rare events, and virologists associated a viral genome with a defined nucleotide sequence, as still implied today in the contents of data banks [ ] . the cloud nature of qβ was understood as a consequence of its high mutation rate, calculated in − mutations introduced per nucleotide copied [ ] , together with tolerance of individual genomes to accept an undetermined proportion of the newly arising mutations, despite fitness costs. the error rate estimated for bacteriophage qβ has been confirmed, and is comparable to values calculated for other rna viruses [ , ] . high mutation rates and quasispecies were verified for other rna viruses based on dissection of viral populations by molecular or biological cloning, and sequence analysis of individual clones. john holland and colleagues were the first to recognize that a rapidly evolving rna world inserted in a dna-based biosphere had multiple evolutionary and medical implications [ ] [ ] [ ] . genome plasticity of rna viruses had been suspected for many decades. key early observations were variations in viral traits described by findley in the 's, the studies of granoff on transitions of plaque morphology of newcastle disease virus, or the high frequency of conversions between drug resistance and dependence in coxsackie a virus, among other studies with animal and plant viruses in the middle of the th century (for a historical overview and references, see ) . when put in the context of present day knowledge, we realize that these observations on phenotypic changes were the tip of the iceberg of an extremely complex reality of viral populations. high mutation rates and population heterogeneity characterize rna viruses, with consequences for viral pathogenesis and the control of viral disease. detailed studies on quasispecies dynamics in vivo have been performed with human immunodeficiency virus type (hiv- ) and hepatitis c virus [ ] [ ] [ ] . the first mathematical formulation of quasispecies was deterministic; it assumed steady state mutant distributions in equilibrium without perturbations derived from modifications of the environment or population size [ ] . these conditions are common in initial theoretical formulations of complex phenomena because they confer mathematical tractability. since then, several extensions of the theory to non-equilibrium conditions with stochastic components have been developed, with the aim of finding general solutions for multi-peak fitness landscapes. these objectives approximate quasispecies to the real case of rna viruses, which are compelled to deal with dramatic variations in population size and environment (reviewed in [ ] ). research on quasispecies has proceeded through several theoretical and experimental avenues that include continuing studies on evolutionary optimization and the origin of life, rna-rna interactions and replicator networks, the error threshold in variable fitness landscapes, consideration of chemical mutagenesis and proofreading mechanisms, evolution of tumor cells, bacterial populations or stem cells, chromosomal instability, drug resistance, and conformation distributions in prions (a class of proteins with conformation-dependent pathogenic potential; in this case the quasispecies is defined by a distribution of conformations) [ , ] . new inputs into experimental quasispecies research have come from deep sequencing to probe viral and cellular populations, recognition of interactions within mutant spectra, models of viral population dynamics related to disease progression and pathogen transmission, and new teachings from fidelity variants of viruses (the several theoretical, experimental and practical facets of quasispecies have been reviewed in several chapters of [ ] ). here we summarize the main aspects of quasispecies dynamics, and recent developments relevant to virus evolution and pathogenesis. the molecular basis of high error rates is the limited template-copying fidelity of rnadependent rna polymerases (rdrps) and rna-dependent dna polymerases (also termed reverse transcriptases, rts). in addition, these enzymes are defective in proofreading) [ ] because they lack a ' to ' exonuclease domain present in replicative cellular dna polymerases [ ] . also, postreplicative-repair pathways, abundant to correct genetic lesions in replicating cellular dna, appear as ineffective for double-stranded rna or rna-dna hybrids. the presence of a proofreading-repair activity in coronaviruses increases their copying accuracy in about -fold [ ] . this and other repair activities, that may act on standard rna or retroviral genomes [ ] [ ] [ ] [ ] , do not prevent the formation of mutant spectra, although their amplitude may be lower than for other rna viruses, at least in populations close to a clonal (single genome) origin. quasispecies dynamics will operate in any viral or cellular system in which due to high mutation rates (as a result of low fidelity nucleic acid polymerases or environmental alterations) mutant spectra are rapidly generated [ , [ ] [ ] [ ] [ ] [ ] . studies with different virus-host systems have established some general observations on the mechanisms of mutant generation, and implications of quasispecies dynamics [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in rna virus genetics when we speak of "a mutant" the entity we handle is a cloud of mutants in which the specific mutation to which we direct our attention is present in all (or the great majority of) individual genomes. there is no such a thing as "a" wild type or "a" mutant virus. they are always clouds of mutants. changes in the relative dominance of components of mutant spectra are particularly severe during in vivo infections, with complex dynamics of intra-host heterogeneity and variations. bioinformatic procedures have been developed to unveil the relationships among different but closely related genome types that may suggest some hierarchical order of mutation acquisition or identification of transmission clusters (examples are partition analysis of quasispecies, paq [ ] or quasispecies evolution, network-based transmission inference, quentin [ ] ). the crux of the matter regarding quasispecies implications is that at any given time, the viral population includes a reservoir not only of genotypic but also of phenotypic variants, conferring upon the population some adaptive pluripotency. accumulating laboratory and clinical evidence renders untenable that minority components of mutant spectra should be dismissed on the grounds of their being neutral. they can participate in selective processes and cannot be excluded from interpretations of virus behavior. variation universally involves point mutations and it can also include recombination (in its replicative and non-replicative modes), and genome segment reassortment [ ] . all modes of molecular variation are compatible, only restricted by the scope of mechanisms accessible to the replicative machinery, and for the need of viral genomes to remain functional. david evans and colleagues identified many recombination events associated with enterovirus replication, and only a few recombinants made their way towards continued replication [ ] . recombination can mediate adaptability and virulence [ ] . high mutation and recombination rates have led to the conceptual distinction between mechanistically unavoidable and evolutionarily relevant variation, in connection with the issue of clonal versus non-clonal nature of virus evolution (microbial evolution in general) [ , ] . only a minority of the nascent variation during replication can be successfully propagated. within limits that are set by biological constraints, each population is made of an array of variant genomes, with a total number which is commensurate with the virus population size. to infect a plant, animal or cell culture with infectious units can have very different consequences than to infect with infectious units, not only because the host defense systems may be overwhelmed by the high infectious dose, but also because the mutant repertoire that engages in adaptive explorations is larger. part of the variants of a mutant spectrum, either in isolation or in consortium with others [ ] , may perform better than other members of the same population in the event of an environmental change. selective pressures favor replication of some components of a mutant spectrum over others, despite all of them being interconnected by mutation. differential performance can be at the level of viral genomes (during replication, intracellular gene expression, interaction with host factors, etc.) or viral particles (for thermal stability, entry into or exit from cells, to withstand neutralizing antibodies, etc.) [ , [ ] [ ] [ ] , [ ] [ ] [ ] . adaptability of rna viruses is linked to parameters that facilitate exploration of sequence space: genome size ( . to kb), population size (variable but that can attain an impressive individual genomes in an infected host at a given time), replication rate, mutation rate, fecundity (yield of viral particles per cell), and number of mutations required for a phenotypic change (surprisingly low for several relevant traits) (see [ ] ). mutant spectrum dynamics has been depicted in different ways, and we have chosen one that encompasses frequent events in natural populations and research designs, such as virus isolation from an infected host, adaptation to cell culture for studies on experimental evolution, or adaptation to alternative hosts in vivo (fig ) . despite the complexity conveyed by the figure, the reality is even more complex, given the large population sizes, with an indeterminate proportion of genomes actively replicating at any given time (sometimes equated with the effective population size in general genetics), and harboring multiple mutations per genome. the scenarios suggested by current experimental data defy our imagination. the relative frequency of individual mutations fluctuates in an unceasing exploration of sequence space [ ] [ ] [ ] , with phenotypic changes (not only genotypic changes) being far more frequent than previously thought. the experimental evolution design that consists of passaging viral populations for long time periods (many sequential infections) is often extremely revealing. in foot-and- mouth disease virus (fmdv) such a design led to a remarkable phenotypic diversification into subpopulations of colonizers and competitors, that modulated virulence of the mutant ensemble [ ] . in hcv such a design unveiled continuous mutation waves and a more accurate understanding of the types of fitness landscapes occupied by high fitness viruses [ , ] . the nucleotide sequence of an individual genome from a population (no matter which the degree of population complexity might be), can be determined either following a biological or molecular cloning event or by deep sequencing of entire viral genomes, in a manner that mutation linkage (assignment of different mutations to the same genome molecule) can be established. each of these procedures implies some limitations: biological cloning can bias the representation in favor of infectious genomes, while molecular cloning can introduce noninfectious (defective) genomes in the analysis [ , , ] . whole genome quasispecies description is still technically challenging due to the artifactual introduction of mutations. most current deep sequencing platforms yield sequences of short reads for a given amplicon (sequence under analysis); minority mutations in an amplicon cannot be reliably linked to mutations in a different amplicon of the same genome; at most, statistical inferences on linkage can be proposed. despite these limitations, control experiments and improvements of bioinformatic procedures support that the majority of sequence heterogeneity analyzed in viral populations indeed reflects differences in the natural template populations. if mutation linkage can be solved on a routine basis, a new wave of molecular information relevant to epistatic interactions will enter the picture. there are additional levels of indeterminacy in the sequential analysis of viral populations, in particular those replicating in vivo. components of the mutant spectrum represented at a given time in the sample taken for sequencing may differ from those in the next time point, due either to sampling uncertainties or bona fide fluctuations of genome frequencies. it is not justified to accept a rough similarity because even a single mutation in a given sequence context may affect biological properties [ ] . in the words of john holland and colleagues: "it is important to remember that every quasispecies genome swarm in an infected individual is unique and "new" in the sense that no identical population of genomes has ever existed before and none such will ever exist again" [ ] . on top of the fleeting nature of any mutant distribution, the standard methods available for quasispecies characterization provide genomic sequences of a minority of the population (estimated in − to − for molecular cloning-sanger sequencing, and in − to − for deep sequencing; reviewed in [ ] ). we can only have an approximate representation of viral populations and their dynamics, as evidenced by many experimental studies [ , , , , , , , ] . the points summarized in previous sections fully justifies addressing analytical tools towards the mutant spectrum rather than ignoring it or considering its presence a side issue. use of consensus sequences to describe the genome of a virus isolate, despite being warranted by the difficulties of conveying the information recapitulated in a mutant spectrum, blurs and enfeebles biological interpretations. experimental results have demonstrated that minority genomes from a mutant spectrum (that cannot be identified by examining the consensus sequence) can include mutations that confer resistance to antiviral inhibitors, neutralizing antibodies or cytotoxic t cells, or that can alter the capacity to induce interferon (ifn) or to respond to ifn, virulence or particle stability, among other phenotypic traits [ , , , , [ ] [ ] [ ] [ ] [ ] . mutant spectra can also mediate cyclical adaptation to different cell types [ ] . a mutant spectrum defines a consensus but the consensus is an abstraction; it may not be represented in the population. many events in viral pathogenesis and evolution are due to mutant spectrum modifications or interactions which cannot be properly interpreted solely on the basis of consensus sequences [ , [ ] [ ] [ ] , , , , , , , , ] . mutant spectra are not mere aggregates of mutants acting independently. they are often engaged in collective responses of two major types: those that depend on the presence of sets of variants, and those that rely on intra-mutant spectrum interactions. behavior of reconstructed quasispecies. in some cases of sweeping selection (very strong selection for a trait), an individual (or a limited number of individuals) that encodes signatures prone to be selected, may approach dominance while becoming the founder of a mutant cloud (because formation of a cloud is inherent to replication). conditions for dominance (in this case in response to selection) are that the genome senses the selective sweep and that its replication in the new selective environment is permitted. in other cases, a collection of mutants is selected. this was illustrated with a fmdv quasispecies that was reconstructed in the laboratory with multiple antigenic variants (each at low frequency) that belonged to two different categories, and shared resistance to the same monoclonal antibody [ ] . one category included mutants with an amino acid substitution that affected receptor recognition (since the antigenic determinant overlapped with the integrin receptor recognition site); in the other category, the substitutions affected the antigenic determinant but not the receptor recognition site. passages of the virus in absence of the monoclonal antibody resulted in dominance of antigenic variants that maintained the receptor recognition capacity, but the dominant variants were surrounded by a cloud of mutants of the other antigenic variant category. conversely, passages in the presence of the antibody led to selection of variants with altered receptor recognition, surrounded by a cloud of antigenic variants that maintained receptor recognition. the results underlined the role of mutant clouds in selective events, and unveiled a new mechanism of antigenic flexibility [ ] . quasispecies memory. quasispecies memory is a type of molecular memory dependent on the recent history of the evolutionary lineage and the integrity of the mutant spectrum [ , ] . the search for memory was prompted by the complex adaptive system behavior of a viral quasispecies, suggested by the presence of core information (considered the one that defines viral identity) despite variation of constitutive elements (the mutant spectrum). a wellknown example is memory in the immune system that mobilizes and expands minority components in response to stimuli previously faced by the system [ ] . in the experiments designed to identify memory in viral quasispecies, members of the mutant spectrum increased in frequency as a consequence of their replication during a selection event that drove them towards dominance. when the selective constraint was withdrawn, memory genomes remained at levels that were -to -fold higher than the basal levels attributable solely to their generation by mutation, as documented with independent fmdv genetic markers, and with hiv- in vivo [ , , , ] . thus, memory is a history-dependent, collective property of the quasispecies that confers a selective advantage to respond to environmental changes previously experienced by the same evolutionary lineage. it can be manifested only if the mutant spectrum maintains its completeness, since memory is lost when the population undergoes a bottleneck event that excludes minorities. a relevant example of the consequences of memory occurs in antiviral pharmacology with the administration for a second time of the same or a related antiviral agent (capable of evoking shared resistance mutations) used in a previous treatment. the second intervention may face inhibitor-resistant memory genomes from the earlier treatment, thus contributing to virus escape [ ] . this is an aspect that has not received adequate attention in the planning of antiviral interventions for patients who fail a first treatment and have to be subjected to a second treatment. individual genomes surrounded by a cloud of related mutants can be either suppressed to be kept at low frequency, or helped to be maintained in the population. the two alternative fates are dependent on several factors, one being the surrounding mutant spectrum in those steps of the infectious cycle in which an effective competition among variants is established, for example within replication complexes. this important concept was first derived theoretically [ , ] , and then approached experimentally with several viruses. in an early study, juan carlos de la torre and john holland described suppression of high fitness vsv by mutant spectra of inferior fitness [ ] . suppressive effects have since been documented with standard and mutagenized viral populations. some examples are: • suppression of high fitness antigenic variants of fmdv by low fitness antibody-escape mutants [ ] . [ ] . • suppression of pathogenic lymphocytic choriomengitis virus (lcmv) (that cause growth hormone deficiency in mice) by non-pathogenic lcmv variants [ ] . • suppression of fmdv by a mutagenized fmdv population [ ] . • suppression of fmdv by capsid and polymerase fmdv mutants [ ] . • suppression of drug-resistant viral mutants during antiviral therapy [ , ] . opposite to suppression is maintenance of a mutant either by a favorable position in a fitness landscape or by interactions of complementation or cooperation with members of the mutant spectrum. the position in a fitness landscape influences vulnerability to mutations, as popularized with the terms "advantage of the flattest" or "survival of the flattest", indicating that a variant located at the top of a sharp fitness peak has higher probability to decrease fitness as a result of new mutations than the same variant located at a fitness plateau [ ] [ ] [ ] . survival of the flattest has been also proposed as an ingredient in some models of the error threshold [ ] . collective behavior of viruses was documented with mutant rna viruses resistant to nucleotide analogues. the study of this class of mutants has been instrumental for the understanding of the molecular basis of template copying fidelity, and the consequences of fidelity alterations in the adaptive capacity and pathogenic potential of rna viruses [ ] [ ] [ ] . in the first mutant studied, amino acid substitution g s in the pv polymerase resulted in about four-fold increase in template-copying fidelity. this modification reduced pv adaptability and infective potential in vivo [ , ] . the mutant in isolation did not replicate efficiently in the brain of susceptible mice, but it did when its mutant spectrum was broadened by -fluorouracil mutagenesis or when it was co-inoculated with wild type pv [ ] . complementation (often occurring when a functional protein encoded by a set of genomes is used by another set of genomes whose encoded protein is not functional) may underlie some collective responses of quasispecies such as fitness of individuals isolated from a population being inferior to fitness of the population [ , ] . complementation was described between two truncated fmdv genomic forms [ , ] . the genomes with internal deletions became detectable upon high multiplicity passage of a clonal population of standard fmdv, a virus with a monopartite single stranded rna genome. infectivity was generated by complementation of the two truncated forms, in absence of standard, full length fmdv genomes. for complementation to be effective, prior exploration of sequence space through point mutations was a requirement [ ] . the system underwent a remarkable evolutionary transition akin to genome segmentation. drastic genetic lesions in viral genomes are difficult to observe unless a mechanism such as complementation comes into the rescue of the deviant genomes. additional examples of complementation among rna viruses have been reported ( [ ] [ ] [ ] ; for review see [ , ] ). complementation is a means to maintain defective genomes at detectable frequencies in viral populations. a distinction has been made between complementation and cooperation, in which two different genomes give rise to a new phenotype through the interaction between two variant proteins [ ] . an example of cooperation was characterized during studies with measles virus on membrane fusion which is essential for virus entry into cells. for this virus fusion is mediated by two proteins termed h and f. a truncated h was deficient in cell fusion but the activity was regained when the truncated h was accompanied by two forms of f but not one of the forms individually [ ] . therefore, complementation, cooperation, interference and suppression can emerge from interactions among components of mutant spectra that have their origin in random mutations. selection acts on whatever sets of mutants can provide a useful trait, to turn random occurrences into biological meaning. a means to interrupt the participation of individual genomes in interactions with their mutant spectrum is for the quasispecies swarm to undergo drastic reductions in population size that isolate one or few individual genomes from their surroundings. such reductions are termed bottlenecks (fig ) , and they have an important participation in shaping evolutionary lineages for all kinds of organisms, and also for viruses. bottleneck events are also depicted in fig ( plaque-to-plaque transfers in box at the left). they occur frequently not only upon host-to host transmission but also inside infected hosts [ ] [ ] [ ] , and they can perturb positive and negative selection events in processes that are difficult to identify and characterize. drastic bottleneck events have been reproduced with laboratory populations of viruses in the form of plaque-to-plaque transfers [ , ] (depicted in fig ) . this design served to verify experimentally the operation of müller's ratchet, or fitness decrease by the irreversible incorporation of mutations in asexual organisms in absence of compensatory mechanisms [ ] . the serial bottleneck transfers unveiled the presence of rare mutations, not seen in standard laboratory or natural viral populations. in absence of forced bottleneck events, such rare mutations would be lost by negative selection because of the fitness cost they inflict [ ] . the investigation of how fmdv clones debilitated by müller's ratchet regained replicative fitness revealed several alternative molecular pathways for fitness recovery [ ] . the implications of this observation went largely unnoticed until recent results with hepatitis c virus (hcv) have also suggested the accessibility of multiple pathways for fitness gain [ , ] . also, extensive passage of a biological clone of fmdv in bhk- cells conferred the capacity to infect several human cell lines in addition to the expected fitness increase for multiplication in bhk- cells [ ] . thus, several lines of evidence suggest that fitness gain in a specific environment may paradoxically broaden the phenotypic potential of a virus. it will be interesting to investigate whether focused adaptation of other viruses to a specific environment may also entail a broadening of diversity, with many phenotypic variants attaining similar fitness levels. if generalized, this broadening of phenotypic space would provide a new interpretation of the molecular basis of adaptation, and explain why adaptation to alternative environments may not lead to attenuation. deprivation of an individual virus from possible suppression, complementation or cooperation, may represent a liberation to initiate a new evolutionary process, or a condemnation to extinction. if liberated from suppression, the isolated genome must replicate and be able to reconstruct a mutant cloud to regain adaptive capability. this has led to the suggestion that high mutation rates evolved to allow such mutant spectrum recovery following bottlenecks. other models attribute high mutation rates to adaptive optimization independent of bottlenecks, or to a mechanistic consequence of rapid replication (reviewed in [ ] ). whatever their ultimate origins, high mutation rates serve the purpose of adaptation in multiple circumstances, not only following bottlenecks. a founder virus can introduce a different phenotype for the ensuing evolution. evolution of viruses in nature and as disease agents can be viewed as succession of mutant spectrum alterations, subjected to expansions and reductions of population size in a continuous interplay of positive and negative selection and random drift. while short-term (for example, intra-host) evolution is observable and measurable, viruses may appear to be relatively static in the long term for decades (as seen with antigenic variants of fmdv [ ] ) or longer. intra-host evolution is generally more rapid than inter-host evolution, as documented with viruses [ ] and other biological systems [ ] . apparent invariance may be the result of selection for long-term survival of populations that have previously frenziedly tested evolutionary outcomes in short-term processes [ ] . soon after quasispecies was evidenced for viruses, some medical implications were made explicit [ , ] . several specific or general points (reviewed in [ , , ] , and in several chapters of [ ] ) can be succinctly exposed as follows: • high mutation rates and population heterogeneity endow viruses with the potential to escape immune pressures (including those due to vaccination) and antiviral inhibitors used in therapy. it is an open question if vaccination can promote long-term evolution of antigenic determinants. • attenuated rna virus vaccines can revert to virulent forms. rna viruses released in nature for pest control purposes can mutate to new phenotypes. • virus attenuation and virulence is dependent on viral genetic traits. variant forms of a given virus may display increased virulence or atypical disease. • components of a mutant spectrum can exhibit a different cell tropism or host range than most genomes in the same population, with implications for the emergence and re-emergence of viral disease. • viral pathogenesis is influenced by microevolutionary processes in which some viral subpopulations are replaced by others to persist or to invade new cell types, tissues or organs. • the larger the actively replicating (effective) population size and the replication rate, the most effective is exploration of sequence space for phenotypic expansions that favor survival and persistence. • there is a connection between four parameters that caracterize viruses during infection processes: replication rate (the rate at which viral rna or dna is synthesized intracellularly for viral progeny production), viral load (the total amount of virus quantified in an infected host or host compartment), genetic heterogeneity, and replicative fitness (the yield of infectious particles that can contribute to the next generation). they can influence disease progression, and any of them can be targetted for disease control. in all interactions conductive to disease, the host cells individually and as groups in tissues and organs play decisive roles. the consequences of a viral infection are always host-dependent. however, the virus itself poses a major challenge that a deeper understanding of quasispecies dynamics is helping to confront. there is an increasing perception that darwinian principles should assist in the planning of antiviral designs [ ] . the aim of vaccination is to evoke a protective response that either prevents virus replication or disease. the aim of an antiviral pharmacological intervention is to inhibit virus replication to provide the immune system with an opportunity to clear the virus. expressed simply, the direct danger for vaccination and treatment is that virus can escape through selection of mutants resistant to vaccine-triggered defense components or to the externally administered inhibitors. this has led to several proposals to confront viral disease, that can be summarized as follows (reviewed in [ ] ): vaccines should include repertoires of b cell and t cell epitopes to evoke an ample immune response. the broad response should minimize selection of escape mutants that may be present as minority components in mutant spectra, as repeatedly documented experimentally [ , , , ] . with the current types of available vaccines, those that best comply with the multiple epitope requirement are, in the order of expected efficacy to confer protection against highly variable viruses: attenuated > inactivated whole virus > several expressed proteins > one expressed protein > multiple synthetic peptide antigens > single peptide antigen. the scarcity of effective synthetic vaccines for rna viral pathogens despite huge scientific and economic efforts is a reflection of the underlying problems. antiviral monotherapy (use of a single antiviral agent) is to be avoided. the following recommendations have been made and in some cases successfully implemented: • inhibitors used in combination should target different viral gene products. • splitting a treatment into two steps: first an induction regimen, and a second maintenance regimen. drugs administered in the two steps should be different. • targetting of cellular functions needed for the virus life cycle. • use of innate immune response-stimulating drugs (for example, inhibitors of enzymes involved in pyrimidine biosynthesis). • combined use of immunotherapy and chemotherapy. • lethal mutagenesis or virus extinction by excess of mutations introduced during viral replication. these strategies (whose supportive theoretical and experimental evidence has been reviewed in [ , ] ) have as their main objective to avoid selection of treatment-escape mutants by multiple selective constraints that cannot be surmounted by the virus. control is effective either because exploration of sequence space cannot reach the required multiple mutations (even when recombination is available) or because the multiple mutations inflict a severe fitness cost [ ] . vaccines exposing multiple epitopes and combination therapies follow the same strategy whose aim is to limit possible escape routes to viral quasispecies in the face of the suppressive constraint. lethal mutagenesis is the process of virus extinction at the error rate at which a virus can no longer maintain its genetic information [ , , , , , , , ] . application of lethal mutagenesis as an antiviral strategy deserves attention in the context of the present article because its origins lie in quasispecies theory, in the form of the error threshold relationship (fig ) . both the error threshold and lethal mutagenesis are highly fitness landscape-dependent, but both can occur in complex fitness landscapes as those pertinent to viral populations [ ] . the term lethal mutagenesis was coined by lawerence loeb and colleagues [ ] , and it is now widely used to describe the antiviral activity of base and nucleoside analogues that increase the viral mutation rate. although several models have been proposed to account for virus extinction by excess mutations [ ] , an extension of the violation of the error threshold stands as a likely mechanism ( [ ] ; recent review in [ ] ). interestingly, some antiviral agents licensed for human use, initially thought to act only as inhibitors of viral replication, may actually exert their antviral activity against some rna viruses at least partially by lethal mutagenesis. this is the case of favipiravir (t- ; -fluoro- -hydroxy- -pirazinecarboxamide) and ribavirin ( -β-d-ribofuranosyl- -h- , , -triazole- -carboxamide) that are currently being intensively investigated as lethal mutagens [ ] . defense mechanisms based on genome modification of invading genetic parasites such as editing cellular activities that are recruited as part of the innate immune response (adar, apobec, rip, etc; reviewed in [ ] ) represent a natural counterpart of the principle utilized by lethal mutagenesis. applicability to pathogenic cellular elements is a real possibility, and lethal mutagenesis to control tumor cells is an active field of investigation [ , ] . thus, the recognition of quasispecies dynamics has suggested some fundamental guidelines for disease prevention and control that are gradually permeating clinical practice. this is in line with the recognized need to apply darwinian principles to the control of infectious disease. the main concepts covered in the present article and their domains of applicability are summarized in table . the adequacy of quasispecies theory (versus other formulations of evolutionary dynamics [ ] ) as a framework for the error-prone replication of viruses and its consequences stems from its including mutation as an integral part of the replication process table . summary of main concepts related to quasispecies and their implications a. implications for virology references limited template-copying fidelity leads to formation of dynamic mutant distributions. they mediate virus adaptability. [ , , , ] phenotypic reservoir mutant spectra are a phenotypic reservoir for selection to act upon [ , , ] adaptive parameters viral quasispecies adaptability relies on six parameters: genome size; population size; replication rate; mutation rate; fecundity; and number of mutations required for a phenotypic change [ , , ] mutant spectra are not mere mutant aggregates. emergent behavior can result from positive interactions of cooperation or complementation or negative interactions of interference among components cf the mutant spectrum [ , , [ ] [ ] [ ] , [ ] [ ] [ ] quasispecies memory a record of past genome dominances that prepares a viral population to respond to a selective constraint previously experienced by the same lineage. bottlenecks erase quasispecies memory. [ , , , ] the total number of genomic sequences available to a virus. adaptation is a movement towards a favorable region of sequence space. de-adaptation (i.e. lethal mutagenesis) is a movement towards unfavorable regions of sequence space. [ , , , ] population bottleneck a drastic reduction in population size. it promotes random drift in evolutionary outcomes. the diversifying effect of bottlenecks is accentuated by the cloud nature of viral populations. [ , , , ] biological constraints high mutation rates expand sequence space occupation. biological constraints impose negative selection on many newly generated mutants. constraints contribute to maintenance of virus identity. [ , , ] progress of an infection is often associated with virus adaptation to host environments. variants of the same virus can differ in disease potential (virulence). [ , , ] quasispecies and long-term evolution short-term evolutionary rate based on reorganization of mutant spectra is faster than long-term evolutionary rate. conceptual links between quasispecies and phylodynamics at the epidemiological level are needed. [ ] aconcepts are listed in the order relevant to the topics covered in the text, and serve to underline key points and some supportive studies. the concepts are expanded in the text, with additional references. [ ] . quasispecies dynamics poses a great challenge for the molecular interpretation of shortterm evolutionary events that bear on virus-host interactions and disease processes. a rewarding aspect of progress in having captured the meaning of the challenge (at least partially) is that we can exclude some of the prevention and control strategies that once were considered an option. a significant example is the historical rejection of combination therapies as contrary to the established canons of pharmacology, while now monotherapy is considered a risky practice (for discussion of this point, see [ ] ). we understand now that focused antiviral barriers that involve a single constraint (one inhibitor, one monoclonal antibody, one peptide antigen as vaccine) have a large probability of failing. likewise, attempts to produce "universal" drugs, vaccines or diagnostic tools are unlikely to succeed given the intra-population diversity (present and potential) of the pathogenic agents to be controlled. a viral genome region which is conserved among types, subtypes, isolates may be so only regarding a consensus sequence but not the underlying mutant spectrum. methods are now available to identify low level mutations that may predict escape from selective constraints. on a related note, it remains extremely unlikely to predict the emergence and re-emergence of viral diseases for a number of interconnected reasons, not the least important being pathogen adaptability [ ] . the outlined limitations to predict the occurrence of viral diseases and to control them are based on our current understanding of viral complexity and dynamic change of such complexity. obviously, we may be wrong, and in science we are open to the unexpected. quasispecies poses also a challenge for the annotation of the events we witness. reality is far more complex than the means we have developed to describe viral populations in continuous change. how could the problem we approached? procedures to organize and relate sequences for phylodynamic purposes need updating to exploit increased computing power to handle mutant spectra rather than one or few (consensus or other) sequences from each biological sample. the expanding number of sequences expected to become available including complete genomes (that will allow mutation linkage to enter the picture) if handled properly, may inform of the molecular basis of new phenotypes. the penetration into mutant spectra has also modified the concept of rare mutation since what in terms of consensus could be catalogued as rare may in fact be frequently occurring but rarely observed. a mutation which has low frequency at one time point or in a given environment may rise to high frequency at another time point or in a different environment. rare mutations that populate mutant swarms may belong to defective genomes exerting relevant host interactions [ ] . on a more general note, recognition of viral quasispecies was premonitory of the impressive diversity in the biological world that the metagenomic approaches are currently unveiling, including cellular heterogeneities whose biological implications remain largely unexplored. supporting information s text. version history of the text file. 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drug resistance. handbook of antimicrobial resistance lethal mutagenesis of hiv with mutagenic nucleoside analogs the increasing impact of lethal mutagenesis of viruses error catastrophe and antiviral strategy perspective: apobec mutagenesis in drug resistance and immune escape in hiv and cancer evolution lethal mutagenesis: targeting the mutator phenotype in cancer human cancers express mutator phenotypes: origin, consequences and targeting unifying evolutionary dynamics microbial threats to health. emergence, detection and response the defective component of viral populations evolutionary virology at we thank colleagues and students who have participated in research on viral quasispecies and its conceptual origins. we dedicate this article to the memory of manfred eigen ( eigen ( - . key: cord- -g skuik authors: johnstone, jennie; majumdar, sumit r.; fox, julie d.; marrie, thomas j. title: viral infection in adults hospitalized with community-acquired pneumonia prevalence, pathogens, and presentation date: - - journal: chest doi: . /chest. - sha: doc_id: cord_uid: g skuik background the potential role of respiratory viruses in the natural history of community-acquired pneumonia (cap) in adults has not been well described since the advent of nucleic amplification tests (nats). methods from to , adults with cap who were admitted to five hospitals were prospectively enrolled in the study, and clinical data, cultures, serology, and nasopharyngeal swabs were obtained. nats from swabs were tested for influenza, human metapneumovirus (hmpv), respiratory syncytial virus (rsv), rhinovirus, parainfluenza virus – , coronaviruses (oc , e, and nl ), and adenovirus. results a total of patients were included; the median age was years, % of patients were male, and % of patients had severe cap. overall, patients ( %) had a pathogen identified. of these pathogens, were viruses ( %), were bacteria ( %), were mixed ( %), and the rest were “unknown.” influenza (n = ), hmpv (n = ), and rsv (n = ) accounted for most viral infections; other infections included rhinovirus (n = ), parainfluenza (n = ), coronavirus (n = ), and adenovirus (n = ). streptococcus pneumoniae was the most common bacterial infection ( %). compared with bacterial infection, patients with viral infection were older ( vs years, respectively; p = . ), were more likely to have cardiac disease ( % vs %, respectively; p = . ), and were more frail (eg, % with limited ambulation vs % of bacterial infections; p = . ). there were few clinically meaningful differences in presentation and no differences in outcomes according to the presence or absence of viral infection. conclusions viral infections are common in adults with pneumonia. easily transmissible viruses such as influenza, hmpv, and rsv were the most common, raising concerns about infection control. routine testing for respiratory viruses may be warranted for adults who have been hospitalized with pneumonia. community-acquired pneumonia (cap) is one of the most clinically important diseases in adults, affecting to per , adults per year. of these, at least to % will require hospitalization for the treatment of their pneumonia.f cap management guidelines have been influenced by older cap etiology studies," which helped to direct empiric therapeutic antimicrobial choices for therapy against bacterial pathogens such as streptococcus pneumoniae, haemophilus influenzae, and "atypical" bacwww.chestjoumal.org teria, including chlamydophila pneumoniae, mycoplasma pneumoniae, and legionella pneumophila. although cap guidelines acknowledge respiratory viruses as a "cause" of pneumonia, few recommendations are made regarding management, largely due to the paucity of data regarding prevalence, clinical presentation, and outcomes. furthermore, viral etiology studies in pneumonia are difficult to interpret as noninvasive viral detection methods are often considered to be only markers of infection rather than the cause of pneumonia." clearly, much better knowledge of the potential role of respiratory viruses present in patients with pneumonia is needed. most published studies's? of respiratory viruses have relied on tests with relatively poor sensitivity such as serology and direct fluorescent antigen (dfa) tests. such tests are limited in the sample type to which they can be applied and are not suitable for a broad range of respiratory viruses. more recently, the introduction of highly sensitive nucleic acid amplification tests (nats) has dramatically improved our ability to detect multiple viral pathogens such as influenza, respiratory syncytial virus (rsv), rhinovirus, parainfluenza, and adenovirus. such tests can be undertaken using a small single sample of respiratory secretions with results available with rapid turnaround times. - in addition, these tests have allowed us to detect emerging respiratory viruses such as human metapneumovirus (hmpv) and coronaviruses, viruses that are difficult to grow in cell culture. [ ] [ ] [ ] to date, there have been few studies, , , [ ] [ ] [ ] , reported in patients with pneumonia using nats to detect viral infection, and these studies have either not included clinical data . . or have not tested for all potentially important respiratory viruses in a comprehensive manner.lv-!? better knowledge of the role of infection with respiratory viruses in adults with pneumonia may lead to better management. thus, we performed a prospective study in consecutive adults who had "from the department of medicine (drs, johnstone, majumdar, and marne), faculty of medicine and dentistry, university of alberta, edmonton, ab, canada; and the department of microbiology (dr. fox), provincial laboratory for public health, calgary, ab, canada. dr. majumdar was supported by the alberta heritage foundation for medical research (health scholar) and the canadian institutes of health research (new investigator). this project was funded in part by an establishment grant (to dr. marrie) from the alberta heritage foundation for medical research. funding sources had no role in study design, data collection, data analysis or interpretation, or writing of the report. all authors participated in the study conception, design, analysis, interpretation of results, and revision of the manuscript, and approved the final version of the manuscript. dr. johnstone drafted the initial manuscript. dr. marrie acquired the data, obtained funding for the study, and will act as guarantor, the authors have reported to the accp that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article, been admitted to the hospital with cap, and sought to describe their pathogens, clinical presentation, and outcomes. from january to january , consecutive adults (~ years of age) who had been admitted to five hospitals in edmonton, ab, canada, with cap were enrolled in a prospective study of pneumonia, patients were excluded from the study if they had received antibioties or been hospitalized within the prior weeks, were unable or unwilling to provide informed consent, or had the following conditions: immunocompromised (ie, had received > mg of prednisone per day for > month, other immunosuppressives, had cancer with reeent chemotherapy, or had hiv with a cd count of < cells/u.l), tuberculosis; bronchiectasis, cystic fibrosis; or pregnancy. all patients gave written informed consent, and the health research ethics board of the university of alberta approved the study. we did not record data on patients who were unable to provide consent or who did not meet the enrollment eriteria. pneumonia was defined as an acute lower respiratory tract illness with two or more of the following symptoms or signs: cough; productive cough; fever; chills; dyspnea; pleuritic chest pain; crackles, and bronchial breathing plus an opacity or inflltrate seen on a chest radiograph that was interpreted as pneumonia by the treating physician. to characterize the severity of the pneumonia itself, we calculated the pneumonia severity index (psi) using the methods of fine et al. is clinical, radiographlc, and laboratory data and short-term outcomes were collected by a trained research nurse; the nurse was masked to microbiology results at the time of data collection. patients were followed up throughout their hospital stay until discharge, routine blood culture, sputum specimens, nasopharyngeal (np) swabs, and serum samples were processed for each patient according to the study protocol. np swabs that were submitted for the detection of viral pathogens first underwent d fa testing for influenza a and b, rsv, and parainfluenza virus - (imagen; dakocytomation ltd; ely, uk), in addition, expanded testing of np samples was undertaken for a range of respiratory pathogens by nats using extraction and amplification methods that have been described previously'! i briefly. nats were designed to amplify and detect influenza a and b, hmpv, rsv, rhinovirus, parainfluenza - , coronaviruses (oc , e, and nie ), and adenoviruses. all the nats utilized in this study have been published, and the assay parameters evaluated. .i . . o laboratory validation of these assays confirmed a limit of detection of :s copies (cloned target or synthetic rna) or one or fewer tissue culture infectious dose of % (for culturable viruses). the specificity of all assays was confirmed using samples and spiked materials 'containing high loads of alternative respiratory pathogens. (further details on viral nats are available from j.d.f. on request [also see references , , , and ] .) bacterial infections were identified using standard laboratory protocols, acute and convalescent serum samples were collected on the day of hospital admission and were repeated to weeks later, serum samples were tested for the presence of c pneu-numiae and chlamydia psittaci igm and igg by a mieroimmunofluorescence assay," m pneumoniae igm enzyme immunoassay (platelia, biorad; hercules, ca), coxiellabumetii phase i and phase ii titers by indirect immunofluorescence.w and l pneumophila titers by indirect immunofluorescence.s-m pneunumiae and l pneumophila were also tested using nats, and were validated as above but with a limit of detection of :s; copies (cloned target or synthetic rna) or :s; cfu. a diabtj sis of respiratory viral infection was made if a virus was detected by nat or dfa and a coexisting bacterial pathogen was not identified. a diagnosis of bacterial infection was made if a viral pathogen was not detected, and the following criteria were met: ( ) isolation of a respiratory pathogen from purulent sputum (defined as an adequate quality sputum sample with > leukocytes and < epithelial cells per x magnification field) or blood culture , ,,; ( ) a fourfold rise in igg titers for c pneumoniae (> : ) and c psittaci (> : ) ; ( ) a single increased igm titer for m pneutnoniae (> : ) or c pneunwniae (> : ) ; ( ) an antibody titer of> : , to l pneumophila in a serum specimen obtained during either the acute or convalescent phase', ( ) a fourfold rise in antibody titer to> : or a fourfold rise in antibodies to c bumetii', ( ) a single titer of > : to a phase ii c bumetii antigen'. or ( ) the detection of m pneutnoniae or l pneumophila by nat. i a mixed infection was defined as the presence of both respiratory virus and bacteria, as defined above, last, if no pathogens were detected, based on the tests used in the study protocol, we classified this as "unknown." patient characteristics and outcomes according to pathogen were compared using x test, fisher exact test, student t test, or mann-whitney u test, as appropriate. although we present data for .ji pathogen categories, our primary analyses compare viral infection to bacterial infection. the few viral cases (n = ) in our sample precluded attempts at multivariable analyses. all data were analyzed using a statistical software package (spss, version . ; spss inc; chicago, il). three hundred patients were enrolled into the study, and patients ( %) had evaluable np swabs. the reasons for nonevaluable np specimens included insufficient sample (n = ) and missed collection (n = ). because the primary purpose of the study was to evaluate for the presence of viral pathogens, we excluded those patients without np swabs. there were essentially no differences between those with evaluable np swabs and those without for either clinical characteristics or outcomes, with the following exceptions: impaired functional status ( % vs %, respectively; p = . ); lobar pneumonia seen on a chest radiograph ( % vs . %, respectively; p = . ); and median length of stay ( vs days, respectively; p = . ). we considered the patients with evaluable np swabs to be our final study sample. sputum and blood cultures were requested for all patients, but these were not performed in some patients due to their inability to produce a sputum specimen (n = ), their refusal of a blood draw (n = ), or death (n = ). convalescent serum samples were not obtained in patients because they did not return for follow-up blood work (n = ) or died (n = ). overall, the median age of patients was years (interquartile range [iqr], to years), % were male, and % had severe cap (psi class or ). in total, patients ( %) had a respiratory pathogen identified, whereas pathogens were classified as "unknown" in patients ( %). of those patients with a pathogen identified, ( %) had a viral infection, ( %) had a bacterial infection, and ( %) had a mixed viral and bacterial infection. of the patients with a viral infection, the most common organisms were influenza a (n = ), influenza b (n = ), hmpv (n = ), and rsv (n = ). other organisms included coronavirus (n = ), rhinovirus (n = ), parainfluenza (n = ), and adenovirus (n = ). three patients had two viruses detected. of patients with viral infections, ( %) had influenza, hmpv, or rsv as a cause (table ) . of the patients with bacterial infection, infections ( %) were caused by s pneumoniae and infections ( %) were caused by common atypical pathogens (table ) . patients with viral infections were older than those without viral infections (median age, vs years, respectively; p = . ), were more likely to have underlying cardiac disease ( % vs %, respectively; p = . ), and tended to be more frail (eg, % had severely limited ambulation vs % of those with bacterial pneumonia; p = . ). other differences included the presence of chest pain, which was far less common in those patients with a viral infection than in those with a bacterial infection ( % vs %, respectively; p = . ) [ table ]. in terms of laboratory findings, those with viral, infections were far more likely to have a normal leukocyte count than those without viral infection ( % vs % leukocytes, respectively; p < . ). all cases of viral infection occurred between the months of october and may, with one exception (one episode of rhinovirus infection occurred in july), whereas bacterial infections occurred year round (fig ) . although the importance of s pneurrwniae and atypical bacterial pathogens is well understood in patients with cap, in this prospective cohort study we have now demonstrated the significant potential contribution of respiratory viruses in patients presenting with pneumonia. indeed, fully one sixth of all cases ( %) in this cohort of adults who were hospitalized with pneumonia had a respiratory virus identified; alternatively, more than one third of those there were no significant differences in outcomes according to the pathogens identified. specifically, there were no differences in median length of hospital stay (patients with viral infection, days [iqr, to days]; patients with bacterial infection, patients ( %) with a pathogen identified had a respiratory viral infection. influenza, hmpv, and rsv comprised almost two thirds of all cases of viral infection and, in our study, were acquired during the influenza season. there were some differences in presentation between those patients with a viral infection and those without, including the following: older age; presence of cardiac disease (but in the near absence of chest pain on presentation); and greater frailty. of note, patients with a viral infection were far more likely than patients with bacterial pneumonia to have a normal leukocyte count. despite these apparent differences, given that the majority of our cohort never had a respiratory pathogen identified, it is obviously very difficult to distinguish the presence or absence of viral infection in patients with pneumonia. this is further borne out by the fact that outcomes were virtually identical irrespective of the pathogens involved. adult cap etiology studies-" conducted prior to the use of nats estimated viral involvement in . to % of all cap cases. testing was generally based on serologic conversion or positive dfa test results for influenza a or b; rsv; parainfluenza virus , , and ; or adenovirus. in our cohort, viral infection without evidence of bacterial coinfection was detected % of the time, which falls within the commonly reported range. a study by marcos et al.!? which used nats, reported a similar prevalence of viral infection in spain. however, the study by marcos et al lo differed from ours in several noteworthy ways, as follows: they did not test for hmpv; and they included immunocompromised patients in their study. our results also differ from those of jennings et al, as follows: they documented a viral infection % of the time in their cohort of adults with cap, but, surprisingly, more than a third of infections were attributed to rhinovirus, and almost one fifth were mixed infections. the impact of rhinoviruses in our study may be underestimated as data have indicated that the picornavirus family of viruses is much more variable than originally thought. it is extremely difficult to design and validate assays to pick up all divergent rhinoviruses, and the original assay design that we utilized in this study would not identify all those that have been reported.f this is an inherent limitation in the type of study undertaken; as we identify more novel respiratory pathogens and variants, it is inevitable that some will have been missed. most older etiology studies-" have reported influenza infection in patients with pneumonia to % of the time, followed by rsv. influenza was the most common virus identified in our study, affecting % of patients; however, we found hmpv to occur as commonly as influenza, and more frequently than rsv. this important finding has not been widely documented as most respiratory virus studies , , , o have not included testing for hmpv, largely due to the difficulty in its identification in the past. to our knowledge, only two previous etiology studiess-!? used nats for detecting hmpv. one study.!? which was restricted to copd patients with pneumonia, found hmpv as a pathogen in . % of cases; another study" from new zealand found no cases of hmpv. the strengths of this study include its prospective nature and the thorough collection of data from a cohort of consecutive patients who had been admitted to the hospital with cap. there are also several limitations to the study. first and foremost, despite our best efforts and a detailed study protocol, a number of bacterial investigations iie, blood culture, sputum culture, and convalescent serum specimens) www.chestjournal.org were missed, thereby potentially underestimating the number of cases of bacterial pneumonia and (potentially) underestimating the number of mixed infections. the number of missed bacterial investigations may be the reason for our % rate of unknown infections, although our rate of recovery is similar to other studies , -- that have reported to % unknown infections, second, we excluded patients without evaluable np swabs from our analyses. not obtaining specimens for conducting a nat was a study protocol violation in % of patients ( of patients). we speculate that either np swabs were not collected when patients transitioned from the emergency department to the wards, or that there was a miscommunication with the reference laboratory regarding when or where to send the study-related swabs, that said, there were few important clinical differences between patients with and without evaluable np swabs, with two excep-chest/ / / december, tions. those patients without evaluable np swabs were more likely to have lobar pneumonia, which, according to our data, would bias the results toward bacterial infection. those patients without evaluable np swabs were also more likely to be functionally impaired, which would bias the results toward viral infection. third, there is potential for both falsepositive and false-negative np results, although testing with nats has been reported to have excellent sensitivity and speciflcity.p as noted above, sequence divergence for the rhinoviruses (and, potentially for the other virus groups) may also have led to some underestimation of the number of viral infections. fourth, we detected viruses in the upper respiratory tract using np specimens, which does not necessarily equate with the causation of pneumonia. however, the purpose of this study was to describe the potential role of respiratory viral infection in those patients with pneumonia; a study describing "confirmed" viral pneumonia would require lung tissue samples from all enrolled patients. last, our overall sample size might be considered small by some, and our cohort was drawn from only one health region in canada, which might limit the generalizability of the results to some degree. in our study, patients with pneumonia and respiratory viral infection were older and more frail than those without evidence of viral infection. differentiating between patients with viral infection and those without based on clinical findings and routine laboratory test results remains a challenge. indeed, although we were unable to perform a multivariable logistic regression analysis due to the small sample size, it seems unlikely that any constellation of symptoms, signs, and routine laboratory findings will ever reliably differentiate between the presence or absence of a virus. , o, , current guidelines:] recommend empiric antibiotic therapy targeted against common bacterial pathogens for patients who are admitted to the hospital with pneumonia. how to manage patients with pneumonia and a respiratory viral infection, without a documented coexisting bacterial pathogen, is far less clear. future research, similar to that found in the pediatrics literature, is needed to help answer whether empiric therapv with antibiotics can be discontinued in this clinic~ seenario.f' perhaps most importantly, the inability to identify patients with a respiratory virus without comprehensive respiratory viral testing is a concern from the perspective of infection control. the presence of a respiratory viral infection can result in nosocomial outbreaks. for instance, outbreaks due to influenza have been well documented.s" and cases of rsv and hmpv nosocomial transmission are increasingly recognized. i , . the nosocomial spread of respiratory viruses among adults poses the biggest threat to immunocompromised patients, including frail elderly patients: , i- the current infection control guidelines recommend placing patients with a suspected respiratory viral infection in private rooms or cohorting them with patients with the same viral infection as a way to prevent transmission." given the % prevalence of viral infection in adults in our study, and the indistinguishable presentation from typical bacterial pneumonia, our results suggest routine isolation (with droplet and contact precautions) of all adults with pneumonia, from the time of hospital admission until respiratory viral infection is ruled out, should be considered to help prevent the nosocomial transmission of respiratory viruses. this suggested approach should become logistically feasible when the turnaround time for nat results is < h and as the price of testing with nats decreases over time. this will be facilitated by emerging commercial viral identification assays that are both accurate and relatively inexpensive.p conclusion infections with respiratory viruses are common in patients who are hospitalized with pneumonia, com-prising % of all identified pathogens and %of all patients in our study. influenza, hmpv, and rsv were the most common respiratory viruses identified. in patients presenting with pneumonia, it remains difficult to differentiate patients with viral infection from those without viral infection. our findings suggest that routine testing for common respiratory viruses may be warranted for all adults hospitalized with pneumonia. community-acquired pneumonia: clinical features and outcomes bts guidelines for the management of community acquired pneumonia in adults infectious diseases society of america!american thoracic society consensus guidelines on the management of communityacquired pneumonia in adults microbiological profile of community-acquired pneumonia in adults over the last years incidence and characteristics of viral community acquired pneumonia in adults lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults improved diagnosis of the etiology of community acquired pneumonia with real-time polymerase chain reaction polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and b viruses, respiratory syncytial virus and parainfluenza viruses , , and the role of viruses in tbe aetiology of community acquired pneumonia in adults enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests nucleic acid amplification tests for detection of respiratory viruses newly identified respiratory viruses a newly discovered human pneumovirus isolated from young children with respiratory tract disease a previously undescribed coronavirus associated with respiratory disease in humans comparison of multiplex reverse transcription pcr enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens human metapneumovirus infection in adults with community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease a prediction rule to identify low-risk patients with community-acquired pneumonia diagnosis and epidemiological studies of human metapneumovirus using real-time pcr detection of a broad range of human adenoviruses in respiratory tract samples with a sensitive multiplex real-time pcr assay the microimmunofluorescence test for chlamydia pneumoniae infection: technique and interpretation evaluation of four commercial immunoglobulin g and immunoglobulin m specific enzyme immunoassays for diagnosis of mycoplasnw pneumoniae infections detection and persistence of specific igm antibody to coxiella bumetii by enzymelinked immunosorbent assay: a comparison with immunofluorescence and complement fixation tests reactivity of serum from patients with suspected legionellosis against antigens of legionellaceae and legionella-like organisms by indirect immunofluorescence assay patients admitted to hospital with suspeeted pneumonia and normal chest radiographs: epidemiology, microbiology, and outcomes community acquired viral pneumonia masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during viral pneumonia in older adults etiology of communityacquired pneumonia in hospitalized patients in chile viral communityacquired pneumonia in nonimmunocompromised adults viral lower respiratory traet infection in the elderly: a prospective inhospital study pneumonia: etiology. epidemiology, and outcomes at a teaching hospital in argentina etiology of severe pneumonia in the very elderly microbial etiology of acute pneumonia in hospitalized patients impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility a summer outbreak of human metapneumovirus infection in a long-term care facility viral infections in immunocompromised patients: what's new with respiratory viruses? a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients guidelines for preventing health-care-associated pneumonia nucleic acid amplification tests for detection and analysis of respiratory viruses: the future for diagnostics? acknowledgment: thanks to carol mangan, our research nurse, for her invaluable contribution, and to the provincial laboratory for their comprehensive laboratory help. thanks also go to sipi garg and meagan rosenthal for help with preparing the database for analyses. key: cord- - qh pblc authors: quah, jessica; jiang, boran; tan, poh choo; siau, chuin; tan, thean yen title: impact of microbial aetiology on mortality in severe community-acquired pneumonia date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: qh pblc background: the impact of different classes of microbial pathogens on mortality in severe community-acquired pneumonia is not well elucidated. previous studies have shown significant variation in the incidence of viral, bacterial and mixed infections, with conflicting risk associations for mortality. we aimed to determine the risk association of microbial aetiologies with hospital mortality in severe cap, utilising a diagnostic strategy incorporating molecular testing. our primary hypothesis was that respiratory viruses were important causative pathogens in severe cap and was associated with increased mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. methods: a retrospective cohort study from january to july was conducted in a tertiary hospital medical intensive care unit in eastern singapore, which has a tropical climate. all patients diagnosed with severe community-acquired pneumonia were included. results: a total of patients were in the study. microbial pathogens were identified in ( . %) patients. mixed viral-bacterial co-infections occurred in ( . %) of patients. isolated viral infections were present in patients ( . %); isolated bacterial infections were detected in patients ( . %). hospital mortality occurred in ( . %) patients. the most common bacteria isolated was streptococcus pneumoniae and the most common virus isolated was influenza a. univariate and multivariate logistic regression showed that serum procalcitonin, apache ii severity score and mixed viral-bacterial infection were associated with increased risk of hospital mortality. mixed viral-bacterial co-infections were associated with an adjusted odds ratio of . ( % ci . – . , p = . ) for hospital mortality. conclusions: respiratory viruses are common organisms isolated in severe community-acquired pneumonia. mixed viral-bacterial infections may be associated with an increased risk of mortality. the microbial aetiology of severe community-acquired pneumonia (cap) remains varied throughout the world, with influences due to seasonal climate change, outreach of vaccination programmes and pathogen endemicity [ , ] . two decades ago, it was thought that viruses played a minor role in the pathogenesis of severe cap, notwithstanding influenza epidemics [ ] . recent literature contradicts this and suggests that viruses are frequently found in severe cap [ , ] . advances in molecular techniques have improved the sensitivity, accuracy and turnaround time of microbial diagnostic tests [ , ] . the availability of highly multiplexed commercial tests kits for common viral and bacterial pathogens has enabled these tests to be performed in large numbers of patients simultaneously, and across a variety of clinical settings [ , ] . multiple respiratory viruses may be present concurrently, or co-exist with bacterial pathogens to cause disease [ ] [ ] [ ] . the reported incidence of viruses in severe cap resulting in respiratory failure ranges . % to % [ , ] . this variation in the detection rate of viruses reflects potential limited availability of test assays and heterogeneity of physician practices in viral microbial diagnostic tests performed [ , ] . postulated prohibitive factors against the routine performance of viral diagnostics tests in patients with severe cap may include a lack of clear clinical guidelines, perceived low cost-effectiveness and the paucity of effective anti-viral therapies for respiratory viruses other than influenza. the primary aim of our study was to determine the risk association of microbial aetiologies with hospital mortality in severe cap, utilising a diagnostic strategy that incorporated molecular testing. our primary hypothesis was that respiratory viruses were important causative pathogens in severe cap and was associated with increased mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. this was a retrospective cohort study performed in an -bed medical intensive care unit of a -bed tertiary teaching hospital, in singapore. ethics approval was obtained from the singhealth centralised institutional review board (cirb / /fp), with waiver of consent. adults above the age of admitted to the medical intensive care unit with severe cap from january to july were included. written and electronic medical records were reviewed. cap was defined as an acute infection of the lung parenchyma associated with acute chest radiographic infiltrates and or more of the following: fever of °c or higher or hypothermia of °c or less; a new cough; dyspnea not explained by other reasons; worsening cough or change in respiratory secretions in a patient with pre-existing chronic cough. these symptoms should have been present at the time of, or within h of, hospital admission. chest radiographs reported by the hospital radiologists were obtained to confirm the presence of radiographic pulmonary infiltrates. patients with shock requiring vasopressor support, mechanically ventilated or who have out of minor criteria for pneumonia severity as defined by the infectious diseases society of america/american thoracic society, are considered to have severe cap [ ] . the minor criteria include: respiratory rate of breaths per minute or greater; pao /fio ratio equal or less than ; multi-lobar pulmonary infiltrates; confusion or disorientation; blood urea nitrogen equal or greater than mg/dl; leukopenia with white blood cell count of less than , cells/mm ; hypothermia indicated by core body temperature less than °c; hypotension requiring aggressive fluid resuscitation. all patients with prior hospitalisation within days of study enrolment; on active chemotherapy for neoplastic diseases; receiving renal replacement therapy by haemodialysis and prisoners were excluded. immunocompromised patients were not excluded as they were likely to have cap microbial pathogens as well as opportunistic infections. the time of admission to the intensive care unit was verified with electronic records. all patients had at least set of aerobic and anaerobic blood cultures performed within h of admission. routine collection of endotracheal aspirates for gram stain and semi-quantitative bacterial cultures occurred within h of intubation. viral and atypical pathogen polymerase chain reaction (pcr) amplification tests were collected from endotracheal aspirates. sputum samples were sent for bacterial aerobic cultures, while nasopharyngeal swabs were performed for atypical pathogen and viral pcr amplification tests for subjects who did not require intubation. urinary samples were tested for the presence of urinary streptococcus pneumoniae antigen and legionella pneumophilia serogroup antigen in patients without anuria. where indicated, acid-fast staining and mycobacterial cultures of respiratory samples were performed. nucleic acid was extracted from swabs or respiratory samples using ez virus mini kit (cat no. , qiagen, germany) performed on a semi-automated system (ez , qiagen, germany), and stored at - °c for more detailed molecular testing. besides routine standard clinical testing, additional multiplex real-time pcr testing was performed for each extracted sample. anyplex written and electronic medical records were reviewed for clinical data and laboratory indices. the most severe value was recorded for analysis if any blood test was repeated more than once within h after intensive care admission. serum c-reactive protein was measured by particle enhanced immunoturbidimetry (cobas® crpl ) and serum procalcitonin was measured using sandwich immunoassay (cobas® elecsys brahms pct). variables such as the acute physiology and chronic health evaluation ii (apache ii) severity score, presence of shock, mechanical ventilation, intensive care unit length of stay, and mortality were captured prospectively as part of administrative clinical care audits. empirical antibiotics were deemed to be adequate if it adhered to the hospital antimicrobial guidelines. combination therapy with beta-lactams and macrolides were recommended. where melioidosis was suspected, carbapenems were preferred to other classes of beta-lactams. categorical variables were expressed as number (percentages) and normally distributed quantitative variables were expressed as mean (± standard deviation). categorical variables were compared with chi-square test or fisher's exact test, and quantitative variables were compared with t-test or mann-u whitney test. the primary outcome measure was all-cause hospital mortality. demographic and disease variables were included in the univariate and multivariable logistic regression model. pneumonia symptoms were excluded from regression analysis as there were no known associations with mortality in severe cap. the variable for number of co-existent pathogens was excluded from regression analysis due to significant overlap with mixed viral-bacterial co-infections, which was included in the multivariate model. the following severity indicators: shock, altered mentation and ventilator support were not included as these variables are incorporated in the apache ii severity score. mycobacterium tuberculosis was classified as a bacterium for the logistic regression. demographic and disease variables were then compared between patients with and without virus infections, for identification of potential risk factors associated with acquisition of respiratory viral infections. a p-value of < . was considered significant. missing data was excluded from univariate and multivariate analysis. stata special edition version . (statacorp llc, texas, usa) was used for statistical analysis. one hundred seventeen patients were admitted to the medical intensive care unit for severe cap within the study period. baseline characteristics of patients who suffered in-hospital mortality (n = , . %) were compared with patients who survived, in table . of note, the patients who did not survived were older compared to survivors ( . years vs . years, p = . ). other baseline demographic and co-morbidity variables were not significantly different. blood cultures, respiratory specimen cultures and respiratory specimen pcr testing for viruses and atypical bacteria were performed in all patients. results of microbiological tests are presented in table . causative microbial aetiologies were identified in . % of patients with specific organisms presented in table . majority of pathogens identified were respiratory viruses and bacteria. viruses were found in . % of patients (n = ) with the most common virus being influenza a. . % (n = ) of patients with influenza a received empirical oseltamivir on the basis on clinical suspicion. bacterial infections were found in . % of patients (n = ). . % of patients (n = ) had mixed virus and bacterial co-infections. patients ( . %) had only viral pathogens and patients ( . %) had only bacterial pathogens, found as the microbial aetiology for severe cap. using hospital mortality as the primary outcome, univariate logistic regression was performed (table ). patients who did not survive had a higher apache ii score and higher serum procalcitonin levels. the microbiological aetiology that was significantly associated with increased hospital mortality was the detection of mixed viral-bacterial co-infections. on multivariate analysis, apache ii severity score, serum procalcitonin and mixed viral-bacterial co-infections remained significantly associated with increased adjusted odd ratios for hospital mortality. while apache ii severity score is known to be predictive for mortality in severe cap, the study found that the presence of mixed viral-bacterial co-infections was associated with increased hospital mortality by an adjusted odds ratio of . ( % confidence interval . , . , p = . ). the patients with respiratory viruses detected (both isolated viral pathogens and mixed viral-bacterial co-infections) were compared with patients who did not have any respiratory virus infections, in table . the mean serum c-reactive protein was found to be greater ( . ± . mg/l vs. . ± . mg/l) in patients without respiratory viruses compared to patients with detection of respiratory viruses (table ). respiratory virus infection as a cause of severe acute respiratory distress syndrome is well-established in literature data was missing for patient [ ] [ ] [ ] . however, its significance as a contributory pathogen in the outcomes of severe cap is uncertain. in this study, we showed that respiratory viruses were as commonly found as bacteria ( . % vs . %), as an aetiological pathogen. mixed viral-bacterial co-infections occurred in . % of patients and was associated with an adjusted odds ratio of . for hospital mortality. the impact of respiratory viruses on the prognosis of severe cap remains unclear with recent studies demonstrating contradictory results. fisher et al., in a prospective -year microbiologic survey of nosocomial pneumonia and cap complicated by respiratory failure, showed that . % of patients had viral infections, which was associated with a hospital mortality of . % [ ] . however, siow et al., found that viral infections were independently associated with lower hospital mortality compared to other microbial aetiologies, with an adjusted odds ratio of . (ci . - . ; p = . ) [ ] . in light of these findings, accurate characterisation of the impact of microbial aetiology on the outcomes of severe cap is required to influence future development of rapid molecular diagnostics assays and novel antimicrobial therapies that would target both viruses and bacteria [ , ] . piralla et al. reviewed the microbiological data of severe cap in northern italy during winter-spring seasons over years and found that . % of patients had one or more respiratory viruses identified [ ] . the most common viruses isolated were influenza a and rhinovirus, similar with our findings. while our study was performed in a tropical country, local microbiological surveillance has shown that influenza epidemics occur twice annually [ ] . this would mean that influenza seasons occurred over the course of this study. the molecular mechanisms in the viral pathogenesis of severe pneumonia are most well studied in influenza a and streptococcus pneumoniae co-infections. viral infections alter host immune responses that increase susceptibility to bacterial infection through viral-induced interferons [ ] [ ] [ ] . on clinical suspicion alone, only . % (n = ) of patients with influenza a in this study received empirical oseltamivir. the authors postulate that incorporation of early influenza a diagnostic tests may decrease the time to effective anti-viral therapies. the second most common virus detected in our study was rhinovirus (n = ). the association of rhinovirus with severe pneumonia has previously been shown in a surveillance program for middle east respiratory syndrome coronavirus in saudi arabia [ ] . its genotypes a to c are associated with severe pneumonia, with in-hospital mortality rates from . to . % [ ] . patients who are immunocompromised or who have chronic lung disease are most at risk [ ] . there are several reasons why viral infections may have been less prominent as a cause of severe cap in prior decades. firstly, grève et al. performed a prospective observational study on physician practices in the use of respiratory virus diagnostics demonstrating that despite clinical guideline recommendations on testing of respiratory viruses during influenza season, less than half of patients admitted to the intensive care unit with pneumonia were tested for viral pathogens [ ] . this may have led to under-recognition of the true significance of viral pathogens and mixed viral-bacterial infections, on outcomes in severe pneumonia. other factors which may have contributed to underdiagnosis of viral pneumonias include the unavailability or cost of molecular diagnostic assays, and the lack of effective anti-viral therapies [ ] . however, the authors argue that in this age of globalisation, highly virulent respiratory viruses have the potential to spread rapidly. constant surveillance is required to detect outbreaks and for the implementation of isolation precautions in a timely manner [ , ] . understanding the significance of respiratory viruses in the pathogenesis of severe cap would guide administrators with resource allocation when implementing vaccination programs for at-risk populations. the high rates of compliance with performing respiratory aerobic cultures, blood aerobic and anaerobic cultures in this study, were in accordance with sepsis guidelines [ ] . the most common bacterial pathogen found was streptococcus pneumoniae ( . %), which is consistent with the known epidemiology of cap globally [ ] [ ] [ ] . gadsby et al., in a prior study, was able to demonstrate a bacterial yield of . % when respiratory specimens from patients with cap were tested with bacterial multiplex pcr [ ] . incorporating the use of bacterial multiplex pcr in future studies, may increase the rate of bacteria detection, and shed light on potential molecular synergisms between specific viruses and bacteria in the pathogenesis of severe cap. pulmonary tuberculosis is endemic in the region where this study was performed. in our study, patients had tuberculosis, one of whom had concomitant adenovirus infection while another had streptococcus pneumoniae. the initial presentation of pulmonary tuberculosis with clinical features consistent with severe cap has been described by tseng et al. [ ] , where % of patients with pulmonary tuberculosis presented with respiratory failure or septic shock. the authors postulate that pulmonary tuberculosis may play a role in increasing host susceptibility to severe infection with cap organisms. the authors recognise that there were several limitations to this study. firstly, while first-dose antibiotics would have been administered as soon as sepsis is identified, we are unable to accurately define the time between administration of antibiotics and collection of specimens for microbiological assessment. this may affect the yield [ , , ] . secondly, the inherent retrospective nature of this study increases the risk of bias in data collection. however, as part of pre-established intensive care unit clinical audits and with the availability of national health records, clinical data such as participant demographics, co-morbid illnesses and severity indicators such as apache ii were established at the time of intensive care admission and stored prospectively. thirdly, given the high population density of the country ( rd in the world) where this study was performed, the microbial epidemiology of severe cap may only be extrapolated to urban settings. the study is a single-centre survey conducted in of the acute general hospitals serving a population of . million in a land area of . km . hence, the epidemiology may lack generalisability when extrapolated to other tropical countries. the fourth limitation is that we included patients who were immunocompromised with typical cap organisms and survived. they may potentially have had opportunistic infections that were not detected, however, as they did not contribute to the mortality outcomes, we felt that the microbiological data contributed by these patients were useful in the understanding of the prevalence of various data are presented as number (%), mean ± standard deviation apache acute physiology and chronic health evaluation a data was missing for patients. b data was missing for patient classes of cap organisms. lastly, another potential limitation was that vaccination records could not be retrieved, and we were unable to ascertain its influences on microbial aetiologies of severe pneumonia in our study population. the main strength of the study is the characterisation of the epidemiology of microbial pathogens in severe cap. we were able to show that in a tropical environment, the viral and bacterial pathogens associated with severe cap were similar with regions with a seasonal climate. despite a lower-than-expected mortality rate for severe cap in our study ( . %) compared with international data [ ] [ ] [ ] [ ] , we were able to demonstrate that mixed viral-bacterial co-infections were independently associated with hospital mortality. respiratory viruses are important causative pathogens in severe cap and are associated with increased risk of mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. abbreviations apache ii: acute physiology and chronic health evaluation ii; cap: community-acquired pneumonia; pcr: polymerase chain reaction etiology and outcome of severe community acquired pneumonia in immunocompetent adults etiology of community-acquired pneumonia and diagnostic yields of microbiological methods: a -year prospective study in norway epidemiology of community-acquired respiratory tract infections in adults the intensive care global study on severe acute respiratory infection (ic-glossari): a multicenter, multinational, -day inception cohort study viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia molecular diagnosis of respiratory viruses the filmarray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens rapid viral diagnosis for acute febrile respiratory illness in children in the emergency department clinical utility of on-demand multiplex respiratory pathogen testing among adult outpatients burden of respiratory viruses in patients with acute respiratory failure community-acquired polymicrobial pneumonia in the intensive care unit: aetiology and prognosis clinical characteristics and outcomes in hospitalized patients with respiratory viral co-infection during the h n influenza pandemic management of severe communityacquired pneumonia: a survey on the attitudes of physicians in iberia and south america clinical practice of respiratory virus diagnostics in critically ill patients with a suspected pneumonia: a prospective observational study infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards virus-induced acute respiratory distress syndrome: epidemiology, management and outcome influenza a (h n ) vs non-h n ards: analysis of clinical course a prospective one-year microbiologic survey of combined pneumonia and respiratory failure the use of polymerase chain reaction amplification for the detection of viruses and bacteria in severe communityacquired pneumonia the past decade in bench research into pulmonary infectious diseases: what do clinicians need to know? laboratory diagnosis of pneumonia in the molecular age frequency of respiratory viruses among patients admitted to intensive care units in seven consecutive winterspring seasons ( - ) in northern italy characterization of influenza activity based on virological surveillance of influenza-like illness in tropical singapore the immunology of influenza virusassociated bacterial pneumonia integrated clinical, pathologic, virologic, and transcriptomic analysis of h n influenza virus-induced viral pneumonia in the rhesus macaque kinetics of coinfection with influenza a virus and streptococcus pneumoniae etiology of severe communityacquired pneumonia during the hajj-part of the mers-cov surveillance program clinical and molecular characterization of rhinoviruses a, b, and c in adult patients with pneumonia human rhinoviruses and severe respiratory infections: is it possible to identify at-risk patients early? viral pneumonia and acute respiratory distress syndrome burden of acute respiratory disease of epidemic and pandemic potential in the who eastern mediterranean region: a literature review global threat of animal influenza viruses of zoonotic concern: then and now surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock global and local epidemiology of communityacquired pneumonia: the experience of the capnetz network global changes in the epidemiology of community-acquired pneumonia community-acquired pneumonia requiring hospitalization among u.s adults comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia empirical use of fluoroquinolones improves the survival of critically ill patients with tuberculosis mimicking severe pneumonia impact of antibiotic therapy in severe community-acquired pneumonia: data from the infauci study predictive factors of mortality in severe community-acquired pneumonia: a model with data on the first h of icu admission viral infection in patients with severe pneumonia requiring intensive care unit admission etiology of severe pneumonia in the very elderly assessment of prognosis in patients with community-acquired pneumonia who require mechanical ventilation the authors would like to thank the following: ms. carmen kam, the resident biostatistician for verification of the study statistical analysis; nurses ms. wang xi qin, ms. goh yuan xuan, ms. lim qian ru for assistance in data collection; senior medical technologist ms. heng ying xuan, ms. lee hui zi for assistance in performance of pcr tests; dr. tay tunn ren for her tutelage in manuscript writing. the study was performed with a grant awarded from changi general hospital research grant in (grant reference number chf . ), for an amount of $ singapore dollars (equivalent to usd , conversion usd = sgd . ). the datasets analysed during this current study are available from the corresponding author on reasonable request. authors' contributions jlq contributed to the design, analysis, interpretation of the study; drafting and revision of the manuscript. bj contributed to the design, interpretation and microbiological analysis of the study. pct contributed to the design and data acquisition of the study. cs contributed to the design of the work, drafting and revision of the manuscript. tyt contributed to the conception, analysis and interpretation of the study, drafting and revision of the manuscript. all authors have given final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.ethics approval and consent to participate ethics approval was obtained from the singhealth centralised institutional review board, singapore. reference number: cirb / /fp. waiver of consent granted for this study. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -c uhcatg authors: costa, lusmaia d.c.; costa, paulo sucasas; camargos, paulo a.m. title: exacerbation of asthma and airway infection: is the virus the villain? date: - - journal: jornal de pediatria doi: . /j.jped. . . sha: doc_id: cord_uid: c uhcatg abstract objective to review the available literature on the association between acute viral respiratory tract infection and the onset of asthma exacerbations, identifying the most prevalent viruses, detection methods, as well as preventive and therapeutic aspects. sources a search was conducted in pubmed, lilacs, and scielo databases, between the years and , using the following descriptors: asthma exacerbation, virus, child, and acute respiratory infection. summary of the findings a total of original articles addressing the identification of respiratory viruses during episodes of asthma exacerbation were selected, mostly cross-sectional studies. there was a wide variation in the methodology of the assessed studies, particularly in relation to the children's age and methods of collection and viral detection. the results indicate that, in up to . % of exacerbations, a viral agent was potentially the main triggering factor, and human rhinovirus was the most frequently identified factor. the pattern of viral circulation may have been responsible for the seasonality of exacerbations. the association between viral infections and allergic inflammation appears to be crucial for the clinical and functional uncontrolled asthma, but few studies have evaluated other triggering factors in association with viral infection. conclusions respiratory viruses are present in the majority of asthmatic children during episodes of exacerbation. the involved physiopathological mechanisms are yet to be fully established, and the synergism between allergic inflammation and viral infection appears to determine uncontrolled disease. the role of other triggering and protective agents is yet to be clearly determined. asthma is a chronic, genetically-determined disease, whose prevalence in the pediatric population ranges between . % and . % among brazilian adolescents and schoolchildren, respectively. from the physiopathological viewpoint, it is characterized by chronic inflammation with the involvement of several cell types, associated with airway hyperresponsiveness, with episodes of reversible airflow limitation. it is clinically manifested by recurrent exacerbations, also called ''asthma attacks'' or, more appropriately, acute asthma, characterized by progressive worsening of dyspnea, coughing, wheezing, chest tightness, or a combination of these. the loss of clinical and functional asthma control usually occurs gradually, but it can occur abruptly in a subgroup of patients. it is one of the main causes of emergency consultations, having been responsible, in , for deaths in children younger than years in brazil. public policies have been developed to promote both scientific knowledge about the disease and its management, as well as to organize assistance programs in public health, which include, among others, the dispensing of medications. however, exacerbations continue to represent a significant number in statistics, with great impact on public and private healthcare systems. the multifactorial origin of the clinical-functional lack of disease control is well known; since the early s, respiratory viruses have been associated with the triggering of asthma exacerbations in adults and children. in the s, the development of more sensitive and specific molecular techniques allowed for the increase in respiratory virus detection and therefore, ways to better explain this association. studies using reverse transcriptase polymerase chain reaction (rt-pcr) as the detection technique, isolated or combined with traditional methods, observed positivity for respiratory viruses in up to . % of episodes of acute asthma exacerbation in children. considering the possibility of a causal relationship between respiratory virus infection and the triggering of asthma attacks in children, the implications of this association, as well as the possibility of specific prophylaxis and therapy for these agents, special attention to this subject is justified. therefore, this literature review aimed to analyze articles, published between and , assessing the association between asthma exacerbation and acute viral airway infection. a search was conducted in the pubmed, lilacs and scielo databases, using the descriptors: ''asthma exacerbation'', ''viral infection'', and ''child'', resulting in a total of references for that period. after selecting the articles published in portuguese, english, spanish, or french, articles remained. after reading the titles and abstracts, original articles that assessed respiratory tract viral infection in asthmatic children during exacerbation were selected. some articles of historical importance or review articles that included the three descriptors were added to generate the bibliography of this review. the list of references was inserted into endnote x (thompson corp., ca, usa), a bibliographic citation management software. the most frequently identified respiratory viruses in association with asthma exacerbation were human rhinovirus (hrv), respiratory syncytial virus (rsv), human adenovirus (hadv), influenza (flu), parainfluenza (pflu), human metapneumovirus (hmpv), and human coronavirus (hcov). of the listed viruses, most have rna as the nucleic acid; their biological characteristics and taxonomy are described in table . the main transmission methods for these viruses are through contaminated fomites, droplets, aerosols, or direct contamination. by infecting the nasal epithelium cells, these agents trigger an immune response, which involves mainly the dendritic cells and natural killer (nk) cells, inducing the release of a number of pro-inflammatory cytokines and chemokines by the infected epithelial cells, such as interferon (ifn- ) and tumor necrosis factor alpha (tnf␣), among others ( fig. ) . in asthma patients, the viral infection causes an imbalance in the immune homeostasis of the respiratory system. several mechanisms related to viral infection and allergic inflammation, as well as their role in triggering acute asthma, have been proposed; among them, the deficient function of the epithelial barrier caused by the virus, which has been implicated as a predisposing factor by some. however, both asthma and atopy are associated with epithelial damage, which may contribute to increased susceptibility to infections, including viral diseases and sensitization by aeroallergens. another evaluated factor was mucus production as an airway defense mechanism; in mice studies, it was demonstrated that allergic inflammation and viral infection act synergistically increasing mucus production, which can lead to airway impaction and obstruction in asthma patients. virus-induced alterations in interferon production have also been observed. for instance, in vivo and in vitro studies in epithelial cells from healthy adults and asthma patients infected with hrv demonstrated a decreased production of type i interferon (␣ and ␤) in the latter, making them more susceptible to infection associated with viral exacerbation. , similar results were obtained in studies performed with children, where the production of interferon and th cytokines by bronchial epithelial cells was assessed after hrv- infection. lower interferon production and higher concentrations of viral rna have been demonstrated in children with asthma, regardless of their atopic status, and in atopic children without asthma, suggesting that an impaired immune response to viral infection occurs not only in asthma patients, but in children with other disorders associated with th lymphocytes. however, other studies failed to demonstrate the same reduction in interferon production; others even found an increase in its production in exacerbated asthma. , the bronchial epithelium produces some cytokines, including interleukin and , as well as thymic stromal lymphopoietin, which promotes the differentiation of innate lymphoid cells into th . the latter can be induced by viral infection, and its production can be increased by interleukin- (il- ), suggesting that the interaction between viruses and allergic airway inflammation may enhance the inflammatory th response and potentially reduce the antiviral response. , collection and viral detection methods viral detection is highly dependent on the quality of the collected sample, on the time of symptom onset to the time of collection (ideally within hours), and on transportation and storage of the sample before testing. the analysis for respiratory viruses should be performed in material from the airways. upper airway secretion is used in most cases, and several methods are employed for this collection, such as nasopharyngeal aspirate (npa), nasopharyngeal swab (nps), nasopharyngeal lavage, and the combined nasal-oral swab; the first technique is considered the gold standard. recently, new flocked swabs (copan, brescia, italy) were developed, and presented better performance during data collection. recent studies using this type of swab presented a sensitivity comparable to that of npa when the detection is performed by pcr, suggesting that this swab can be used in epidemiological research and surveillance studies, due to its greater technical simplicity. , there are few data to support the use of combined oralnasal swabs for virus detection, and its sensitivity is lower than that of the nasopharyngeal swab or aspirate, which can be explained by the lower viral load in the oropharynx than in the nasopharynx. the collection can also be performed on material from the lower airways, such as induced sputum and bronchial lavage. the methods for detection of respiratory viruses are varied and include rapid tests for antigen detection, culture, direct and indirect immunofluorescence, and nucleic acid amplification reactions, such as rt-pcr, which can detect a single agent (monoplex) or perform multiple detections (multiplex). the sensitivity of the latter is higher, and it was used in most recent studies. , immunofluorescence reactions have lower cost, are faster to perform, and are also able to detect multiple viruses. a panel of seven viruses (iflu a and b, pflu to , hadv, and rsv) is generally used. some viruses, such as hrv and bocavirus, can only be detected through nucleic acid amplification reactions. several authors have performed studies aiming to detect viruses in respiratory secretions of exacerbated asthma patients, showing a prevalence of viral identification that varies with several factors, such as patient age, time of the year, method of sample collection, and method of viral detection. table presents the original articles that demonstrate results of viral testing in children with exacerbated asthma. most studies found prevalence rates between . % and . %; in these cases, the most frequently identified virus was hrv. an investigation of respiratory viruses in children aged between three and years hospitalized for asthma exacerbation was performed over a period of months in buenos aires, argentina. due to the possibility of other diagnoses for wheezing episodes in infants, the definition immune process involved in response to respiratory viruses and their association to allergic inflammation. respiratory viruses infect bronchial epithelial cells (becs) through tool-like receptors (tlrs). during replication, they trigger an inflammatory process with induction of cytokine and chemokine production by becs, among them interferon (ifn- ), tumor necrosis factor alpha (tnf-␣), interleukins (il- , il- ), and thymic stromal lymphopoietin (tslp). the dendritic cells (dcs), components of the innate immunity, are directed to secondary lymphoid organs after capturing viral antigens, where they stimulate the lymphoid cells, the protagonists of the specific immune response. in asthma patients, the production of ifns is reduced, allowing for greater viral replication and under stimulation of tslp, there is a deviation from lymphoid profile to helper t lymphocyte (th ), promoting lower antiviral response and increased allergic inflammation, with bronchial hyperreactivity and increased production of mucus, causing bronchial obstruction and asthma exacerbation. of asthma was based on the criteria of castro-rodriguez for children younger than years, and on the global initiative for asthma (gina) criteria for those older than . immunofluorescence and pcr were performed in nasopharyngeal secretions of the children and showed a positive rate of . % for viruses in general; hrv and hrsv were the most frequently identified types. there was also . % of dual detection, with the involvement of all analyzed viruses. in méxico, the frequency of the viral positivity at the immunofluorescence was higher in children with asthma ( . %) than in a control group of wheezing children without asthma ( . %). hrv was not included in that study, and iflu, pflu, and hadv were the most frequently identified virus in the group of asthma patients. in japan, respiratory viruses were detected by multiplex pcr in . % of children with exacerbated asthma, with a mean age of . months. the hrsv was related to a single episode of wheezing (p < . ). hrv was more frequently observed in patients with a history of asthma (p < . ). a group of french children with exacerbation treated at home was compared to stable asthmatic children. immunofluorescence, pcr, and serology for viruses (mycoplasma pneumoniae and chlamydophila pneumoniae) detected a pathogen in . % of samples, with significantly higher frequency in cases than in controls ( . %). viral detection tests were positive in % of cases, and hrv was the most common ( . %). in . % of cases, the serologic tests were positive for both atypical pathogens. another series of children with exacerbation, compared to stable children, was studied by turkish authors and showed positivity of . % in the cases and . % in controls, through rt-pcr reaction. hrv was the most commonly found virus in . % of the samples. in japan, children with acute asthma were compared to stable asthmatic children and children without asthma. using an antigen detection kit and rt-pcr, respiratory viruses were detected in . % of nasal aspirate samples in exacerbated asthmatic children, and hrv was the most common ( . %). in parallel, the assessment of inflammatory markers showed a significant elevation (p < . ) of interleukins il- , , , and in serum and in the nasal aspirates of patients in exacerbation, as well as an increase in serum eosinophilic cationic protein (ecp) levels (p < . ). flu, although less frequently associated with these episodes, appears to be responsible for increased morbidity in patients with an underlying chronic disease, including asthma. of , children aged to years admitted with a diagnosis of infections by fluv-a and b between and in the united states, . % were asthma patients, and complications were more significantly associated with fluv-a (p < . ). other viruses were not assessed in that population. another study compared exacerbated children treated in hospitals (n = ) with those treated at home (n = ). immunofluorescence for flu, hadv, hrsv, and piv was performed, as well as pcr for bocavirus. a . % rate of viral detection was obtained, but no difference was observed regarding the viral profile between inpatients and outpatients. the most frequently observed viruses were rsv ( . %) and bocavirus ( . %), but hrv was not included in the viral panel of this study. a group of australian children aged up to years had their nasal secretions collected in three periods between and , and were compared with a control group of non-asthmatic children with upper respiratory tract infection (urti) in the same period and another group of children with controlled asthma, assessed during routine consultations. hrv and hmpv were screened by rt-pcr and a panel of seven viruses (fluv-a and b, piv- to , hadv, and hrsv) was studied by immunofluorescence. hrv infection accounted for . % of the urti of non-asthmatic children, and co-infection was common, especially with the hrsv, especially in children younger than years. children with symptomatic asthma had the highest rates of hrv infection ( . % vs. . % among all children). finally, children with controlled asthma had the lowest rates of hrv identification ( . % vs. . %). studies conducted in and aimed to the identification of hrv in exacerbated asthmatic patients through rt-pcr, found an overall frequency of viral identification of . % and . %, respectively. the first study used a group of comparison consisting of stable asthma patients, in which the identification rate was lower ( . %) than in the case group ( . %). both studies found a greater association between exacerbations and the presence of hrv c. another important issue in the complex association between viruses and asthma is related to the intensity of the association of exacerbations with viral infection. in this sense, several studies , --- presented inconclusive results, although hrv was associated with increased severity or worse response to treatment. , , the association between viral infection and acute asthma severity was evaluated in children aged to years. a positivity rate of . % for the presence of virus was observed by direct immunofluorescence (dif) and multiplex pcr; hrv was detected in . % of cases, and type c was observed in half the cases and was associated with greater severity. fifty-eight asthmatic children aged to years were monitored for a period of five weeks between april and september of . they had nasal lavage samples collected weekly for multiplex pcr analysis, in addition to a symptom diary, peak expiratory flow, and notes on rescue medication use. a virus was detected in . % to . % of the specimens; hrv was identified in . % to . % of the positive samples, and was associated with greater symptom severity. nonetheless, viral testing by multiplex pcr for pathogens in children with exacerbated asthma compared with controlled asthma patients, performed in hong kong between and , showed no association between the presence of the virus and exacerbation severity. one virus was identified in . % of cases, and this detection was, in general, more associated with exacerbations (or . ; % ci: : to : ; p < . ). when analyzed individually, no virus was associated with exacerbation, although hrv was the most frequent, being identified in . % of exacerbated and in . % of controlled asthma patients, but with no significant difference (p = . ). nasopharyngeal aspirate samples of asthmatic children aged between and years collected during episodes of exacerbation were referred for viral identification by pcr. the positivity rate was . %;the most frequently observed were hrv ( . %), followed by hrsv ( . %). there was no association with exacerbation severity. a study compared the response to treatment with bronchodilators between exacerbated children with viral respiratory infection symptoms (n = ) and a group without such symptoms (n = ). the mean age was . years, and exacerbation severity did not differ between groups. children with viral symptoms had poor response to bronchodilators, requiring more doses of beta-agonists after , , and hours. the viral screening was conducted in . % of cases; hrv was the most frequently found virus ( . %). in another study, exacerbated children were treated at the hospital and compared to asymptomatic adults. multiplex pcr reactions for eight respiratory viruses and monoplex pct for enterovirus, hrv, and bocavirus detected the presence of respiratory viruses in . % of cases; hrv was once again the most frequently observed virus ( . %). genotyping showed a higher frequency ( . %) of type c hrv and association with type a showed a worse clinical outcome. asthma exacerbations have seasonal distribution, occurring cyclically in both adults and children, and can be explained by the viral circulation pattern or change in the level of pollutants and aeroallergens. one example is what occurs in temperate countries, where a higher rate of occurrence is more likely to be observed in the fall and spring among schoolchildren. a combination of factors may explain this phenomenon, such as increased circulation of hrv in late summer and early autumn, increased circulation of pollutants and aeroallergens, and the return to school after the summer vacations. the influence of the return to school activities may be explained by lower adherence to maintenance treatment during the vacation period. the circulation of other viruses has been reported in other countries in the northern hemisphere, especially hrsv during autumnwinter, flu in winter, piv- and in the fall, and piv- in the spring. , in brazil, data on viral circulation were collected from the brazilian system of epidemiological surveillance on flu viruses and their counterparts in the period between and . samples obtained from nasopharyngeal swabs of patients in different sentinel units distributed throughout the country were analyzed by immunofluorescence. they showed a predominance of fluv and hrsv, with circulation throughout the year, with peaks for the latter between march and june, and between may and august for fluv. no data were located concerning the movement of hrv in brazil. few published data regarding the seasonality of exacerbations were found. to make a parallel to virus circulation and the occurrence of exacerbations, the authors analyzed data obtained in some studies, such as the study conducted in the federal district, which observed a higher frequency in the month of march. still in the midwest region, in the state of goiás, an increased frequency of respiratory symptoms, not specified as asthma, was observed in winter. an observation regarding the distribution of the occurrence of asthma in the state of minas gerais also showed higher concentrations in fall-winter, between may and july, indicating a predominance of respiratory and/or asthma symptoms in the brazilian fall-winter seasons. in addition to the seasonal variation of the virus, other factors involved in the genesis of asthma exacerbation may explain this variation, such as aeroallergens and pollutants, which also vary throughout the different seasons. it is likely that the combination of these and other factors result in the observed seasonal peaks in exacerbations. in the month of april of the years and , a study was conducted in korea aiming to monitor viral infection and to identify sensitization to aeroallergens in children with acute asthma or diagnosis of a cold, whose mean age was . years. children with allergic sensitization presented the same number of viral infections, but with more symptoms than those nonsensitized. in another study, conducted in manchester, england, children hospitalized for exacerbation were compared to children with stable asthma and children hospitalized for non-respiratory disease. the authors concluded that the association between viral infection and allergen exposure increased the risk of hospital admission by . -fold. in brazil, camara et al. investigated the role of viral infections, sensitization, and exposure to aeroallergens as risk factors for wheezing in children aged up to years. in those younger than years, the frequency of viral positivity was significantly higher in cases ( . %) than in controls ( . %). in older children, there was no significant difference: . % of cases and . % of the positive controls. they concluded that in children younger than years, the risk factors associated with wheezing were viral infection and a family history of atopy; among older children, sensitization to inhalant allergens was the most important event for the onset of crises. the effect of air pollutants is usually disregarded in the presence of viruses or allergens. however, there is evidence that acute exposure to specific pollutants may contribute to the symptoms and severity of exacerbations. for instance, cigarette smoke induces a model of non-eosinophilic inflammation with relative resistance to corticosteroids. passive smoking is quite common in homes of asthmatic children, causing a negative impact on disease control. in scotland, the legislation that banned smoking in public places reduced hospitalizations for asthma by . %. other pollutants appear to contribute to asthma exacerbations, such as those resulting from the combustion of natural gas and engine oil, such as nitrogen dioxide (no ). children spend most of their time outside and breathe in a greater amount of pollutants per kilogram of weight when compared to adults, and the increased levels of no are associated with the severity of virus-induced exacerbations. this emphasizes a potential synergism between these two inflammatory stimuli. moreover, controlled exposure in asthma patients demonstrated that no increases the response to inhaled allergens. a cohort of asthmatic children aged between and years were monitored for symptoms, measurement of peak expiratory flow, measurement of exposure to no , and presence of virus in nasal secretion during a period of months. one or more viruses were detected in % of the reported episodes of respiratory symptoms; it was demonstrated that exposure to high concentrations of no in the week before the onset of a viral respiratory infection was related to the exacerbation severity. a longitudinal study conducted in the united states measured exposure to cigarette smoke in , children with asthma and no in a subset of of them, over a period of nine months. they demonstrated increased symptoms in those exposed to no , but only among non-atopic children, with a relative risk of . ( % ci: . to . ). there was no association between symptoms and increased cigarette smoke exposure. two cross-sectional studies compared children exposed to different levels of cigarette smoke and showed that those exposed to high levels had higher symptom scores (p < . ), nocturnal symptoms (or . ; % ci: . to . ), and need for relief (p = . ) and control (p = . ) medications. a study in which children aged between and years were randomized to intervention with environmental education guidelines aimed at reducing exposure, showed a reduction in exposure in the group that received instructions for a period of months. the intervention group had fewer days with symptoms (p < . ) after one year of followup, in addition to a decrease in the levels of dust mites (dermatophagoides pteronyssinus and dermatophagoides farinae) and cockroach antigens in the home environment. fungal sensitization is prevalent in children with asthma, although few studies have addressed this issue, compared to studies related to dust mites. one study demonstrated that children with a positive skin test for fungi had more days of symptoms when compared to those with negative tests ( . vs. . for two weeks, p = . ). during the study period, fungi were grown from the intra-and extra-domestic environment; increased exposure to fungi was associated with increased days of symptoms and unscheduled physician visits for asthma. the preponderance of virus participation among the infectious agents in exacerbations makes the indiscriminate prescription of antibiotics in this situation pointless in most cases. previous studies suggest that chest radiography is improperly and unnecessarily used in children and adults with acute asthma treated in emergency rooms. due to the alterations that are usually found in patients during asthmatic crises, such as hyperinsufflation, fluid extravasation, and atelectasis associated with hypoxemia, the misinterpretation of these findings as a sign of pneumonia is common and, consequently, unnecessary prescription of antibiotics. a multicenter study of asthmatic patients treated in emergency rooms evaluated the request for additional tests, in this case, chest radiography and blood tests. severely ill patients, those under year, and those with a comorbidity were excluded. a total of ( . %) children underwent additional tests, such as chest radiography ( . %) and blood tests ( . %). after excluding febrile or hypoxic patients, . % were still subjected to at least one of the exams. despite the lack of brazilian data, the routine of pediatric emergency care services in the country appears to adhere to this rule. in order to prevent the dissemination of viral agents, due to the high capacity of viral spread through droplets and fomites, hand washing, and the use of respiratory masks are simple strategies that have been proven to be effective. staying away from situations that favor clusters of people during periods of increased viral circulation has been recommended, although there are no studies that proved the effectiveness of this strategy. , the use of substances such as herbal preparations including echinacea and vitamin c has been evaluated, but double-blind, placebo-controlled studies failed to demonstrate their benefit. the prevention of viral infections through vaccines has been the most effective way to control diseases caused by viruses. in the case of respiratory viruses, the only vaccine available is for flu, although there are ongoing studies for the development of vaccines for other respiratory viruses, especially hrv. however, their great antigenic diversity hinders research success; recent studies have tried to establish a more adequate antigenic target in the viral structure. , specific rsv immunoglobulin has been successfully used in reducing hospitalizations for viral bronchiolitis, and new perspectives for the treatment of exacerbations triggered by viral infections have emerged from studies directed to synthetic agonists of tlr receptor, ifn-␤ agonist, and il and il -antagonists, among others. , there is no specific treatment for most respiratory viruses. some antivirals have been successfully used, as in the case of flu infection, such as amantadine, rimantadine, oseltamivir phosphate, and zanamivir; the latter is not indicated for patients with asthma. ribavirin is indicated for the treatment of severe infections caused by rsv. other antiviral agents are being studied and have not yet been approved for clinical use, such as pleconaril, vapendavir, pirodavir, and rupintrivir. glucocorticoids have potent anti-inflammatory effects and have been successfully used in maintenance treatment in patients with persistent asthma, controlling inflammation and preventing exacerbations. some studies have assessed its effect on virus-induced asthma. the suppression of the release of pro-inflammatory mediators induced by hrv infection in vitro in bronchial epithelial cells, such as ccl , ccl , cxcl , and il , as well as the reduction of factors associated with remodeling, was achieved after the use of budesonide. other in vitro studies documented the action of other corticosteroids alone or in combination with bronchodilators or leukotriene antagonists in reducing the release of several inflammatory molecules, with potential modulation of the deleterious effects of viruses on the asthmatic population. , despite the proven benefits of inhaled corticosteroids in the control of asthma triggered by multiple factors, their action on virus-induced exacerbations is unclear. the use of low-to-moderate doses of inhaled corticosteroids as maintenance therapy cannot prevent intermittent viral-induced wheezing. , however, better results have been obtained with the intermittent use of inhaled corticosteroids at high doses. the use of viral detection techniques with high sensitivity and specificity has increased the identification of some respiratory viruses in children with asthma exacerbation. the direct or indirect immunofluorescence reactions still have great practical importance, as they can detect a panel of seven viruses (fluv-a and b, piv- - , hadv and hrsv), being an affordable and fast method, with good sensitivity, especially in children. it is currently used by the brazilian ministry of health for the screening of respiratory viruses in the diagnosis of severe acute flu-like illness and flu-like illness in sentinel units. the techniques for nucleic acid amplification (rt-pcr) are more expensive, but more sensitive; thus, they are used in research and by the brazilian ministry of health for the identification and genotyping of flu. furthermore, it allows for the identification of some viruses with significant clinical and epidemiological importance, such as hrv and bocavirus, not identified by immunofluorescence. , as for the method used to obtain the sample, it is worth mentioning the controversial issue of nasopharyngeal swab in viral research. although its use has been consolidated for bacterial infections (s. pneumoniae and s. aureus), its role in viral infections still deserves some consideration. the authors agree that, from the practical point of view, it is more feasible, eliminating the use of suction systems, probes, and more specialized training, when compared to aspirate or lavage samples. however, only those with more advanced technology (flocked swab), which provides best capture and release of cells and, therefore, of the virus, are equivalent to the aspirate in terms of sample quality. nevertheless, this swab is not routinely used in services and researches in brazil. regarding the association between viral infection and asthma exacerbation, a wide variation was observed concerning the methods of studies that assessed viral infection in exacerbated asthmatic children in the studies included in this review. for instance, sample size varied from to , cases and the age ranged from to years. this finding is important, given the difficulty in defining asthma in children younger than years, which should be considered in case selection. , moreover, it is known that there is a considerable difference between the age groups and the most prevalent viruses, such as the hrsv. regarding the methods of respiratory secretion collection in the included studies, there was no uniform means of collection; the aspirate was used in . % of studies, the swab in . %, both in %, and the flocked swab was not used. there was a wide variation regarding the detection methods and in relation to some outcomes. in addition to the differences in sample collections, all these studies were cross-sectional, which does not allow for the establishment of a cause-effect association between viral infection and the onset of exacerbation, but suggest such an association. in relation to other factors known to be associated with uncontrolled asthma, such as allergens and irritants, most studies did not include these variables in the evaluation. when the inflammatory process typical of asthma is associated with a viral respiratory infection, there is a tendency to greater severity and duration, as well as a poorer response to conventional treatment of the acute episode. , the involved mechanisms still need to be fully elucidated, evidencing the synergistic effect between viral infection and allergic airway inflammation in the pathogenesis of exacerbations. , another pertinent issue is the role of inhaled corticosteroids in attenuating the inflammation triggered by the virus, also seldom mentioned in these studies. its action in the control and reduction of morbidity associated with asthma is well established, but it is still a controversial subject regarding the prevention of viral-induced wheezing. its effectiveness in the inflammatory process triggered by a virus has been demonstrated in in vitro studies, , , but studies evaluating its clinical benefit have yet to reach conclusive results. , regardless of the direction of virus-allergen interaction, the present findings strongly suggest that an adequate strategy to prevent virus-induced exacerbations should focus on two courses, namely the improvement of antiviral response and the reduction of allergic sensitization or inflammation. the latter can be achieved with appropriate treatment of the asthma patient at risk with medications that reduce airway inflammation. conversely, the preventive measures for viral infection acquisition and its timely diagnosis allow for a proper management of exacerbations, and reduction of the number of hospitalizations and unnecessary additional tests, especially in children who are febrile at the time of assessment. the association between viral infection and asthma in childhood still has several points that need clarification, especially the actual role of viruses in triggering exacerbations and that of inhaled corticosteroids in its attenuation. fundação de amparo à pesquisa do estado de goiás (n • ). the authors declare no conflicts of interest. prevalência de sintomas de asma, rinite e eczema atópico entre crianças e adolescentes brasileiros identificados pelo international study of asthma and allergies (isaac): fase global strategy for asthma management and prevention. global initiative for asthma (gina) proceedings: the role of viral infection in asthma and bronchitis association between human rhinovirus c and severity of acute asthma in children microbiologia médica de jawetz role of deficient type iii interferonlambda production in asthma exacerbations epithelium dysfunction in asthma respiratory syncytial virus in allergic lung inflammation increases muc ac and gob- viruses and bacteria in acute asthma exacerbations-a ga( ) len-dare systematic review deficient antiviral immune responses in childhood: distinct roles of atopy and asthma virus/allergen interactions in asthma interaction between adaptive and innate immune pathways in the pathogenesis of atopic asthma: operation of a 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asthma exacerbations: origin, effect, and prevention use of toll-like receptor agonists against respiratory viral infections budesonide and formoterol inhibit inflammatory mediator production by bronchial epithelial cells infected with rhinovirus budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses epidemiological and genetic characteristics associated with the severity of acute viral bronchiolitis by respiratory syncytial virus effect of continuous treatment with topical corticosteroid on episodic viral wheeze in preschool children preemptive use of high-dose fluticasone for virusinduced wheezing in young children to the laboratory of virology of the instituto de patologia tropical e saúde pública of the universidade federal de goiás. key: cord- -de v e s authors: moens, ugo title: silencing viral microrna as a novel antiviral therapy? date: - - journal: j biomed biotechnol doi: . / / sha: doc_id: cord_uid: de v e s viruses are intracellular parasites that ensure their existence by converting host cells into viral particle producing entities or into hiding places rendering the virus invisible to the host immune system. some viruses may also survive by transforming the infected cell into an immortal tumour cell. micrornas are small non-coding transcripts that function as posttranscriptional regulators of gene expression. viruses encode mirnas that regulate expression of both cellular and viral genes, and contribute to the pathogenic properties of viruses. hence, neutralizing the action of viral mirnas expression by complementary single-stranded oligonucleotides or so-called anti-mirnas may represent a strategy to combat viral infections and viral-induced pathogenesis. this review describes the mirnas encoded by human viruses, and discusses the possible therapeutic applications of anti-mirnas against viral diseases. viruses are common habitants of the human population, where they establish different forms of infection, including an acute, a chronic, or a persistent infection with production of low levels of virions. some viruses can exist in a true latent state in which infectious particles are only produced upon reactivation stimuli. viruses that reside harmlessly in their host can under certain conditions or in immunocompromised persons be responsible for malignant and nonmalignant diseases, which may even lead to the death of the host. a causal role for human polyomaviruses (hpyv), papillomaviruses (hpv), herpesviruses (hhv), hepatitis b virus (hbv), hepatitis c virus (hcv), and human t-cell lymphotropic virus type-i (htlv-i) and cancer is accepted (for recent reviews see [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). it is estimated that oncoviruses are associated with % of the human cancers [ ] , while nonmalignant infections from human immunodeficiency virus (hiv), hbv and hcv alone cause more than million deaths annually worldwide [ ] . other viral infections (hiv no included) were responsible for the death of more than patients in japan in , ∼ individuals in the usa in , and people in united kingdom in according the statistics of the world health organization [ ] . thus the pathogenic properties of viruses necessitate the development of efficient antiviral therapies. viruses attempt to create a favorable cellular environment allowing viral replication or survival by establishing a lifelong latent infection through evading the immune system of their hosts. viruses can hide within a cell by restricting their activity to a minimum so as not to conceal their presence to the immune system and at the same time they will also try to avoid apoptosis. for these purposes, viruses have developed different strategies, one of which includes the posttranscriptional regulation of both cellular and viral gene expressions through modulating the host's rna-interference (rnai) machinery. viruses can suppress the rnai pathway by viral microrna (vmirna) targeting cellular or viral transcripts, or by viral proteins (e.g., human immunodeficiency virus tat protein, influenza virus ns /ns protein, ebola vp protein, and vaccinia virus e l protein) or viral rna (adenovirus va transcripts) that counteract the host's rnai machinery (for recent reviews see [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). this review summarizes the recent findings on virus-encoded mirnas and their described functions and briefly discusses the potential of antiviral mirna as a novel therapeutic strategy in combating virus infections. anti-mirna oligonucleotides (amos) are chemically modified synthetic oligonucleotides that are complementary to their target sequence and this will silence the action of the target. amos are modified with the dual purpose to stabilize them and to improve their affinity for their targets. one modification is the ' sugar modification which implies a chemical modification of the '-o of the ribose residue (figure (a) ). the '-o -methyl amos have a methyl group linked to the '-o of the ribose residue, while the '-o -methoxyethyl amos contain a methoxygroup. this modification provides improved rnase resistance and binding affinity to rna compared to unmodified antioligonucleotides. however, '-o -methoxyethyl amos possess a higher affinity and specificity to rna than their '-o -methyl amos. other ' sugar modifications that have been used include '-fluor and locked nucleic acid (lna). in lna-modified oligonucleotides, the '-ooxygen is bridged to the '-position via a methylene linker to form a rigid bicycle, locked into a c '-end (rna) sugar conformation (figure (a) ). lnas give very strong duplex formation with their target sequences and they display excellent mismatch discrimination, hence avoiding off-target effects (for recent reviews see [ ] and [ ] ). lna injections against mir- , a cellular mirna involved in lipid metabolism, resulted in efficient and long-lasting decrease in plasma cholesterol in african green monkeys without any evidence for toxicities [ ] . a second type of modification is the phosphorothioate backbone which reduces the affinity to the target somewhat, but it confers significant stability to nuclease degradation (figure (b) ). a third generation of antisense oligonucleotides are phosphodiamidate morpholino oligomers (pmo) in which the ribose ring is replaced with a morpholine ring. adding an argininerich peptide (rxr) further increased the stability and tissue retention of the pmo (reviewed in [ ] ). an additional modification can be made to improve the cellular uptake of the amo. krützfeldt et al. linked a cholesterol moiety to their amos and referred to these anti-mirnas as antagomirs. antagomirs should be > nucleotides in length to provide highest efficiency in silencing target mirna [ ] [ ] [ ] . the putative therapeutic potentials of antagomirs were recently demonstrated in treatment of lipid metabolic disease in animals [ ] . an alternative class of amos is peptide nucleic acids (pna), which are synthetic oligonucleotides with n-( -aminoethyl)-glycine replacing the deoxyribose or ribose backbone [ ] . a study published in reported that pna can efficiently block the action of cellular mirnas [ ] . finally, another approach in silencing mirna is the use of so-called microrna sponge, a synthetic mrna that contains multiple binding sites for a particular mirna and that is transcribed from a plasmid containing a strong promoter (reviewed in [ ] ). in conclusion, different classes of amo have been shown to be efficient in silencing mirna and may be useful therapeutic tools (reviewed in [ , [ ] [ ] [ ] ] ). samples and they are referred to as kipyv wupyv [ , ] . this year a novel human polyomavirus, merkel cell polyomavirus (mcpyv), was identified that is associated with merkel cell carcinoma [ ] . the hpyv genome can be divided into three functional regions. the early region encodes the early proteins large t-antigen (lt-ag) and small t-antigen (st-ag), while the late region encodes the capsid proteins vp -vp and the regulatory protein agnoprotein. both regions are separated by the noncoding control region that encompasses the origin of replication and the promoter/enhancer sequences for the early and late genes (reviewed in [ ] ). the sv genome encodes a viral mirna (vmirna) of which both arms are complementary to the early viral mrnas and reduces expression of the early proteins (figure (a); table ). cells infected with mutant sv lacking this mirna or with wildtype sv yielded comparable levels of infectious viruses, but the latter were less sensitive to lysis by cytotoxic t cells and produced less interferon-γ. thus sv -encoded mirna allows the virus to evade the immune system [ ] . the sv mirna is conserved in bkv and jcv and both mirnas generated from the precursor hairpin bind to the same target, that is, the early transcripts. the bkv and jcv mirnas serve the same role as sv mirna, that is, downregulation of early expression. jcv mirna, mir-j , was readily detected in brain samples of pml patients, suggesting a biological role of this mirna [ ] . the group of sullivan has also identified a mcpyv-encoded mirna, mir-m , which does not share sequence identity with the known mirnas of the other polyomaviruses. mcpyv mir-m is located in the early region (figure (a)) and can downregulate early gene expression. in accordance with sv , this may allow the virus to evade the immune system. however, mcpyv is associated with merkel cell carcinoma and the viral genome is integrated in these tumours [ ] . blocking mir-m by, for example, antagomirs will increase the expression of the viral oncoprotein lt-antigen and as such have little beneficial therapeutic effect [ ] . expression of a corresponding or other viral-encoded mirna for the other hpyv wu and ki is lacking so far. human papillomaviruses (hpv) are nonenveloped viruses with a circular dsdna genome of approximately base-pairs. these viruses are associated with benign and malignant lesions of the skin and the genital tract. more than different hpv genotypes have been identified and based on their association with benign warts or cancer, they are classified as low-risk and high-risk variants, respectively [ ] . one study with hpv type failed to clone vmirna from virus-infected cells [ ] . however, this does not exclude that other strains may encode vmirna. moreover, the expression of vmirna may be regulated in a temporal and spatial manner, so that the experimental conditions for capturing vmirna may be tricky. in addition, the high mutation rate of hpv genome sequences may impede the prediction of the presence of putative vmirna. adenoviruses are naked dsdna viruses that can cause mild respiratory, gastrointestinal, urogenital, and ocular disease. more than serotypes have been described in human. although there is no proof for a causative role in malignancies, adenoviruses can induce cancer in animal models and have been extensively studied to scrutinize viral mechanisms for cellular transformation [ ] . adenovirus encodes small noncoding rnas, known as virus-associated rna or vai and vaii rna, which are generated by rna polymerase iii. this noncoding rna plays an important role for viral replication and neutralizes the antiviral action of interferon by blocking the dsrnainduced protein kinase (pkr), which phosphorylates and thereby inactivates the eukaryotic translational initiation factor [ ] . a recent study showed that a minor fraction of vai rna is processed by dicer into functional risc-associated ssrna which can act as mirna ( table ) . blocking of vai-derived small rna by '-o -methyl amo complementary to vai decreased virion production [ ] [ ] [ ] . in an attempt to identify potential targets for vai-derived mirna, computational analysis was used. putative targets include genes encoding apoptosis-related protein napor and pkr-activating protein pact. therefore vai rnaderived mirna may help adenovirus to escape the actions of the host defense mechanisms [ ] . herpesviruses are enveloped dsdna viruses with a genome size ranging between ∼ to ∼ kilobase-pairs. they are divided into three subfamilies denoted α, β, and γ. approximately different herpesviruses have been identified to date, including the human α-herpesviruses herpes simplex virus (hsv- or hhv- ), herpes simplex virus (hsv- or hhv- ), varicella-zoster virus (vzv or hhv- ), the β-herpesviruses cytomegalovirus (hcmv or hhv- ), hhv- a, hhv- b, and hhv- , the γ-herpesviruses epstein-barr virus (ebv or hhv- ), and kaposi's sarcoma-associated virus (kshv or hhv- ). all human herpesviruses are able to establish latent infections with only a small subset of viral genes expressed (reviewed in [ ] ). as will be discussed in the next section, among the viral transcripts that can be detected in latently infected cells are viral-encoded mirnas, which seem to be required to maintain a latent state of infecton, but may also contribute to the pathogenic properties of the virus. herpes simplex virus- (hsv- or human herpes virus ; hhv ) infects the majority of the human population, but remains a latent cohabitant in most people. reactivation of the viruses usually results in cold sores, but it can also cause a spectrum of diseases from sightthreatening ocular infections in immunocompetent adults to more severe infections in newborns and immunosuppressed patients (reviewed in [ ] ). an important gene in hsv latency is the lat gene. this gene encodes the latency-associated transcript (lat), which does not code for a protein. lat seems to promote cell survival of the infected cells [ ] . gupta and coworkers proposed that the antiapoptotic activity of lat was achieved by an mirna-entrapped in the lat (mir-lat), which downregulates the expression of transforming growth factor-β (tgf-β) and smad . the latter is a mediator of the signalling pathway induced by tgf-β, while tgfβ can prevent cell proliferation and induce cell death [ ] . however, a later study revealed that the described mir-lat was not viral encoded, but in fact a cellular mirna expressed in sh-sy- y cells [ ] and the report describing mir-lat was retracted [ ] . work by umbach et al. could also not confirm the existence of this mirna in hsv- infected sh-sy- y cells [ ] . later, it was shown that the hsv- lat exon encodes hsv- mir-lat-icp . , which can be detected in hsv- infected cells [ ] . circa base-pairs upstream in this region, a sequence with % homology to the hsv- mirna mir-i (see further) is present, but no mature mirna was detected in hsv- infected cells. however, the existence of this mirna during hsv- latency in vivo remains to be confirmed [ ] . computational analysis predicted -mirna candidates in the hsv- genome, of which were conserved in hsv- , suggesting they may be functional mirnas [ ] . the authors confirmed the expression of one mature mirna, designated mir-h , in hsv- infected vero cells, where it was expressed late in productive replication. this mirna is encoded approximately bp upstream of the transcription start site of the lat transcript, but a corresponding sequence is not conserved in the hsv- . the function of mir-h journal of biomedicine and biotechnology downregulates the expression of cmv genes involved in its own replication process, for example, transactivators ie and ie ; ul / ; ul mhc class i-related chain b (micb), a cellular ligand for the activating receptor nkg d; downregulation of ie- mir-ul d- mir-us - mir-us - mir-us - mir-us paramyxoviridae (measles virus) predicted [ ] remains to be established and no cellular target mrnas were identified [ ] . umbach and coworkers detected in addition to mir-h five novel viral mirnas in trigeminal ganglia of mice latently infected by hsv- , as well as in hsv- -infected vero cells. these mirnas, that is, mir-h to mir-h , are encompassed in the lat locus ( figure (b)). by quantitative rt-pcr, the authors were able to roughly estimate the number of copies of each mirna during productive infection of vero cells. mir-h and mir-h were expressed at ∼ and copies, respectively, while the other mir-hs were present at less than copies per infected cell. in latently infected trigeminal ganglia, much higher levels were monitored with copies of mir-h , copies of mir-h , copies of mir-h , copies of mir-h , and copies of mir-h . the large difference in numbers of mirna transcripts in latently infected cells compared to cells with productive hsv- infection indicates that mir-hs play an important role in establishing latent hsv- infection. indeed, mir-h expression diminished the protein levels of icp , a viral transcriptional activator that promotes viral replication, while mir-h inhibits expression of icp , which is required for expression of most hsv- genes during reproductive infection [ ] . hsv- typically infects the genital region and establishes a lifelong latent infection. the prevalence of latent hsv- infection varies between - %. reactivation can cause oro-facial and genital herpes, but hsv- infection can also cause encephalitis and neonatal herpes, and forms a risk factor for hiv acquisition [ ] . the only detectable viral transcript during hsv- latency is the lat, but the molecular function of this transcript remains largely unknown [ ] . hsv- lat exon encodes an mirna, referred to as mir-i, which is expressed during latent, as well as during acute infection. remarkably, several promoters regulate mir-i expression in different stages of the viral life cycle. this hsv- mirna efficiently diminishes the expression of the viral neurovirulence factor icp . , a multifunctional protein required for viral replication in neuronal cells in vivo, and with intrinsic neurovirulent properties [ ] . thus, mir-i may affect the outcome of infection (latent versus productive) by modulating the protein levels of icp . . whether mir-i has other targets remains to be investigated, but an mir-i analogue is also expressed by hsv- , indicating the importance of this mirna for these viruses [ ] . tang and colleagues identified two new hsv- mirnas, mir-ii, which includes mir-ii- p and mir-ii- p, and mir-iii, both encoded by exon of lat. the expression of mir-i, -ii, and -iii increased during infection of cells, but mir-iii displayed slower kinetics than the two other mirnas. similar to mir-i, mir-ii silences the expression of icp . ,while mir-iii functionally resembles hsv-i mir-h in that it can downregulate the expression of icp [ ] . the virus establishes a latent infection, but reactivation leads to herpes zoster, commonly referred to as shingles. acute vzv reactivation may lead to post-herpetic neuralgia [ ] . no putative mirnas could be predicted in the vzv genome [ , ] , but experimental studies to unambiguously proof the existence of vzv mirna are lacking. table and figure (c) ). three viral mirnas, designated as ul - p, mir-ul - p, and mir-us , were identified that are expressed during productive hcmv infection of permissive cells (human foreskin fibroblasts, astrocytoma u mg cells, retinal pigment epithelial cells, and human microvascular endothelial cells). their putative cellular target genes include genes encoding transcription factors (e.g., hnf and tgif ), receptors (e.g., il- receptor precursor; cd ), proteins implicated in t-cell activation (ahnak ), in signal transduction (e.g., rab l), and in biosynthesis of leukotrienes that sustain inflammatory reactions (coactosin-like protein). whether these genes represent bona fide targets as well as the biological relevance of hcmv mirna-mediated silencing of these genes remains elusive [ ] . hcmv usually establishes a lifelong persistent or latent state in healthy individuals by ensuring that infected cells avoid immune recognition. the hcmv-encoded mirna mir-ul - seems to play a central role in helping the virus to hide from the host's immune system. this viral mirna targets mrna for mhc class i-related chain b (micb), and to a lesser extend mica. these proteins are cellular ligands for the activating receptor nkg d, which is expressed on some natural killer (nk) cells, γ/δ t cells, and cd + t cells. during cellular stress (such as viral infection) micb is induced, thus activating nk-cells and t cells that can lead to the killing of infected cells. cells infected with mutant virus lacking this mirna were more susceptible to being killed in an nkg d-dependent manner by nk cells [ ] . the mir-ul - also represses the expression of hcmv genes involved in its own replication process, in part by targeting mrna encoding immediate early proteins. one of them is the viral transactivator protein called immediate early (ie ) that regulates the transcription of viral genes required for acute replication [ ] . ie plays a pivotal role in controlling latency and reactivation. mir-ul- - can thus restrict reactivation of the virus through negative regulation of ie expression [ , ] . two separate studies showed that mir-ul - also inhibited expression of the immediate early protein ie . murphy et al. detected increased ie levels in cells infected with either a virus lacking mir-ul - or with mutations in the seed sequence of the ie gene compared to cells infected with wild-type hcmv [ ] . grey and coworkers demonstrated that addition of mir-ul - rna prior to infection reduced ie protein levels and blocked viral replication [ ] . ie is a crucial protein to ascertain lytic replication of hcmv, thus downregulation of ie may help the virus to establish latency. this strategy may be a common feature for herpesviruses because immediateearly genes may be putative targets for hsv- mir-lat, ebv mir-bart and mir-bhrf - , and kshv mir-k - - p [ ] . yet another target for mir-ul - is the viral protein ul , a homologue of the mammalian dna repair enzyme uracyl-dna glycosylase. ul is required for efficient viral dna replication. hence, mir-ul - mediated downregulation of ul may prevent viral dna replication and favor a latent infection state [ ] . taken together, the actions of mir-ul - seem to be associated with latent viral infection, a state which allows the virus to hide from the immune system. ablating expression of this viral-encoded mirnas by amos may therefore force the virus into a lytic cycle and provide the immune system the opportunity to get ride of the viral infection. -lymphocytes in more than % of the human population. ebv is associated with infectious mononucleosis and has been implicated in the pathogenesis of several malignancies including burkitt's and hodgkin's lymphomas, posttransplant and t-cell lymphomas, x-linked lymphoproliferative syndrome, nasopharyngeal, and gastric carcinomas [ ] . latently ebv-infected cells are classified in stage i, ii, or iii, each of them characterized by distinct ebv gene expression [ ] . among the latency stage-specific ebv transcripts are mirnas. more than different ebv mirnas have been identified that are transcribed during latent infection (table ; [ , , [ ] [ ] [ ] [ ] [ ] ). the ebv mirnas are organized in two major clusters within the ebv genome (figure (d) ). one cluster resides in the bart (abbreviation for bamhi-a rightward transcripts) region. the bart region gives rise to multispliced transcripts and is highly expressed in ebv-positive cancers and in epithelial tissues, while there is low bart expression in b lymphocytes. the exact function of bart mrnas remains obscure [ ] . the intronic region of bart also encodes the mirnas mir-bart to mir-bart . the second region that encodes multiple mirnas is the untranslated region of the gene encoding bhrf (bamhi fragment h rightward open reading frame ), a viral bcl- homologue that prevents apoptosis. bhrf encompasses the mirnas mir-bhrf- - to mir-bhrf- - [ , ] . the expressions of ebv-encoded mirnas in clinical samples and computational analysis to predict putative targets were applied to unravel the biological functions of ebv mirnas. these approaches showed that the mir-barts are abundantly expressed in latently infected epithelial cells, nasopharyngeal carcinomas, ebv-associated gastric carcinoma cell lines and tissues, burkitt's lymphomas latency type i, ebv positive primary effusion lymphomas, and diffuse large b-cell lymphomas, but at a significantly lower level in b cells. this corresponds well with the expression pattern of bart multispliced transcripts (see above). higher levels of bhrf - were measured in latency type iii burkitt's lymphomas and in diffuse large b-cell lymphomas [ , , [ ] [ ] [ ] . another study demonstrated that induction of ebv replication in latency i-infected cells was associated with increased expression of mir-bhrf - , - , and - , but expression levels of mir-bart- and - did not change. on the other hand, induction of ebv replication in latency iii-infected cells did hardly change the expression levels of bhrf - , - , and - [ ] . these observations suggest that ebv mirnas may be implicated in the oncogenic properties of the virus, but also in regulating its replication. moreover, a precise knowledge of the latency state of ebv and the expression pattern of viral mirnas may improve the successful treatment of ebv infection with amos. the function of most ebv vmirna remains poorly understood, but some targets of ebv mirnas have been recently identified. several mi-barts prevent expression of viral latent infection membrane protein (lmp ) protein (see table ). lmp functions as a constitutively active tumour necrosis receptor [ ] , and can activate several signalling pathways including nfκb, ap , jak/stat, mek/erk, and pkc. lmp can also interact with p and affects cyclins, cyclin-dependent protein kinases (cdk), and the cdk inhibitors p and p (reviewed in [ ] ). furthermore, lmp is expressed in all the ebv related malignancies and promotes cellular transformation. its oncogenic property makes lmp an attractive target for ebv therapy. interestingly, overexpression of lmp results in growthinhibitory and sensitization to apoptosis induced by stress or chemotherapeutic agents ( [ ] and references therein). thus amo-mediated neutralization of mi-barts may lead to elevated lmp protein levels and render ebv-positive tumour cells more susceptible to chemotherapy. the viral mirna mir-bart can inhibit expression of viral dna polymerase balf and may thus interfere with viral replication and prevent lytic infection [ , ] . silencing mir-bart could thus allow the virus the complete its life cycle and produce new infectious virus particles, which then could offer the immune system the opportunity to detect and eliminate ebv. using computational prediction programs such as miranda and rnahybrid (reviewed in [ ] ) allowed choy and coworkers to envisage the cellular protein p up-regulator of apoptosis (puma) as a target for mir-bart [ ] . the authors demonstrated that puma levels were decreased in cells expressing mir-bart compared to cells lacking mir-bart . in accordance, when mir-bart was specifically inhibited with an anti-mir-bart oligonucleotide, puma protein levels decreased and apoptosis was triggered. thus, ebv may promote survival of infected epithelial cells by modulating the expression of an apoptotic protein through an mirna-mediated mechanism. this finding may have important implications in the development of anti-ebv agents such as amos directed against mir-bart . fewer studies have been directed to determine the targets of mir-bhrf s. mir-bhrf - is involved in the cleavage of bhrf rna in the cytoplasm, but the biological relevance remains to be determined [ ] . in another study, xia et al. observed that high levels of mir-bhrf - were correlated with low levels cxcl- , a potent interferon-inducible tcell attracting chemokine. mirna-mediated suppression of cxcl- may serve as an immunomodulating mechanism allowing the virus to survive in the host [ ] . on the other hand, enhancing cxcl- expression in ebv-positive tumours by amos against mir-bhrf - may increase susceptibility of the tumour cells to the immune system. in agreement with this, two recent studies reported antitumour activity for cxcl- in animal models [ , ] . kaposi's sarcoma-associated virus, so named because it was detected in kaposi's sarcoma, belongs to the γ-herpesviruses and is also known as human herpesvirus- or hhv- . hhv- is associated with kaposi's sarcoma, as well as with two rare forms of b-cell malignancy: primary effusion lymphoma (pel) and the plasma cell variant of multicentric castleman's disease. like other herpesviruses, kshv can establish a lifelong latent infection characterized by a limited viral gene expression [ ] . a total of kshv mirnas encoded by distinct mirna genes have been reported and their sequences are highly conserved between different kshv genomes in pel cell lines and in clinical samples. however, some polymorphism was observed, particularly in mir-k - and mir-k - [ , , ] . the entire kshv mirna cluster resides within an approximately kilobase-pairs region between open reading frames orf k (kaposin) and orf (figure (e) ). to elucidate the functions of the kshv mirnas, transcriptome analysis was performed from cells stably expressing the mir-k cluster. among the differentially expressed genes were genes encoding proteins implicated in proliferation, immune modulation, angiogenesis, and apoptosis. the gene encoding thrombospondin- was targeted by all ten kshv mirnas, but especially by mir-k - , mir-k - - p, mir-k - - p, and mir-k - . thrombospondin- possesses antiproliferative and antiangiogenic properties. other transcripts that were reduced corresponded to the genes for osteopontin, s calcium binding protein, plasticity related gene product, and integral membrane protein a [ , ] . the mrna for the bcl- interacting protein bclaf was identified as a target for mir-k - . additional inhibition of bclaf expression was obtained in the presence of mir-k , - a, and - b. the exact biological relevance is not yet understood, but sirnamediated depletion of bclaf enhanced the frequency of spontaneous lytic reactivation of kshv. mirna-mediated reduction of bclaf expression would prevent permanent latency of the virus, a type of infection that represents a deadend pathway of viral spreading [ , ] . a kshv mirna that has gained special interest is mir-k - because its seed sequence, known to be critically important for mrna target recognition is % conserved with the cellular mir- , suggesting that these mirnas may regulate common targets. the exact role of mir- remains unclear, but a number of b-cell lymphomas and solid organ tumors overexpress mir- , while mir- transgenic mice develop b lymphomas [ , ] . work by the groups of skalsky and cullen confirmed that mir-k - indeed is an orthologue of mir- , and that they target common transcripts [ , ] . comparing the gene expression profiles in cells stably expressing either mir- or mir-k - revealed that they regulate an analogous set of mrnas. the products of these transcripts include proteins involved in b-cell function (e.g., src-like adaptor or sla), innate immunity (e.g., iκb kinase and phosphoinositide- -kinase), apoptosis (xiap associated factor- ; ldoc ), cell cycle regulation (e.g., fos), and gene expression (e.g., fos and bach ). bach- (btb and cnc homolog ) is a bzip protein that can repress transcription through heterodimerization with the small maf proteins [ ] , while c-fos can heterodimerize with the jun proteins to form the ap- complex. ap- is a multifunctional protein involved in cellular proliferation, transformation, and apoptosis [ ] . for a complete list of mir- /mir-k - regulated genes, the reader is referred to the work of skalsky et al., and of gottwein et al. [ , ] . treatment of latently-infected kshv with an antagomir against mir-k - enhanced fos protein levels about . -fold compared to untreated cells [ ] . computational analyses further revealed seed sequence homology between the viral mirnas kshv mir-k - - p, ebv-bart , and hcmv ul - p with human mirnas mir- a plus , mir- a/b, and mir- , respectively [ ] . both mir- a and mir- are believed to possess tumour suppressor activity and to induce apoptosis by silencing bcl expression, while mir- was demonstrated to be oncogenic [ , ] . kshv-encoded mirnas seem to be crucial both in survival of the virus in its host, but also to play a causal role in viral-associated pathologies. amo-mediated silencing of kshv-encoded mirnas may thus be a strategy to counteract viral infection, but may also undesirably target cellular mirnas with identical seed sequences as the viral mirnas. poxviruses are dsdna viruses that replicate in the cytoplasm and have as such no access to the nuclear proteins involved in the biogenesis of mirna. nevertheless, mirna precursor sequences have been predicted in the genomes of the human poxviruses vaccinia virus and variola virus, but their existence has not been validated [ , ] . whether the other human poxvirus, molluscum contagiosum virus, encodes mirna remains to be established. hepatitis b virus (hbv), an enveloped virus with a circular partial dsdna genome, persistently infects more than million people worldwide. hbv can cause a spectrum of liver diseases ranging from mild liver dysfunctions to chronic hepatitis, cirrhosis, and hepatocellular carcinoma [ ] . this makes efficient anti-hbv therapy highly vital. amo-based vmirna silencing is probably no option since no mirnas could be detected by computational analysis [ ] , and expression of hbv mirnas has not been reported so far. one study identified a putative hbv-encoded mirna, but in vivo expression of this hbv mirna was not tested. computational screening for complementary sequences in the ' untranslated regions of cellular mrna to this hbv mirna did not reveal putative target transcripts [ ] . it therefore seems unlike that this is a bona fide viral mirna. hiv is the causative agent of acquired immune deficiency syndrome (aids), and it is estimated that > million people worldwide are infected with this virus. two species, hiv- and hiv- , infect humans (for a recent review see [ ] ). hiv utilizes reverse transcriptase to convert its ssrna genome into a dsdna provirus. during this process, the ' and ' ends of the viral rna genome are converted into long terminal repeats (ltrs). the ltrs play a pivotal regulatory role in establishing, maintaining, and overriding the latent state of the virus [ ] . the central domain of the ltr is referred to the r region, which encompasses the (transactivation-response region) tar. tar binds the viral protein tat, a transactivator that plays an important role in the transcriptional activation of the provirus genome (reviewed in [ ] ). the tar encodes proven and putative mirnas (figure (f) ). klase and coworkers described an mirna encoded by the hiv- tar element. this mirna causes hdac- to associate with the viral ltr, resulting in diminished viral gene expression. this suggests a role for hiv- mirna in maintaining viral latency [ ] . in another report, ouellet and colleagues demonstrated the expression of two tar element-derived mirnas by northern blotting, primer extension, and rnase protection assay. the mirna derived from the left arm of the tar stem has been named mir-tar- p, while the mirna originating from the right arm was designated mir-tar- p. the latter appears to preferentially accumulate in hiv-positive cells [ ] . the biological role of these mirnas remains to be elucidated, but they may contribute to modulating viral and/or cellular gene expression, with a potential impact on viral replication and/or host antiviral defense efficiency. the mir-tar- p described by ouellet overlaps with the vmirna no. , while mir-tar- p partially overlaps with vmirna no. described by bennasser and coworkers [ ] . they predicted by computer-directed analyses pre-mirnas in the hiv- genome, which in principle could yield mature mirnas. their expression has not been validated, nor has their biological role been addressed, but deduction of potential target transcripts resulted in the indentification of cellular genes encoding protein kinases, ion channels, proteins involved in protein synthesis and degradation, growth factors, and dna methylation [ ] . tar dna of the long terminal repeat of hiv- encompasses an antisense rna (hivainr), which encodes hiv proteins, but that can also form a duplex with the ' end of all sense hiv mrna, enabling the virus to control the expression of its gene [ ] . hivainr can potentially code for several mirnas, referred to as haamirnas. putative targets for these mirnas are the mrnas for interleukin (il)- , il- receptor γ chain, human fragile x mental retardation protein (fmrp), and il- receptor-associated kinase (irak ). il- is important in the regulation of t-cell maturation, development and survival of natural killer cells, and survival of long-lived memory t cells, while the il- receptor γ chain is a common component of the receptors for il- , - , - , - , - , and - . aberant expression of this receptor leads to severe t-cell and nk-cell deficiencies. irak is a critical signalling mediator of innate immunity. downregulation the expression of il- , il- receptor γ chain, and irak by hiv mirna would impair the immune system and favor survival of the virus in the host. fmrp is an rna-binding protein that is implicated in protein synthesis and mirna processing. thus hiv could use haamirna to deregulate the host mirna mechanism to dispose the virus by depleting fmrp ( [ ] and references therein.) although the existence of these haamirnas has not been proven, it is tempting to speculate that, in accordance with other viruses, hiv encodes mirnas allowing hiv to survive in the host. a recent study examined the possibility of hiv- tar to encode mirnas. two putative mirnas, mir-tar - p and mir-tar- p were identified, but their expression awaits validation [ ] . besides tar, other regions of the hiv genome have been shown to contain mirna sequences. the nef gene of hiv- is located at the ' end of the viral genome and is highly expressed during the early stages of virus replication. nef is a multifunctional accessory protein that is important for viral replication, but that also plays a key role in pathogenesis as nef can downmodulate cd , cd , and the class i major histocompatibility complex [ ] . hiv- encodes a nef -derived mirna referred to as mir-n (figure (f) ). unlike classical mirna, this mirna does not affect gene expression at the post-transcription level, but rather at the transcription level by promoter interference. mir-n suppresses hiv- promoter activity via a negative responsive element in the '-long terminal region and via nef sequences in the ' untranslated region [ , ] . future studies are required to elucidate the precise mechanism by which mir-n represses hiv- promoter activity. downregulation of nef expression may suppress hiv- replication and allow persistently low pathogenic or latent viral infection [ ] . as the nef gene is conserved in hiv- , hiv- may also apply a similar mechanism to maintain a low profile in the host. the identity and action of hiv mirnas remains to be scrutinized before amos-based therapy can be considered as anti-hiv drugs. however, computational alignment of the potential hiv- mirnas with specific human t-cell mrnas identified potential cellular targets including genes encoding cd , cd and interleukin- , il- , and il- [ ] . viral mirna-caused inhibition of the expression of these proteins seems advantageous for the virus, and therefore counteracting vmirna by amo may help the host to clear hiv infection. htlv-i persistently infects - million humans worldwide and is the etiological agent for adult t-cell leukaemia [ ] . one study reported that t cells persistently infected with htlv- did not express viral micrornas [ ] , but meticulous studies should be performed to rule out the existence of htlv-i mirna. none of these viral genomes seem to contain putative mirna sequences, except for measles virus and yellow fever virus, which each possesses a single putative mirna. however, the mirna in yellow fever virus could not be validated, while the existence of mirna in measles virus was not tested [ , ] . intriguingly, the liver-specific mir- facilitates the replication of the oncovirus hcv, but the mechanism for this function of mir- in hcv replication is still unknown [ , ] . numerous human diseases are caused by viral infections, but the intimate relation with the host makes the development of antiviral drugs difficult. vaccination has been proven to be very succesful to combat some viral infections, but mutations and diversity of virus strains has hampered the development of efficient vaccines against other viruses. new antiviral treatments are based on drugs that inhibit specific viral activities such as viral proteases or polymerases (for recent reviews see [ , ] ). viral-encoded mirna that may be implicated in the viral life cycle and the pathogenic properties of the virus offers a novel attractive target for antiviral therapy. silencing the action of viral mirnas may enable the host cell or the immune system to gain control over the virus and even to eliminate the virus. the idea of targeting viral transcripts is not new, and rna interference has been demonstrated to efficiently mediate inhibition of replication of human pathogenic viruses such as hiv- , hcv, dengue virus, severe acute respiratory syndrome (sars) coronavirus, poliovirus, human rhinovirus, influenza a virus, hepatitis d virus, hbv, hsv- , hpv, jcv, ebv, and cmv in cell culture (reviewed in [ ] ). besides recent studies have proven the potential of this rna interference as antiviral therapy in animal models [ , ] , and even in clinical trials such as alnylam against respiratory syncytial virus and nuc b against hbv (reviewed in [ , ] ). however, anti-hiv rna interference studies revealed that escape virus variants could appear which could evade the inhibitory action of sirna [ , ] . journal of biomedicine and biotechnology the use of amo to neutralize viral mirna adds a new twist to rna interference. amos are easy to produce and relatively cheap, and easy to administer locally (but not systemically). moreover, they possess low toxicity and are highly specific. most viral mirnas identified so far have little homology to each other and to known host cell mirnas (reviewed in [ ] ). this reduces the risk of offtarget effects of anti-mirna oligonucleotides and increases the therapeutic potentials of mirna silencing. the mirna silencing action of both lna and antagomirs is sensitive to single nucleotide exchanges. for antagomirs, it was shown that this effect depends on the position of the mismatch. nucleotide substitutions at the very ' end or in the centre did not prevent downregulation of mir- [ ] . these data indicate that changes in the ' end of the antagomir may abrogate its ability to destroy target mirna and should be taken into account when designing and testing antagomirs. another advantage of amos is that they probably can be used against all serotypes of a specific virus. although not meticulously investigated, mirna sequences between different viral strains seem to be conserved because of their importance for the viral life-cycle (see e.g., kshv-encoded mirnas; [ , , ] ). however, polymorphism in viral mirnas has also been observed (see next paragraph). although amos may provide an attractive novel antiviral therapy, practical problems and other pitfalls may hamper the use of them. for example, antagomirs directed against mir-ul- - could drive the virus towards acute replication and disrupted the inhibition of mcib expression, resulting in possible clearance of the virus by the immune system. however, there is a potential risk of severe pathological effects caused by acutely replicating hcmv, especially in immunocompromised patients [ ] . another disadvantage of amos may be off-target effects. as mirna do not require full complimentarity to bind their target sequence, it can be imagined that an amo not only binds to its predicted mirna but also to other mirnas and even mrnas. in addition, ssrna oligonucleotides may interact with toll-like receptors and thereby stimulating the immune system. similar side effects have been reported for sirna (reviewed in [ ] ). polymorphism in viral-encoded mirnas has been described in viral-infected cell lines and in clinical samples. for instance, mir-k - of different kshv isolates contains mutations, which can affect maturation and biological activity of this mirna [ , , ] . thus the mirna may not be expressed, in which case the amo will have no effect or the amo may not bind because of the mutations in its target mirna. another problem facing the use of amos in antiviral therapy is that the expression of a specific gene may be regulated by several viral mirnas. for example, translation of the transcript of thebachgene is prevented by three kshv viral mirnas: mir-k - , mirna-k - , and mir-k - . thus the effectiveness of an amo against, for example, mir- - can be compromised. indeed, treatment of latently infected kshv virus with a specific antagomir against mir-k - alone only modestly increased the amount of bach protein [ ] . a cocktail of different amos directed against distinct viral mirna may help to overcome this problem. so far, such studies are lacking, but a recent study successfully applied sponge mirna to silence hbv transcripts. an expression vector encoding multiple mirnas targeting hbv hbsag mrna strongly reduced the expression of this protein [ ] . another challenge is to improve in vivo delivery of the amos to viral infected cells and obtain long-lasting action of the antagomirs. aerosol delivery devices similar to the ones used for delivery of asthma therapeutics could be used for respiratory viruses [ ] . other delivery strategies include intravenal or systemic injection, viral vectors, and lipid-and polymerbased vehicles [ ] [ ] [ ] [ ] . recently, sustained inhibition of hcv replication in cell-culture was obtained when celldegradable multilayered polyelectrolyte film (mpf) coated with sirna was delivered to infected cells. by this approach, a single regime of mpf-mediated sirna treatment was sufficient to inhibit hcv replication for days. moreover, mpf-mediated delivery of sirna also protected uninfected cells from hcv infection. another advantage is the very low toxicity of mpf [ ] . these promising observations in cell culture put mpf-based delivery of amos forward as an efficient antiviral tool. another limitation of anti-mirnas is the site of application. studies with antagomirs against mir- in mice revealed that when injected into tail veins, antagomirs were incapable of silencing mir- , whereas local injection into the mouse cortex efficiently induced degradation of the target mirna [ ] . another drawback of the use of amos is that the chemical modification can exert antiproliferative or other off-target effects such as been demonstrated for the phosphorothioate backbone, which can associate with cellular proteins [ ] . antisense oligonucleotides such as lna and pmo have proven to efficiently inhibit rna and dna virus replication in cell culture and animal models, without toxicity for the cell or animal. however, these pmo were directed against viral protein-encoding mrna, and studies of pmo-mediated silencing of viral mirna have not been reported so far (see e.g., [ , [ ] [ ] [ ] ). future viral mirna research is faced with important challenges before amos may enter the clinic. our comprehension on the functions of viral mirna and the interplay between viral infection and cellular mirna expression is just beginning to emerge. studies aimed at the identification of viral mirna and elucidation of their functions should be pursued. difficulties facing computational-based prediction are false positives, but also the shortcoming to detect genuine mirnas. moreover confirmation of expression of mirna by, for example, northern blot may fail to monitor mirna. expression levels of vmirna may be cell-specific, for example, ebv mir-bhfr - had considerably lower expression levels in jijoye cells than in b - cells [ ] . dose-and time-dependent studies are required to determine the optimal therapeutic regime. such pharmacokinetics and pharmacodynamic studies are largely lacking [ ] , but recent in studies in mice revealed that a single cerebral or tail-vein injection of mg/kg body mass anti-mir had a silencing effect for at least days and as long as days in the tissues examined [ , ] . in another study, a five-week regime of two intraperitonal or 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rnai, and gene silencing strategies for therapy: mission possible or impossible? vector design for liver-specific expression of multiple interfering rnas that target hepatitis b virus transcripts sustained delivery of sirnas targeting viral infection by cell-degradable multilayered polyelectrolyte films inhibition of replication and transcription activator and latencyassociated nuclear antigen of kaposi's sarcoma-associated herpesvirus by morpholino oligomers lna derivatives of a kissing aptamer targeted to the transactivating responsive rna element of hiv- controlling morpholino experiments: don't stop making antisense key: cord- -si btsab authors: beard, philippa m.; griffiths, samantha j.; gonzalez, orland; haga, ismar r.; pechenick jowers, tali; reynolds, danielle k.; wildenhain, jan; tekotte, hille; auer, manfred; tyers, mike; ghazal, peter; zimmer, ralf; haas, jürgen title: a loss of function analysis of host factors influencing vaccinia virus replication by rna interference date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: si btsab vaccinia virus (vacv) is a large, cytoplasmic, double-stranded dna virus that requires complex interactions with host proteins in order to replicate. to explore these interactions a functional high throughput small interfering rna (sirna) screen targeting druggable cellular genes was undertaken to identify host factors (hf) influencing the replication and spread of an egfp-tagged vacv. the experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of vacv. the screen revealed pro- and anti-viral hfs that strongly influenced vacv replication. these hfs were investigated further by comparisons with transcriptional profiling data sets and hfs identified in rnai screens of other viruses. in addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in vacv replication. this revealed, as anticipated, that many pro-viral hfs are involved in translation of mrna and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several dna repair pathways possess anti-viral activity. multiple components of the ampk complex were found to act as pro-viral hfs, while several septins, a group of highly conserved gtp binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral vacv hfs. this screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. this advancement in our understanding of the vacv life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics. vaccinia virus (vacv) is a large double-stranded dna virus with a complex cytoplasmic life cycle. it is the prototypical member of the orthopoxviridae genus of the poxviridae family which includes variola virus (the causative agent of smallpox), monkeypox virus and ectromelia virus. vacv was used as a vaccine in the successful global eradication of smallpox in the th century and closely related attenuated strains such as modified vaccinia virus ankara (mva) are now some of the most frequently used recombinant vaccine vectors against a variety of human and animal diseases including hiv, malaria and tuberculosis [ ] . understanding the vacv life cycle is therefore important since it provides the base for the development of efficient and safe novel vaccines. vacv, like all other viruses, harnesses the cell to enable its replication. it turns off or subverts multiple crucial anti-viral pathways including cytokine production, toll-like receptor pathways, nf-kb activation and the dsrna pkr response [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition vacv suppresses both intrinsic and extrinsic proapoptotic pathways [ ] and activates numerous anti-apoptotic, pro-survival pathways including the pi k/akt pathway [ , ] , the mek/erk pathway [ , ] , the p mapk pathway [ ] and the mapk/jnk pathway [ , ] . modulation of so many different signalling pathways prevents viral-induced premature cell death and contributes to the ability of poxviruses to replicate in a wide range of cell types. to investigate this complex pathogen-host relationship further, a rnai screen of druggable host targets was carried out to analyse the effect of cellular protein depletion on vacv replication, using a multi-cycle vacv infection assay that monitors all stages of virus replication including virus spread. the screen identified a range of previously identified hfs, but also novel hfs and pathways influencing vacv infection that may facilitate the development of broadly effective anti-viral strategies and the optimisation of poxviral-based vaccine vectors. a schematic diagram of the workflow used in the rnai screen is shown in figure . sirna smartpools ( sirnas per gene, dharmacon) were diluted to . mm and dispensed in ml volumes using a rapidplate liquid handler (qiagen) into eight black -well plates (corning). these were stored at uc until needed (maximum h). on the day of transfection, plates were thawed and ml transfection reagent (dharmafect , dharmacon) diluted in hank's buffered saline solution (hbss, thermo-fisher) was added to each well containing sirna using a multidrop (thermofisher), to give a final transfection reagent concentration of . %. plates were incubated for min at room temperature to allow formation of transfection complexes. during complex formation, low-passage (p - ) hela cells (ecacc) from approximately % confluent flasks were washed in pbs and trypsinised in trypsin-edta (lonza) before diluting in phenol red-free, antibiotic-free transfection medium (dmem/f- : with % fcs, mm hepes and l-glu; gibco). cells in a volume of ml were added to each well using the multidrop . plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (dmem + . g/l dglucose, l-glu and pyruvate with . % fcs and penicillinstreptomycin) or ml media containing vacv strain wr with egfp tagged a protein [ ] diluted to moi . , was added using the multidrop . plates were incubated at uc for h before ml of media was added to each well, the plates inverted to remove the media and virus, and a final volume of ml of media added to the plates before they were returned to the incubator. after h the plates were inverted to remove the media and ml of % buffered formal saline added to fix the cells. fluorescence levels were measured using a polarstar optima plate reader (bmg labtech). data from eight replicates was used for analysis. background intensity correction was carried out by subtracting the median value of uninfected wells and the data was normalised using the robust z score method [ ] , and corrected for the number of cells in each well. the correction for the number of cells in each well was carried out by estimating the linear correlation coefficient between the level of fluorescence (phenotype score) and the number of cells (toxicity score) using least squares optimization. this coefficient was used to linearly adjust the phenotype scores. one replicate of the screen was imaged by a high content screening system. the buffered formal saline was removed from the cells by inverting the plates, and cells were washed in ml of room temperature pbs before permeabilising for min at room temperature in ml of . % tritonx- diluted in pbs. plates were inverted and ml of a : dilution of alexafluor- phalloidin (invitrogen molecular probes) diluted in pbs + % bsa was added and incubated for min in the dark. the phalloidin was removed by inversion and ml of dapi ( mg/ml) diluted in pbs was added and left on. cells were analysed by automated microscopy using an opera high content screening system (perkin elmer) and acapella high content imaging and analysis software. to identify sirna smartpools which exerted significantly toxic effects the number of cells in each well was counted and converted to a z-score. a z-score is equivalent to the number of standard deviations away from the mean. sirna treatments that reduced the cell number by two or more standard deviations below the population mean (z-score of or less) were removed from further analysis. a z-score of was equivalent to cells, compared to a population mean of . selected sirna smartpools were diluted to . mm in x sirna buffer and dispensed in triplicate in -well plates (corning). to this, ml dharmafect diluted in dmem to give a final concentration of . % was added using the multidrop . following a min incubation to enable complex formation, . hela cells in ml transfection media were added and plates were transferred to a uc humidified incubator with % co . after h, medium was removed and cells rinsed in pbs before lysing in ml trizol (invitrogen). triplicate wells were combined, and rna extracted by purelink (tm) rna mini kit (life technologies). mrna levels were determined by either taqman qpcr with gene-specific primers and probes from the universal probe library (roche), or by sybr green qpcr, using the appropriate one-step rt-qpcr kits (thermofisher). expression levels were normalised to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and normalised expression levels averaged. the phenotype observed in the primary screen was confirmed for a subset of candidate genes with deconvoluted sirna smartpools. the four individual sirnas targeting different regions of each gene were diluted to . mm in x sirna buffer and dispensed to -well plates in triplicate. transfection and, h later, infection with vacv-a egfp was carried out as described above. at and h post infection fluorescence was measured using a synergy ht plate reader (biotek). the experiment was carried out three times to produce a dataset of three biological replicates each containing three technical replicates. the data were analysed using mixed models [ ] fitting gene, time-point gene*time interaction and first time-point value as fixed effects. values observed at the first time-point were fitted as a 'baseline covariate' in order to increase the sensitivity of the analysis. the repeated experiments were fitted as random effects, causing the variation in results between the repeated experiments to be taken into account when testing statistical significance. differences between the amount of fluorescence present in wells treated with each sirna and wells treated with a non-specific sirna (targeting the hsv- gene vp ) were tested within the mixed models using t-tests. groups of genes were tested on separate plates and each of the groups were analysed using a separate mixed model. a phenotype was considered confirmed if two or more of the four sirnas resulted in a p-value of . or less. six wells of a well plate were transfected with a sirna smartpool and, h later, infected with vacv-a egfp as described above. at , and h post infection cells were scraped into the overlying media, collected and then frozen and thawed three times and sonicated for seconds (misonix sonicator ). the resultant lysate was titrated on bs-c- cell monolayers and virus titre quantified as plaque forming units (pfu) per ml [ ] . enrichment analysis was performed with respect to pathwayand go-based gene sets defined in msigdb [ ] , as well as with respect to gene sets derived from protein complexes curated in the corum [ ] and pin [ ] databases. specifically, the genes were sorted based on the screening data, and then the propensity of gene sets towards pro-or anti-viral activities were sought out using rank-sum tests with multiple testing [ ] . the three gene expression data sets used in the analyses were data set a (gse , a microarray of vacv infected hela cells (http://www.be-md.ncbi.nlm.nih.gov/projects/geo/query/acc. cgi?acc = gse )), data set b (gse , a microarray of macrophages, monocytes and fibroblasts [ ] ), and data set c (sra , a rna-seq based analysis of gene expression in vacv infected hela cells [ ] ). for comparison of the rnai hit list with the two hela cell based cellular expression data sets, the differential expression-based rank for each gene (g) was obtained in each condition (i.e. a timepoint in a data set) and then a ''summerisation q-value'' q g was calculated which reflects how unexpectedly high the ranks are (using order statistics; in particular the rank of the third -quantile). therefore q g quantifies how unexpectedly often g is among the most strongly regulated genes (up or down). to identify hfs that influence vacv replication we used a druggable genome small interfering rna (sirna) library (dharmacon) in a high-throughput screen. this library targets genes that are considered potential candidates for therapeutics ( figure a) . briefly, smartpool sirnas (a mix of sirnas per gene) targeting genes were distributed into -well plates and reverse-transfected into hela cells. cells were infected h post-transfection at a low multiplicity of infection (moi . ) with the vacv strain vacv-a egfp. after h (thus allowing multiple complete virus replication cycles), egfp fluorescence was quantified as a measure of infection and compared to controls in order to determine the effect of individual gene depletion on vacv replication. two positive sirna controls known to downregulate vacv-a egfp growth (targeting prk-ab and egfp), two negative controls (mock transfection and rscf sirna which is not processed by the risc machinery) and two nonspecific sirnas (targeting vp or vp / from herpes simplex virus type ) were included in duplicate in each plate (figure b) . to confirm that the measurement of virus-expressed fluorescence was a reliable marker of viral replication, fluorescence was correlated to virus-titre, as determined by standard plaque assay, over a range of time-points post-infection after treatment with control or inhibitory sirnas (figure c) . this resulted in a pearson product moment correlation coefficient of . , confirming that fluorescence was a reliable determinant of virus replication. the entire druggable screen was repeated four times in duplicate to generate a robust primary data set of eight replicates. pairwise agreement comparing the levels of fluorescence across the eight replicates revealed good reproducibility (median spearman's coefficient . ). one replicate of the vacvinfected cells was analysed by automated microscopy using an opera high content screening system and acapella high content imaging and analysis software to quantify the number of cells present in each well. a total of sirna pools ( % of the total) were associated with a significant reduction in cell number (figure a) . these were removed from further analysis and are listed in table s in file s . the fluorescence data from the remaining wells in the primary screen was normalised platewise using the robust z-score method [ ] . a summary value was calculated for each gene by taking the mean across the replicates, and these values were converted to zscores which were corrected for the number of cells in the well to produce the level of fluorescence per cell for each sirna. a negative z-score indicated a reduction in vacv replication and a positive z-score indicated an increase in vacv replication (figure d ). the two positive controls (sirna targeting prkab and egfp) produced strongly negative z-scores as expected. the median level of fluorescence (z-score of ) was very close to the level of fluorescence seen in wells transfected with the non-specific sirna (negative control), indicating that roughly half of the sirna pools caused an increase in fluorescence and half caused a decrease. a ''hit'' was defined as a sirna pool which generated a z-score of $ or #- . using these criteria, a hitlist of hfs ( . % of the total) was generated, consisting of pro-viral hfs which inhibited replication upon depletion and anti-viral hfs which increased replication upon depletion ( table s in file s ). to confirm the effect of the sirna smartpools on mrna levels, a subset of smartpools were transfected into hela cells and after h, total rna extracted and subjected to a quantitative rt-pcr to determine the level of mrna of the targeted gene. genes out of tested ( %) had their transcript level reduced by % or more, indicating that the majority of the smartpools functioned as expected ( table s in file s ). to confirm the effect of gene depletion on vacv replication a subset of hfs, chosen on the basis of their potential for further investigation, were tested using the four individual, deconvoluted sirnas of each smartpool. the level of viral fluorescence was compared to that seen with non-specific sirna and a statistically significant reduction or increase in fluorescence (p, . ) induced by at least individual sirnas from the original smartpool was required for confirmation of the hit. overall ( %) out of candidate genes tested by this method were successfully confirmed ( table s in file s ). this level of validation is commensurate with similar rnai screens of viral replication which have reported confirmation of between % and % of primary screen hits [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . within our deconvoluted dataset the validation level of putative pro-viral hfs was notably higher than that for the anti-viral hfs. only % of the anti-viral hits ( / ) were successfully validated in comparison to % of the pro-viral hits ( / ) and % of the sirna pools with no effect in the primary screen ( / ). one potential reason for the lower validation rate of the anti-viral hits might be that the dynamic range of the virus replication assay is such that inhibitory effects are more easily demonstrated, as under normal replication conditions virtually all cells in the well became infected by h post infection (data not shown) suggesting the system reached near saturation. to examine this further, the effect of selected sirnas on traditional viral growth curves was tested and correlated with their perturbation of fluorescence in the primary screen. hela cells were transfected with each one of sirna smartpools (upregulatory map k , control vp , and downregulatory trip, ppap a, vps and cct ) and infected with vacv-a egfp at an moi of . after h. analysis of virus titre at h intervals by plaque assay found the endpoint titres correlated very closely with the z-score obtained from the primary rnai screen (figure ) , further validating the robustness of the primary screen. notably, however, whilst the inhibitory sirnas decreased the maximum virus titre by between -(cct ) and -fold (vps ) the sirna treatment directed against a candidate antiviral hf (map k ) only increased peak virus titre by -fold. thus the response of the system is more suited to detecting inhibitory perturbation, with relatively reduced sensitivity for detection of individual anti-viral hfs. the list of individual hfs was analysed for genes already known to influence vacv replication. numerous examples were found including clathrin and proteins involved in golgi vesicular trafficking, both of which are required for the production of enveloped vacv forms [ , ] , as well as multiple components of the ampk complex which has been shown to aid vacv entry [ ] . in addition the signalling pathway regulating protein traf was identified in the screen as a pro-viral hit; further work demonstrated that it promoted rapid vacv entry [ ] . the identification of known host factors for vacv and our follow-up identification of the role of traf in vacv replication supports the reliability and significance of this rnai screen dataset. overall, eight replicates of a genome-wide rnai screen of multiple vacv replication cycles identified cellular genes, consisting of hfs that positively support vacv replication and hf with anti-viral effects. to prioritise investigations of the potential vacv hfs, the candidate genes were compared to the hit lists of other viral rnai screens, including two recently published vacv screens [ , ] . the methodology in the previously published vacv screens varied considerably; mercer et al [ ] measured the growth of a thymidine-kinase-deficient vacv (strain western reserve) after only h of infection, thereby identifying cellular proteins involved in the initial stages of virus replication but excluding analysis of viral spread. they reported pro-viral hf but no anti-viral hfs. a second screen by sivan et al [ ] used the vacv strain ihd-j (which has a point mutation that accelerates the release of progeny virions from the cell surface) to identify genes which influenced viral replication after h of infection, thus measuring the entire replication cycle with emphasis on viral spread. they reported pro-viral and anti-viral hfs. the overlap between the hit lists reported by the three vacv rnai studies (mercer et al, sivan et al, and this study) is depicted in figure a and b and the hfs common to two studies are listed in table s in file s . the number of overlapping hits between two of the screens ranged from to and no hfs were common to all three vacv studies. a small number of common hits between sirna screens of the same virus is a frequent finding [ , ] and, given the variation in methodology between the three vacv screens (including viral strain, infection time, and data analysis), is not surprising. however, comparison of the enriched functions and pathways identified in each of the three vacv screens revealed marked similarities (discussed below), demonstrating the power of comparative screening approaches to identify significant cellular pathways involved in virus replication. genome-scale sirna screens have been carried out for many viruses other than vacv, including hiv- [ ] [ ] [ ] , west nile virus (wnv) [ ] , hepatitis c virus (hcv) [ , ] , vesicular stomatitis virus (vsv) [ ] , borna disease virus [ ] , enteroviruses [ ] , dengue virus [ ] , herpes simplex virus (hsv- ) [ ] and influenza a virus [ , , ] . host factors common to two or more of these screens could represent broadly acting cellular proteins with a generalised effect on viral replication. a [ , , ] and three published hiv rnai screens with a total of hits [ ] [ ] [ ] . doi: . /journal.pone. .g comparison of the vacv hfs identified in this screen with those identified in other viral screens found a small overlap with wnv, vsv, borna disease virus and dengue virus, whilst vacv hfs were shared with hsv- , with influenza a virus and with hiv- (figure c) . a list of overlapping genes can be found in table s in file s . amongst the factors in common, the nucleocytoplasmic transport factor nup was identified as a proviral hit in the vacv screen reported here as well as hiv, hsv- and influenza a virus screens [ , , , ] . it is located at both the cytoplasmic and the nuclear faces of the central channel of the nuclear pore complex (npc) [ ] , is involved in rev-dependent rna export during hiv infection [ ] , and has been shown to play both proand anti-viral functions during influenza a virus infection [ , , ] . nup was a somewhat unexpected proviral hit in our screen since poxvirus replication and assembly occur in the cytoplasm. however the two vacv rnai screens published recently also identified a number of nuclear pore proteins as proviral, with one screen demonstrating that knockdown of nup strongly inhibited viral morphogenesis [ ] . the number of nuclear pore proteins now identified as pro-viral hfs strongly suggests poxviruses require functional nuclear membrane transport for efficient replication. another hf that affects both vacv and influenza a virus replication is map k (also known as mkk and mek ), which activates the p mapk signalling pathway and is involved in low ph-dependent entry of influenza virus and vsv [ , ] . vacv also has a low ph-dependent entry mechanism [ ] which may be similarly reliant on map k . alternatively, it may be required to activate the p mapk pathway to promote cell survival post infection [ ] . in contrast, ifitms (interferon inducible transmembrane proteins) have been identified in functional genomic screens as mediating resistance to influenza a virus, dengue virus and west nile virus infection in vitro and in vivo [ , ] as well as marburg and ebola viruses, sars-coronavirus [ ] and hiv [ ] . these proteins prevent entry of viruses at the plasma membrane, endosomes and lysosomes [ ] , however none had an effect on vacv replication in our screen, suggesting vacv is resistant to the repressive effect of this protein family (figure d ). to determine whether the expression of hfs identified by rnai is modulated by vacv, the rnai hit list was compared to previously published transcriptional profiling data sets performed on cells infected with vacv. three available expression profiling data sets (designated a, b and c, see materials and methods for sources) which used different cells, virus isolates and time points were compiled and compared. correlation was mainly limited to intra-data set measurements (i.e. different time points or cell lines within a data set) with some examples of agreement between data sets a and c, both of which used hela cells. to test whether there was a general propensity of hfs to be differentially expressed in virus-infected cells the distribution of the rnai hits was compared to that of the whole rnai screen in these three transcriptional profiling data sets. no significant tendency of proviral hfs to be up-regulated or anti-viral hfs to be downregulated was detected (data not shown). subsequently the expression of individual hfs was examined using the transcriptional profiling data of the two hela-based studies (a and c). this revealed specific examples of hfs which are differentially expressed in virus-infected cells (figure ) . four pro-viral hfs (runx , eif c, hbegf, and adm) were significantly upregulated in vacv-infected hela cells (q, . ), suggesting that vacv might promote expression of these proteins to assist viral replication and spread. runx is a subunit of the transcription factor cbf which regulates critical processes in both myeloid and lymphatic haematopoiesis. chromosomal translocations and mutations of runx are among the most frequent genomic abnormalities in different types of leukaemia [ ] . furthermore, a genome-wide association study found a genetic polymorphism in runx to be associated with the serological response to vacv vaccination (dryvax vaccine, wyeth laboratories) in african-americans. this suggests that the runx polymorphism may influence replication and viral gene expression of the live-attenuated vaccine in vivo which causes differences in the strength of the adaptive immune response [ ] , a hypothesis supported by our identification of runx as a pro-viral vacv hf. in addition to upregulated pro-viral hfs, opposite examples were also identified, with two anti-viral hfs (angptl , rbak) significantly upregulated and one pro-viral hf (sap ) downregulated in vacv-infected cells. this lack of correlation between functional hfs and gene expression at the transcriptional level serves to underscore the complexity of virus-host interactions and highlights the need for further follow-up studies. to assess further the role of candidate hfs and associated functions and pathways in vacv replication, an overrepresentation analysis of the complete vacv rnai data set was performed with respect to pathway- (figure a ) and go- (figure b ) based gene sets as defined in the msigdb database, as well as from curated protein complexes defined in the corum [ ] and pin [ ] databases (figure c) . translation was the most strongly enriched theme in all these analyses, particularly the eif complex. poxviruses are known to utilise the host translation machinery for production of viral proteins therefore the enrichment of translation as a pro-viral theme is a validation of the screening method. the cellular translation machinery has been highlighted in other viral rnai screens as essential for vsv [ ] and hepatitis c virus [ ] and hsv- [ ] . more interestingly, transcriptional initiation and general rna polymerase ii transcription factor activity were identified in the functional analysis of the rnai screen as significantly overrepresented anti-viral go-based gene sets (figure b) . sevin et al [ ] also reported that interference with dna-dependent rna polymerase ii pathways enhanced vacv spread. our work identified individual anti-viral hfs involved in transcription such as maz (an inflammatory responsive transcription factor also known as saf ), and the transcription factor e f . inspection of individual confocal images taken at h pi of cells depleted of these factors showed notably brighter accumulations of egfplabelled virus, in line with the positive z-scores from the overall primary screen (figure a ). orthopoxvirus infection results in a rapid shut down of cellular transcription, with a marked reduction in the amount of host mrna present as early as h post infection [ , , ] . this effect is believed to result from cessation of host mrna synthesis and degradation of cellular mrna transcripts. the viral proteins involved in this shut-off of host cell transcription have not been identified, although d and d have been implicated [ , ] . in contrast to viral translation which is dependent on host proteins, vacv encodes its own transcription enzymes so is largely unaffected by a general repression of cellular transcription [ ] . therefore vacv-induced downregulation of host transcription prevents the host cell from transcribing antiviral, pro-inflammatory gene programmes, such as the nf-kb cascade, while having a minimal negative effect on viral [ ] and pin [ ] databases. all significantly overrepresented gene sets (-log (q-value). . ) are shown. each row shows the ranks of genes from a particular gene set that were present in the rnai screen. each tick mark denotes the place of a particular gene from that gene set, placed at the appropriate position in the distribution. genes were sorted from left to right from most pro-viral to most anti-viral. the red and blue colours of the ticks are used for visual contrast. a green diamond is used to denote the median rank of the genes in the set. doi: . /journal.pone. .g transcription. whilst vacv has developed mechanisms to shut off host transcription, these data show that virus replication is improved when transcription is impaired prior to infection. thus, despite the efforts of the virus to shut off host transcription, some anti-viral effect of this pathway persists in vacv infected cells. two members of the septin protein family (septin and msf/ septin ) were identified in the rnai screen as anti-viral hfs (figure b) . msf/septin co-purifies from cells with three other septin proteins (nedd /septin , cdc /septin and septin ), suggesting they form a functional complex [ ] . these were therefore grouped in the pathway analysis, resulting in a significant over representation (q = . ) (figure c) . consistent with this, depletion of nedd /septin , septin and septin all increased virus replication although not to the stringent cut-off used here to define a 'hit' (figure b) . deconvoluted sirnas targeting septin mrna confirmed the enhancement of virus replication, although results with the other family members were more variable ( table s in file s ). septin has also been identified as a proviral hit in a recently reported vacv sirna screen [ ] . septins are conserved gtp-binding proteins which act as dynamic scaffolds for recruitment of other proteins. they are involved in actin and microtubule function, cytokinesis, cell movement and vesicle trafficking [ ] . interestingly, they can be recruited together with autophagic proteins to ''cage'' shigella flexneri in the cytosol of infected cells, restricting bacterial dissemination [ ] . the cage assembly is linked with actin polymerisation activity of s. flexneri, suggesting that a similar mechanism may be employed by the host cell to ''cage'' vacv virions (which activate actin polymerisation both early and late in the replication cycle [ , ] ) and thus invoke an anti-viral effect. two groups of genes involved in dna replication and repair were highlighted in the pathway analysis as having anti-viral properties (figure c ). the pcna-mutsa-mutla-dna complex (pcna, msh , pms , and mlh ) and the brca -associated genome surveillance complex (rfc , brca , blm, rfc , msh and mlh ) both promoted virus replication when individual group members were downregulated (figure c ). these pathways are both involved in dna damage signalling and repair [ , ] , a cellular process which is targeted by numerous viruses [ ] . specifically, dna damage signalling pathways act as a host defence mechanism in poxvirus infection, detecting and responding to foreign poxviral dna and inducing intrinsic apoptosis [ ] . the identification of the pcna and brca gene sets as strongly anti-poxviral hfs in the rnai screen suggests they are a part of this, or a similar, defence mechanism. the amp-activated kinase complex (ampk) is a key regulator of energy metabolism. it is activated by a reduction in atp which prompts phosphorylation of many target proteins, resulting in the activation of catabolic pathways and inhibition of anabolic pathways [ ] . it has also been linked to regulation of the actin cytoskeleton [ ] . ampk is a heterotrimer comprising a catalytic a subunit and regulatory b and c subunits. in mammals each subunit has several isoforms (prkaa , prkaa , prkab , prkab , prkag , prkag , and prkag ) [ ] . the druggable rnai screen reported here screened all seven genes and identified three (prkaa , prkab , and prkab ) as promoters of vacv replication whose depletion led to fewer and dimmer accumulations of cytoplasmic egfp (figure d ). this result is in broad agreement with a recently published rnai screen of cellular kinases and phosphatases in a nonpermissive drosophila cell model of vacv infection [ ] , which identified seven hits including three ampk subunits. this study performed a loss of function analysis of hfs involved in vacv infection using rnai. previously identified host pathways and protein complexes which aid vacv replication, such as translation and the ampk complex proteins, were highlighted in the rnai screen. in addition, however, a range of novel host pathways and proteins were identified that influenced vacv infection, such as the dna damage and repair pathways, the septin family of proteins, map k and nup . as many of the genes targeted in this rnai screen have a known drug inhibitor, this work yields a list of hfs that can potentially be targeted by novel therapeutics. file s supporting tables. table s , list of cytotoxic sirnas which caused significant cell death. mva and nyvac as vaccines against emergent infectious diseases and cancer two vaccinia virus proteins structurally related to the interleukin- receptor and the immunoglobulin superfamily a soluble receptor for interleukin- beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection encoding of a homolog of the ifngamma receptor by myxoma virus a r and a r from vaccinia virus are antagonists of host il- and toll-like receptor signaling the e l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase inhibition of ikappab kinase by vaccinia virus virulence factor b the vaccinia virus k l gene product inhibits host nf-kappab activation by preventing ikappabalpha degradation near death experiences: poxvirus regulation of apoptotic death activation of the pi k/akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication role of cell signaling in poxvirus-mediated foreign gene expression in mammalian cells a mitogenic signal triggered at an early stage of vaccinia virus infection: implication of mek/erk and protein kinase a in virus multiplication differential role played by the mek/erk/egr- pathway in orthopoxviruses vaccinia and cowpox biology vaccinia virus protein a r activates p mitogen-activated protein kinase and potentiates lipopolysaccharide-induced interleukin- vaccinia virus b r kinase interacts with jip and modulates c-jun-dependent signaling vaccinia virus cores are transported on microtubules statistical methods for analysis of high-throughput rna interference screens applied mixed models in medicine the vaccinia virus kelch-like protein c l affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles corum: the comprehensive resource of mammalian protein complexes- pindb: a database of nuclear protein complexes from human and yeast statistical significance for genomewide studies stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus simultaneous highresolution analysis of vaccinia virus and host cell transcriptomes by deep rna sequencing the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers discovery of insect and human dengue virus host factors rnai screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand rna viruses a genome-wide genetic screen for host factors required for hepatitis c virus propagation genomewide rnai screen identifies human host factors crucial for influenza virus replication rnai screening reveals proteasome-and cullin -dependent stages in vaccinia virus infection a systematic analysis of host factors reveals a med -interferon-lambda regulatory axis against herpes simplex virus type replication clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread evidence against an essential role of copii-mediated cargo transport to the endoplasmic reticulum-golgi intermediate compartment in the formation of the primary membrane of vaccinia virus a kinome rnai screen identified ampk as promoting poxvirus entry through the control of actin dynamics facilitates vaccinia virus replication by promoting rapid virus entry human genome-wide rnai screen reveals a role for nuclear pore proteins in poxvirus morphogenesis host cell factors in hiv replication: meta-analysis of genome-wide studies the use of rnai-based screens to identify host proteins involved in viral replication identification of host proteins required for hiv infection through a functional genomic screen global analysis of host-pathogen interactions that regulate early-stage hiv- replication genome-scale rnai screen for host factors required for hiv replication rna interference screen for human genes associated with west nile virus infection a functional genomic screen identifies cellular cofactors of hepatitis c virus replication identification of host factors involved in borna disease virus cell entry through a small interfering rna functional genetic screen human host factors required for influenza virus replication nucleoporin nup : a gatekeeper in the eukaryotic kingdoms functional analysis of the interaction of the human immunodeficiency virus type rev nuclear export signal with its cofactors influenza virus targets the mrna export machinery and the nuclear pore complex drosophila rnai screen identifies host genes important for influenza virus replication vaccinia virus entry into cells via a low-ph-dependent endosomal pathway ifitm restricts the morbidity and mortality associated with influenza distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus the ifitm proteins inhibit hiv- infection ifitm proteins-cellular inhibitors of viral entry runx translocations and fusion genes in malignant hemopathies genome-wide association study of antibody response to smallpox vaccine sequence complexity and relative abundance of vaccinia virus mrna's synthesized in vivo and in vitro microarray analysis of a cells infected with rabbitpox virus (rpv): a comparison of wildtype rpv and rpv deleted for the host range gene, spi- down regulation of gene expression by the vaccinia virus d protein characterization of a vaccinia virus mutant with a deletion of the d r gene encoding a putative negative regulator of gene expression vaccinia virus transcription biochemical and cell biological analyses of a mammalian septin complex, sept / b/ the evolution, complex structures and function of septin proteins entrapment of intracytosolic bacteria by septin cage-like structures actin-based motility of vaccinia virus repulsion of superinfecting virions: a mechanism for rapid virus spread mechanisms and functions of dna mismatch repair basc, a super complex of brca -associated proteins involved in the recognition and repair of aberrant dna structures using or abusing: viruses and the cellular dna damage response cytosolic dna triggers mitochondrial apoptosis via dna damage signaling proteins independently of aim and rna polymerase iii amp-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism energy-dependent regulation of cell structure by amp-activated protein kinase the authors thank professor geoffrey smith (cambridge) for the gift of the egfp tagged vacv strain, dr helen brown (the roslin institute) for statistical expertise, and professor paul digard (the roslin institute) for discussion. key: cord- -oxfnt n authors: caricati, c. p.; oliveira‐nascimento, l.; yoshida, j. t.; stephano, m. a.; caricati, a. t. p.; raw, i. title: safety of snake antivenom immunoglobulins: efficacy of viral inactivation in a complete downstream process date: - - journal: biotechnol prog doi: . /btpr. sha: doc_id: cord_uid: oxfnt n viral safety remains a challenge when processing a plasma‐derived product. a variety of pathogens might be present in the starting material, which requires a downstream process capable of broad viral reduction. in this article, we used a wide panel of viruses to assess viral removal/inactivation of our downstream process for snake antivenom immunoglobulin (sai). first, we screened and excluded equine plasma that cross‐reacted with any model virus, a procedure not published before for antivenoms. in addition, we evaluated for the first time the virucidal capacity of phenol applied to sai products. among the steps analyzed in the process, phenol addition was the most effective one, followed by heat, caprylic acid, and pepsin. all viruses were fully inactivated only by phenol treatment; heat, the second most effective step, did not inactivate the rotavirus and the adenovirus used. we therefore present a sai downstream method that is cost‐effective and eliminates viruses to the extent required by who for a safe product. © american institute of chemical engineers biotechnol. prog., : – , humankind has suffered from poisoning because of animal bites since the beginning of times. currently, snakebites represent the main cause of human envenoming, with approximately , , occurrences and , deaths yearly. most of the occurrences happen in asia, latin america, and especially in africa. snake antivenom immunoglobulins (sai) may prevent the referred morbidity and mortality if the correct antidote is administered immediately after the bite. these conditions, however, are impracticable for most regions in need and for some species of snake. , the world health organization (who) endorsed the relevance of sai for global health by including them in the "model lists of essential medicines." all biological health products require a rigorous quality control, including sai. the who recently compiled the quality parameters for this centenary product in "who guidelines for the production, control and regulation of snake antivenom immunoglobulins." the document emphasizes that viral content is an essential control parameter because contaminated sai may infect humans and lead to morbidity or even mortality. indeed, contaminated humanderived immunoglobulin led to several cases of human infections with hepatitis c virus. the phenomenon is so far unreported for animal-derived immunoglobulins. , sai are purified from hyperimmunized plasma of animals, mainly horses, immunized with a pre-determined amount of crude venom from one or more snake species. , ideally, the donor animals should be free of viral pathogens, but this approach fails because of the incomplete understanding of the equine virology and consequent lack of vaccines. therefore, to compensate for this flaw, viral inactivation becomes essential in the downstream process of equine plasma. sai purification diminishes viral load of contaminated plasma, although only process validation guarantees full elimination. , some researchers reported viral elimination throughout sai purification, , but none described a prior plasma selection based on antiviral titers. this selection excludes plasma samples that are positive for antibodies against the model viruses used for validation. without selection, neutralizing antibodies may interfere in the measurement of virus titers, an essential parameter to evaluate the capability of virus removal/inactivation of the steps alone. whenever a whole process fails to complete viral inactivation, modifications like further steps might improve the outcome. some preservatives inactivate viruses cost-effectively, representing a plausible alternative to eliminate viral activity. for example, sai formulations commonly contain phenol that, although known as virucidal, has not been explored to this end for sai production in the literature. to address the above issues, we classified and selected plasma from donor horses (instituto butantan, brazil) according to their antivirus immunoglobulin titers. after selection, we assess viral safety of the sai process developed by instituto butantan and evaluate the efficacy of phenol as a virucidal agent for sai. we chose the venom of crotalus durissus terrificus (south american rattlesnake, cascavel) as a model for immunization. the choice was random because our institute produces several snake antivenoms from equine plasma with the same procedure. the type of venom does not influence the outcome of virus removal. fifteen healthy adult horses were immunized by intramuscular route against the snake venom. these animals were tested for equine infectious anemia virus every months and were considered free from this virus. all animals were also previously immunized against rabies, equine influenza virus, leptospirosis, tetanus, and venezuelan equine encephalitis virus. the immunization scheme to produce antivenom serum consisted in three inoculation days ( , , and ).on each inoculation day, injections of . ml venom solution were applied to different spots within the lumbar region. blood collection was done by the jugular vein from each horse (maximum of % horse weight), weeks after immunization. plasma was obtained by gravity sedimentation of blood ( h/ - c) and tested for sterility/potency, mycoplasma content (mycoplasma pcr elisa, roche applied science), and virus screening. after plasma isolation, blood cells were returned to the donor horses (plasmapheresis). day -crotalus venom ( mg/ml, instituto butantan, brazil) mixed with the adjuvant montanide isa % (mineral oil emulsion, seppic, brazil); day -crotalus venom ( mg/ml) diluted in saline solution; and day -crotalus venom ( mg/ml) diluted in saline solution. the viruses chosen as models for viral inactivation studies are described in table . to replicate these viruses and test their cytopathic effects, five different cell lines were used ( table ). all cell lines were free of mycoplasma (mycoplasma pcr elisa, roche applied science). all cells were cultivated as monolayers in cell culture flasks with dulbecco modified eagle's medium (dmem, sigma-aldrich), supplemented with % fetal bovine serum-fbs (cutlab, brazil), and incubated at c, % co . for subcultivation, passages were performed when a cell monolayer reached % confluence (ratio of : - : ). cell detachment for sub cultivation was achieved by addition of . % trypsin (sigma aldrich) to a . % edta solution (merck). each virus was replicated using the appropriate cell line (table ), while the cell culture was performed as described (item . ). cell culture monolayer at % confluence was infected with virus (multiplicity of infection of : , ) and incubated again. the supernatant was collected after identification of visual cytopathic effect in the monolayer. cell debris were removed by centrifugation ( min/ c/ g) and the remaining supernatant containing the replicated virus was titrated and stored in c freezer. virus storage was performed at a minimum concentration of log measured by tcid %. to achieve the minimum concentration for storage, canine distemper virus and canine adenovirus were treated with peg , ( % v/v) for h/ c followed by centrifugation ( min/ c/ , g), supernatant discharge and pellet resuspension with dmem ( . ml/ ml initial plasma volume). cells for tcid test (table ) were cultivated (item . ) and seeded overnight in -well cell culture plates ( plate per virus, ll containing , cells per well). ten-fold serial dilutions of each virus sample were prepared and inoculated ( ll) into the wells of the respective plates, with eight replicates per dilution. a row with culture medium was used as the negative infection control. the infected plates were incubated for h for virus infection in dmem % fbs ( c/ % co ), followed by medium renewal (dmem % fbs) and new incubation until maximum cytopathic effect (up to days). after incubation, cells were fixed with % formaldehyde and stained with % crystal violet. the infected wells did not stain, whereas the wells containing intact cells stain blue/purple. for each virus experiment, the average of three virus assays were used for tcid , which was calculated by the spierman-karber method. the samples from equine serum immunized against crotalus durissus terrificus were screened for eight different viruses (table ) by direct enzyme-linked immunosorbent assay (elisa). unless otherwise stated, all incubations were performed at c, with washing steps (pbs/tween . %) between incubations. for each virus strain, a -well plate (cat. no. , corning) was coated with ll of virus solution ( lg/ml protein content), diluted in carbonate buffer ( . m, ph . ), and incubated overnight at c. following a h blocking incubation step ( . % gelatin), ll of serum samples serially diluted ( -fold) were added to the plate and further incubated for h. for serum igg detection, rabbit antihorse igg conjugated with peroxidase (sigma, eua) was incubated for h ( ll/well, , times diluted). a further incubation at room temperature was then performed with the substrate o-phenylenediamine (sigma, eua) and hydrogen peroxide (nuclear, brazil) . %. the reaction was stopped after min with sulfuric acid . n. the plate was read at nm (titertechv r spectronic). serum samples that presented an optical density of . (background) for all dilutions were considered free of antibody to the tested viruses. all tested samples were assayed in triplicate and repeated for confirmation. the sai purification process was developed in instituto butantan, brazil, to obtain crotalic antivenom derived from equine plasma. this process is an optimization of a previously developed method. [ ] [ ] [ ] the flowchart (figure ) describes the process for obtaining the concentrated immunoglobulin solution, in which the steps identified with potential for viral inactivation are highlighted. the method and tests were already described in item . . when a routine batch is produced, the concentrated bulk is further diluted, filter sterilized, and packaged. cells (table ) for cytotoxicity were cultivated (item . ) and seeded overnight in -well plates for cell culture ( , cells per well).the test samples were taken immediately after each of the four viral inactivation steps (item . ) and diluted serially (twofold) in culture medium. diluted samples were loaded to the plate for incubation ( h/ c/ % co ). following cell culture medium removal, ll of mtt [ -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide, sigma, eua] solution was added and further incubation was performed for h. after mtt removal, ll of isopropanol was used to dissolve mtt crystals and the resulting optical density was measured at nm (titertechv r spectronic). the average optical density of control wells that did not receive virus represented % viability. all other wells had their viability calculation based on these controls. the capability of viral inactivation was evaluated for the chosen steps following directions from the guidelines and papers published before. , the q a guideline is also related to viral removal/inactivation validation, although not designed for animal plasma derivatives. a purification process without any spiking was performed as a blank. the process (figure ) was conducted routinely until before the chosen step, when the sample was spiked with one model virus. after that, the step was performed as described and the resultant product evaluated by tcid . each model virus was evaluated in a separate experiment for each step. immediately after the spiking and before the evaluated step, duplicate samples were collected and stored ( c) for quantification of initial tcid at the same time as the final tcid . all test samples were stored ( c) until the analyses. the spiking rate used was : for all runs, and the tcid of nonspiked runs were considered the test blank. the viral log reduction factor (lrf) was then calculated for each run, as described below: log reduction factor ðlrfÞ logðv t Þ=ðv t Þ ( ) v volume of starting material, t concentration of virus in starting material (initial tcid ), v volume of material after the evaluated step, t concentration of virus after the step. antiviral immunoglobulin screening of horse-derived plasma samples in order to verify virus elimination in equine plasma, we chose eight strains of viruses as models of all significant viruses reported to infect horses (item . , table ). we then screened plasma samples from donor horses for antiviral immunoglobulins, detected by elisa. the screening showed positive samples out of , considering positive a sample that recognized at least one model virus. viral recognition was independent of the type of genetic material or presence of the envelope, but related with viral origin (figure ) , with the majority of samples reacting with bovine viruses, especially the bhv- and bvdv, followed by canine viruses. because the cited viruses generally do not infect horses, the result probably arouse from cross-reactivity of the antibodies. antibody cross-reactivity may occur with viruses that infect several species of animals, , but no reports broached the subject with bovine or canine and equine viruses. two plasma samples had antibodies against vesicular stomatitis virus (vsv), the only model virus in this work that commonly infects horses ( figure ) . however, all animals were free of vsv (pcr screening, data not shown), leaving previous infection or antibody cross-reactivity as probable causes for positives. researchers found that crude plasma does contain a mixture of immunoglobulins that cross-reacts with various antigens, supporting our results. the positive samples were excluded from further inactivation studies to allow the specific viral elimination step to be tested. potency of sai is defined as the amount of snake venom neutralized by ml of plasma (mg/ml), measured in this work as described before. the four negative samples neutralized at least mg/ml, the minimum potency required for further purification (data not shown). the pool from these samples also surpassed the minimum protein value ( . mg/ml) and constituted the starting material for validation studies. the process to obtain purified sai is described in detail in the methods section (item . , figure ). the main steps are cited in sequential order (a) ammonium sulfate precipitation of immunoglobulins, (b) pepsin digestion to obtain f(ab') fragments, (c) caprylic acid precipitation of nonimmunoglobulin proteins, (d) heat treatment, (e) ammonium sulfate precipitation of f(ab') fragments, (f) tangential filtration, (g) ion-exchange chromatography, (h) tangential filtration, and (i) phenol addition. among all procedures, we chose (b), (c), (d), and (i) for viral inactivation studies because of their reported virucidal capability. , , , ion-exchange chromatography (g) also removes viruses, , but evaluation of this step would require sanitization used between batches. thus, we performed several purification processes of the pooled sample, each one spiked before step (b), (c), (d), and (i) with the eight model viruses individually (item . ). the spiking concentrations (table ) were different for each virus, a consequence of the replication yield obtained: cdv and cav- titers were consistently below six logs and required concentration before spiking, while brv titer decreased slightly above six logs after mixing ( . log) ( table ) . we analyzed cell cytotoxicity of downstream steps before the spiking experiments. samples taken before and after each chosen step were toxic up to . % (diluted in culture medium). therefore, we used . % dilution for cell-based assays. there were no cytotoxicity differences between samples. to assure viral safety, spiking experiments with initial virus titers of six logs should result in undetectable virus load. however, the process still contributes to viral safety if the reduction is between four and six logs, but requires additional viral reduction to meet . european agency for the evaluation of medicinal products (emea) requirements. by contrast, a process step presenting lrfs below four logs generally inactivates an insufficient amount of virus to insure adequate safety. we calculated viral lrfs after each downstream step, and all of them were at the least . log (table ) , which is considered the threshold. the effectiveness of the steps was analyzed by the amount of viruses inactivated that resulted in lrfs greater than logs (effective), - logs (contribute to viral safety), or below logs (ineffective). the collected data pointed out heat treatment as more effective than pepsin or caprylic acid. nevertheless, heat treatment was performed in the presence of residual caprylate from the previous step, which may have influenced lrf values ( - log). all lrfs were about - log and - log for the pepsin and caprylate process steps, respectively, depending on the viruses involved. just the phenol addition step resulted in undetectable viral load. differences in virus susceptibility to pepsin, caprylic acid, and heat treatments enveloped viruses. in our experiments, the most susceptible viruses were bhv- and ccv, with lrfs logs after each treatment. bhv- , as other herpesviruses, generally presents low resistance to antiviral treatments, , whereas heat ( - c, min /h) and low ph might reduce ccv infectivity. we found no reports using both viruses as models for viral inactivation concerning the described purification steps. vsv and bvdv generally resists to inactivation more than most enveloped viruses, , but are extensively neutralized with heat treatment. , indeed, we observed lrf values of bvdv and vsv above six logs when treated with phenol or when heated, but between six and four logs when treated with pepsin or with caprylic acid (table ) . in respect to vsv, other researchers also found lrfs in the same range as we did for pepsin and caprylic acid treatments. , another experiment showed that both treatments reduced approximately . logs of vsv after min, followed by consisted inactivation (> logs) after min. the latter finding suggests that inactivation could be extended for vsv if our process reached h instead of min for pepsin and min for caprylic acid. nevertheless, we have to consider that extending these parameters may also diminish serum potency. because our protocol achieved an extended inactivation in the heat step, as reported before, it is unnecessary to extend steps that might decrease activity to kill this specific virus. concerning bvdv, previous literature showed a similar trend to our results with caprylic acid and pepsin treatment at ph , except for a work reporting high-bvdv resistance with pepsin treatment at ph . the higher resistance found in that work might be because of spiked plasma containing bvdv antibodies. if the plasma contained antibodies bound to bvdv, the initial titer may have been underestimated. the decrease in ph might have destabilized the previous antibody-antigen binding and released infective viruses that were not detected at the beginning. the consequence of these events is an underestimated reduction factor. the four enveloped viruses discussed above were inactivated in a greater extent than nonenveloped viruses, to the known general properties of viruses. , an exception to the trend was the enveloped cdv, which presented lrfs only with phenol addition. this resistance probably arose from animal protein/virus interaction because previous reports described cdv protection from inactivation when calf serum or egg tissue was present. , the later finding is quite relevant, illustrating that viruses easily inactivated in the environment can be quite resistant in biological fluids and therefore should be included in validation panels. nonenveloped viruses. the most resistant virus detected in our experiment was brv- , poorly neutralized by pepsin, caprylic acid, and heat treatments (table ) . brv was also poorly neutralized with acidic treatment (ph - , min). cav- was not effectively inactivated by pepsin and caprylic steps (lrf < ), but it was moderately inactivated after the heat step at acid ph ( - log) in our experiments. yamamoto and colleagues inactivated more logs of cav- with increased pepsin concentration and extensive reaction times, up to h. however, these increments can cause loss of activity, as discussed before. in another paper, petr and jiran completely inactivated cav- in serum-free medium, ph . , after heat inactivation. this increased sensibility to heat probably results from the ph used because cav- is particularly unstable at neutral ph range. caprylic and pepsin treatments are generally considered unsuitable for inactivation of nonenveloped viruses. again, we found an exception to an established trend because the nonenveloped cpv was substantially inactivated after the referred treatments ( - log). we could not find papers using canine parvovirus for validation of the referred treatments in crude plasma samples. we will avoid performance comparison with other parvoviruses or different initial products because of high variability found among the parvovirus group. , , nevertheless, cpv seems to be quite vulnerable to acidic ph, the same condition maintained in our pepsin step. in order to guarantee the "worst-case scenario" for parvovirus inactivation, one would have to test different parvoviruses in the same conditions as we tested cpv. phenol is a common preservative for products derived from animal immunoglobulins. its efficacy against bacteria has been extensively studied, but reports approaching phenol as virucidal are scarce in the literature. addition of phenol to the concentrated bulk resulted in inactivation of all model viruses after just min of exposure, evidenced by undetected values of tcid %. a partial or total inactivation was expected for hbv- , cv, vsv, bvdb, and cdv, because phenolic compounds were described as efficient virucidal agents of enveloped viruses. however, inactivation by phenol of nonenveloped viruses differs with the virus species studied. some model viruses used in this work are also models for human viruses. for instance, bvdv is used as a surrogate model of hepatitis c virus and cpv as a model of human parvovirus b . these viruses are important agents of viral infection through human blood products and, consequently, targets for viral inactivation. , phenol inactivated both viruses, which indicates that it might also be an effective preservative for human-derived immunoglobulins, when it comes to viral safety. concerning phenol safety, its use in high concentrations ( . %) was reported to facilitate formation of igg aggregates and dimmers, contributing to turbidity of the solutions at c. in vivo effects in mice include transient hypotension and disturbance of leukocyte-endothelial interactions. diluted phenol was not reported as toxic or as a destabilizing agent of the formula. the process described here utilizes . % phenol in the concentrated bulk, but after min the batch product is diluted, which abrogates the possibility of the reported side effects. antibody cross-reactivity is a common feature of viruses of the same species or genus that infect different animals. we found several evidences of virus inactivation and thus of possible cross-reaction, showing for the first time that equine plasma recognizes a wide array of typical model viruses, including a substantial amount of bvdv. because this virus commonly belongs to validation panels, especially for hcv modeling, the viral screening becomes essential before evaluating any virucidal step. concerning the performance of the steps, phenol was the most effective one, followed by heat, caprilic acid, and pepsin. this was an expected result because phenol and heat were the only treatments dedicated to eliminate microbes. ideally, phenol should not be used as a complement for bactericidal purposes, but its virucidal properties are quite important when resistant nonenveloped viruses, poorly inactivated by other treatments, are present. that was the case of this protocol because only phenol was able to inactivate all viruses. phenolic compounds were additionally reported as virucidal for equine infectious anemia virus, an essential feature when donor horses are located in endemic regions for this enveloped virus. other processes might remove the resistant viruses, like nanofiltration, but the production costs surpass the budget of the regions in need. we therefore present a sai purification method that is cost-effective and eliminates viruses to the extent required by who for a safe product. in addition, our 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of a manufacturing process who. who j who guidelines for the production, control and regulation of snake antivenom immunoglobulins. who. available at q a: quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin genomic and serological diversity of bovine viral diarrhea virus in japan antibodies against bovine herpesvirus (bhv) may be differentiated from antibodies against bhv in a bhv glycoprotein e blocking elisa a method for measuring specific antibodies in bovine lacteal secretions during the nonlactating period a sensitive and specific immunoassay for the measurement of the antibodies present in horse antivenoms endowed with the capacity to block the phospholipase a -dependent hemolysis induced by snake venoms virus safety of human immunoglobulins: efficient inactivation of hepatitis c and other human pathogenic viruses by the manufacturing procedure turbidity of hyperimmune equine antivenom: the role of phenol and serum lipoproteins purification of intravenous immunoglobulin g from human plasma-aspects of yield and virus safety characterization and viral safety validation study of a pasteurized therapeutic concentrate of antithrombin iii obtained through affinity chromatography note for guidance on production and quality control of animal immunoglobulins and immunsera for human use. european agency for the evaluation of medicinal products (emea) annex : guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products avoiding viral contamination in biotechnological and pharmaceutical processes virus inactivation during production of intravenous immunoglobulin canine coronavirus inactivation with physical and chemical agents inactivation of lipid-enveloped viruses in proteins by caprylate viral safety characteristics of flebogammav r dif, a new pasteurized, solventdetergent treated and planova nm nanofiltered intravenous immunoglobulin partitioning and inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization in the manufacturing process of horse immunoglobulins enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates effect of pepsin digestion on the antivenom activity of equine immunoglobulins inactivation and elimination of viruses during preparation of human intravenous immunoglobulin antimicrobial biocides in the healthcare environment: efficacy, usage, policies, and perceived problems inactivation of enveloped and non-enveloped viruses on seeded human tissues by gamma irradiation. cell tissue bank inactivation of viruses by benzalkonium chloride effect of visible light on canine distemper virus rapid inactivation of rotaviruses by exposure to acid buffer or acidic gastric juice morphological studies of a canine adenovirus the effect of temperature on the hemagglutinin activity of the canine adenovirus (infectious canine hepatitis virus) the effect of ion concentration and ph on the thermal stability of a canine adenovirus viral safety of nanogam, a new nm-filtered liquid immunoglobulin product comparison of the inactivation of canine and bovine parvovirus by freeze-drying and dry-heat treatment in two high purity factor viii concentrates chromatographic removal combined with heat, acid and chaotropic inactivation of four model viruses chemical inactivation of viruses bovine viral diarrhea virus as a surrogate model of hepatitis c virus for the evaluation of antiviral agents the virucidal spectrum of a high concentration alcohol mixture securing viral safety for plasma derivatives inactivation of equine infectious anemia virus by chemical disinfectants we thank the world health organization, instituto butantan, and universidade de sao paulo for all the efforts directed to this work. instituto butantan and who supported this work. key: cord- -ap wfjhq authors: lewis, toby c.; henderson, tiffany a.; carpenter, ashley r.; ramirez, ixsy a.; mchenry, christina l.; goldsmith, adam m.; ren, xiaodan; mentz, graciela b.; mukherjee, bhramar; robins, thomas g.; joiner, terence a.; mohammad, layla s.; nguyen, emily r.; burns, mark a.; burke, david t.; hershenson, marc b. title: nasal cytokine responses to natural colds in asthmatic children date: - - journal: clinical & experimental allergy doi: . /cea. sha: doc_id: cord_uid: ap wfjhq background: the mechanisms by which viruses induce asthma exacerbations are not well-understood. objective: we characterized fluctuations in nasal aspirate cytokines during naturally-occurring respiratory viral infections in children with asthma. methods: sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. the presence of viral infection was ascertained by multiplex pcr. cytokines were measured by multiplex immune assay. mrna expression for selected markers of viral infection was measured by rt-pcr. a cumulative respiratory symptom score was calculated for each day of measurement. generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score. results: the patients completed a total of weeks of assessment ( “well” weeks; self-assessed “sick” weeks). viral infections were detected in three of the “well” weeks and of the “sick” weeks ( rhinovirus, coronavirus, influenza a, influenza b, respiratory syncytial virus, parainfluenza). compared to virus-negative well weeks, nasal aspirate ifn-γ, cxcl /il- , cxcl /ip- , ccl /rantes, ccl /eotaxin- , ccl /mcp- , ccl /mip- β, ccl /mcp- and ccl /mip α protein levels increased during virus-positive sick weeks. only a subset of cytokines (ifn-γ, cxcl , ccl , ccl , ccl and ccl ) correlated with self-reported respiratory tract symptoms. while many aspirates were dilute and showed no mrna signal, viral infection significantly increased the number of samples that were positive for ifn-λ , ifn-λ / , tlr , rig-i and irf mrna. conclusions & clinical relevance: we conclude that, in children with asthma, naturally-occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, ifns and ifn-responsive genes. epidemiological studies have uncovered a strong association between viral infections, especially those caused by rhinovirus, and exacerbations of asthma attacks [ ] [ ] [ ] . however, the mechanisms by which viruses induce exacerbations of chronic airways disease are not well understood. in response to viral infection, the airway epithelium, and to a lesser extent immune cells such as monocytes, macrophages, and dendritic cells [ ] [ ] [ ] [ ] , release pro-inflammatory cytokines, interferons (ifns), and antimicrobial substances, which in turn, promote clearance of microorganisms, and activation of the adaptive immune system. however, in patients with chronic respiratory illnesses, the innate immune response may also be responsible for disease exacerbation [ ] . although the inflammatory response to experimental rhinovirus infection has been monitored [ ] [ ] [ ] [ ] [ ] [ ] , few studies have examined the innate immune response of patients with asthma to natural colds. increases in respiratory tract cxcl /il- , ccl /rantes, ccl / mip- a, il- , ccl /mcp- , and ccl /mcp- have been detected [ ] [ ] [ ] [ ] . to further examine the innate immune response to viral infection in children with asthma, we measured nasal aspirate cytokine levels in asthmatic children before and after upper respiratory tract infections. in contrast to the previous studies, we examined at least three time points before and in association with symptomatic illness. we evaluated the associations between viral infection and cytokine expression, and between cytokine expression and symptom score. we also examined the effects of upper respiratory tract infection on mrna levels of selected markers of viral infection, including ifns. finally, we evaluated a new method of virus detection using a single polymerase chain reaction-ligation detection reaction (pcr-ldr) multiplex assay. we hypothesized that respiratory viral infection of children with asthma causes robust elaboration of pro-inflammatory cytokines and ifns, and that the level of pro-inflammatory cytokines correlates with symptom severity. we conducted an observational cohort study of children with asthma. children were eligible for the study if they received care from a university of michigan physician for asthma, were aged - years, and lived within a mile radius of ann arbor. outpatients were enrolled for - months, with the goal of assessing cytokines at baseline and in association with at least one viral-induced exacerbation during that period. initial consent and first evaluation was conducted at the university of michigan, but all subsequent evaluations were performed in the participant's home. this study was approved by the university of michigan institutional review board (approval number hum ). all clinical investigations were conducted according to principles expressed in the declaration of helsinki (http://www.wma.net). at the initial assessment, parents or guardians completed an extensive questionnaire, which included standardized questions about presence and frequency of asthma symptoms, an inventory of a child's asthma medications and current usage, and queries about environmental exposures, and child and family demographic information. baseline asthma severity was assessed using national asthma education and prevention program guidelines [ ] incorporating an adjustment for asthma controller therapy use [ ] . we performed home measurements of respiratory symptoms and collected nasal secretions (for detection of viral rna by pcr and host biomarkers by pcr and elisa) on days during a week when children were healthy (not reporting upper respiratory tract infection or asthma symptoms), and again during a week when they experienced cold or flu-like symptoms. families were given a calendar and a simple respiratory symptom scale and asked to mark the level of their symptoms. we used a modified version of the respiratory symptom score developed by the child origins of asthma study [ ] , which assessed fever, cough, nasal symptoms, wheezing, difficulty breathing, waking up at night with cough, and interference with usual activities. when the patient experienced a symptomatic respiratory illness, as defined by a symptom score of two or higher on a scale of - , the family notified the study staff and scheduled a visit within h of the beginning of symptoms (defined as day of the illness). information regarding impact of respiratory symptoms on daily activities and recent use of asthma medicines was also collected. as noted above, study technicians visited the home every - days to retrieve additional nasal washing samples, for a total of three visits during the week following reporting of symptoms. the three specific days of the week selected for analysis were based on the convenience of the subjects and laboratory technician. although specimens were most often collected on days , , and of a given week, specimen collection was sometimes compressed into a or -day period. to collect a nasal lavage sample, we utilized a protocol developed by powell and shorr [ ] and modified by james gern (university of wisconsin, personal communication). two squirts of isotonic . % sodium chloride were instilled into the child's nostrils (estimated at < ml per nostril) using a commercially available nasal saline spray (b.f. ascher, lenexa, ks, usa). the subject then blew their nose into a zippered plastic bag, and ml of m rt viral transport medium (remel, lenexa, ks, usa) was added. in general, samples were collected in the presence of study staff and were transported from homes to the laboratory within h of collection in a cooler with ice packs. however, due to logistical issues scheduling home visits, families were also provided with kits for independent collection of nasal washings specimens at home. if a visit could not be scheduled within h, families were instructed to collect the sample, double bag the sample, place in a tightly sealed collection box, and to keep it in the refrigerator until the staff could collect the sample. we required a week interval between sample collections for sequential illnesses. nasal aspirates were homogenized using a handheld homogenizer to allow pipetting of viscous samples and frozen at À °c to allow for batched analysis. viral rna and dna were extracted using a minelute kit (qiagen, valencia, ca, usa). samples were assessed for the presence of viral rna or dna via pcr using the seegene seeplex rv- detection kit (seegene, rockville, md, usa). this kit detects human parainfluenza viruses , , and , human metapneumovirus, human coronavirus e/nl , and oc , human adenovirus, influenza viruses a and b, human respiratory syncytial virus (rsv) a and b, and human rhinovirus a. we also analysed specimens for respiratory viruses using a novel polymerase chain pcr-ldr multiplex assay [ ] . the original system, which was designed to detect influenza viruses, was expanded to include parainfluenza , , , a and b, coronaviruses e and oc , influenza a and b, rhinoviruses a, b, and c, adenoviruses a-e, metapneumovirus, and rsv a and b. we have detected all the viruses included in this multiplex system in clinical samples (i.e. nasal aspirates), with the exception of the adenoviruses, for which we used laboratory positive controls. the viral sequences for the multiplex assay are listed in table . protein levels of ifn-c, cxcl /il- , cxcl /ip- , ccl /rantes, ccl /eotaxin- , ccl /mcp- , ccl / mip- b, ccl /mcp- , ccl /mip- b, and ccl /mip- a were determined using a magnetic bead-based multiplex immune assay (bio-rad, hercules, ca, usa). our interest on the above cc chemokines was prompted by gene array results from rv b-infected, ovalbumin-sensitized, and -challenged mice (data not shown). ifn-a and ifn-b levels were measured using standard elisa (r&d systems, minneapolis, mn, usa). aspirates were homogenized and mrna extracted as described above, using the rneasy or the rneasyplus kit (qiagen). mrna was analysed for cxcl /il- , cxcl / ip , ccl /rantes, ccl /eotaxin, ifn-k , ifn-k / , ifn-a, ifn-b, icam- , tlr , mda- , rig-i, and irf using quantitative real time pcr using specific primers and probes. signals were normalized to gapdh. we assessed feno in exhaled breath on days during a week when the child was well, and on at least days during a viral infection. to measure feno, we employed the niox mino (aerocrine, new providence, nj, usa). because feno measurement can be influenced by diet and exercise, participants were asked to refrain from eating or drinking within h of their exhaled breath assessment. triplicate samples were obtained to assess reliability. mean, standard deviation and interquartile range were used to describe measured values of nasal cytokine levels, nasal mrna, and symptom score before and during viral illnesses. box plots were used to represent the change in biomarkers at different points of the cold relative to the baseline. whiskers of the boxplots represent the minimum and maximum values. distributions of these continuous outcome variables were examined and appropriate transformations taken to achieve normality. the effects of viral illness, as defined by the seegene kit results, on biomarker protein levels, and of biomarker level on symptom score were determined using a generalized mixed models (proc mixed), with an auto-regressive correlation structure using sas statistical software (sas, cary, nc, usa). we evaluated and adjusted for relevant covariates such as age, gender, ethnicity/race, and nasal steroid use. other potential explanatory variables, such as family income, baseline asthma severity, tobacco smoke exposure, and oral antihistamine use were evaluated, but not included in final models as they were not significant predictors. for all mrna outcomes except ifn-b, levels were undetectable in a large number of samples. we therefore categorized our results into detectable/non-detectable levels, and analysed as a binary variable using the generalized estimating equation (gee) approach with the tobit link [ ] . we analysed ifn-b mrna levels as a continuous variable using the gee approach with identity link. unadjusted models are shown. sixteen children with physician-diagnosed asthma were recruited. participants ranged in age from to years old, were % boys, % non-white, and reported symptoms or medication use consistent with persistent asthma ( table ) . participants completed a total of weeks of assessment ( table ). the current report focuses on the weeks where there was concordance between self- reported illness and viral detection using the seegene kit ( virus-positive 'sick' weeks, and virus-negative 'well' weeks). we re-tested eight positive and seven negative samples (by seeplex) using the pcr-ligation detection multiplex assay. twelve of samples were concordant between the two assays. two samples were positive by seeplex and negative by pcr-ldr. one sample showed rsv by seeplex assay and coronavirus oc by pcr-ldr. symptom scores, which qualitatively evaluated the severity of illness, rose during the sick weeks (fig. a) . respiratory symptoms were highest at the first assessment during sick weeks, - days after initial report of illness. children with confirmed viral illnesses experienced significant increases in cough, wheeze, and chest tightness, indicative of asthma exacerbation (table ) . selected nasal aspirate cytokines were measured using magnetic bead-based multiplex immune assay. there were significant increases in all cytokines examined during weeks of reported symptoms, with the single exception of ccl (table , fig. b) . this pattern was more striking when samples with documented viral infections were compared to those confirmed to be virus-negative. unadjusted estimates of the association of confirmed viral infection and cytokine level were highly significant (not shown) and did not change appreciably when adjusted for age, gender, race, and nasal steroid use. changes in cytokines over the course of the illness are shown in fig. . in most cases, cytokines increased during the first days of infection and persisted throughout the week. we also examined the relationship between cytokines and symptom score. unadjusted estimates of the association of confirmed viral infection and cytokine level with symptom score were significant for ifn-c, cxcl , ccl , ccl , ccl , and ccl (table ) . cxcl , ccl , ccl , and ccl cytokine levels did not correlate with symptom score. we also attempted to measure ifn-a and ifn-b protein levels in the nasal aspirates by elisa. ifn-a levels over the lower detection limit ( . pg/ml) were found in only five of specimens (all virus-positive weeks). for ifn-b, levels over the detection limit ( pg/ml) were found in only samples (five virus-negative, seven virus-positive). many aspirates were dilute and a number of specimens showed no mrna signal (table ) . nevertheless, we detected transcripts for icam- , cxcl- , ifn-k , cxcl- , rig-i, mda- , tlr , ifnk / , irf , ifn-a, and ifn-b from patients both before and during viral infections. we could not detect ccl and ccl mrna in any specimens. finally, infection significantly increased the number of samples that were positive for cxcl , ifn-k , ifn-k / , tlr , rig-i, and irf mrna. we measured feno before and during virus-positive sick weeks (fig. ) . there was no significant change in feno measurement with viral infection. viral infections are the most frequent cause of asthma attacks [ ] [ ] [ ] . in theory, chemokine production by virus-infected epithelial cells induces the recruitment of inflammatory cells to the airways, which in turn elaborate cytokines and mediators capable of increasing airway responses. data suggest that immune cells may also be infected by respiratory viruses and produce chemokines [ , [ ] [ ] [ ] [ ] [ ] [ ] . in the present study, we found that, during natural colds, respiratory tract cytokine levels significantly increase in children with asthma. levels of a subset of cytokines correlated with the degree and time course of respiratory symptoms. the chemokines detected are responsible for recruitment of a broad array of inflammatory cells including neutrophils, monocytes, macrophages, and eosinophils. finally, we detected changes in nasal lavage specimens, which were collected at home on a repeated basis, both before and in association with infection. although we did not measure cytokine levels in normal subjects, these data show that children with asthma are apparently capable of a robust cytokine response to viral infection, even during mild exacerbations. in addition, home collection of nasal lavage specimens appears to be a practical tool for studying the natural history of viral infection in children. our results expand upon prior work characterizing cytokine responses detectable in respiratory secretions of asthmatics in association with viral illnesses. pizzichini et al. [ ] found that, compared to days after infection, sputum levels of cxcl , eosinophil cationic protein and fibrinogen from six adult asthmatics were significantly elevated days after the start of a coldinduced exacerbation. furthermore, cxcl levels correlated with the number of sputum neutrophils. teran et al. asthma [ ] found that, compared to asymptomatic periods, levels of major basic protein, ccl and ccl were increased in the nasal aspirates of children during acute viral-induced exacerbations. grissell et al. asthma [ ] found that compared to a group of stable asthmatics, levels of ccl and il- were significantly elevated in the induced sputa of older children and adults suffering from acute viral-induced asthma exacerbations, along with neutrophils and eosinophils. cxcl and ccl were also increased, although not significantly. finally, santiago et al. [ ] found that compared to control samples, ccl and ccl were increased in the nasal aspirates of asthmatic children - h after upper respiratory tract infections. chemokine levels correlated with macrophages in the nasal aspirate and upper respiratory symptoms scores. in the present study, in which patients were used as their own controls, we confirmed the above changes in cxcl , ccl , ccl , and ccl , and provide new data showing significant increases in cxcl , ccl , ccl , and ccl . importantly, we found that increases in these chemokines were sustained throughout the week following viral infection. we also examined the relationship between cytokines and respiratory symptom score. interestingly, we found that some cytokines correlated with symptom score (ifn-c, cxcl , ccl , ccl , ccl , and ccl ) whereas others did not (cxcl , ccl , ccl or ccl ). although we did not examine lower airway cytokine levels, it is tempting to speculate that the former set of cytokines is responsible for the acute asthmatic response to viral infection, whereas the latter set may modulate future immune responses. based on differences in ifn-b and ifn-k production between cells isolated from controls and asthmatic subjects [ ] [ ] [ ] , it has been proposed that patients with asthma are prone to rhinovirus-induced exacerbations due to deficient ifn production on the other hand, other in vitro studies showed no differences in rhinovirus-induced gene expression in epithelial cells isolated from asthmatic and healthy subjects [ , ] , and con-trol and asthmatic subjects experimentally infected with rhinovirus show no difference in respiratory tract viral titre or copy number [ ] . we found that viral infection during natural colds significantly increased the levels of ifn-c protein and ifn-k mrna in the nasal aspirates. viral infection also significantly increased the expression level of rig-i and irf- , each of which are inducible by type i ifns [ , ] . together, these data suggest that asthmatic children are capable of a functional ifn response. on the other hand, we failed to detect ifn-a or ifn-b protein in nasal aspirates, and there was no significant change in mrna levels during natural colds. detection of type i ifns in respiratory secretions is made difficult by the low sensitivity of commercially available assays, physiologically low levels of these cytokines in biological fluids (particularly in fluids without sufficient numbers of plasmacytoid dendritic cells) and presence of natural inhibitors (e.g. soluble receptors). also, we could not compare the results from our experimental subjects to children without asthma. our finding that irf mrna is elevated after respiratory viral infection in children with asthma is validated by a recent study analysing patterns of gene expression in nasal lavage samples from children experiencing picornavirus-induced asthma exacerbations [ ] . consistent with our previous work examining the requirement of irf for rhinovirus-induced gene expression in cultured airway epithelial cells [ ] , coexpression analysis demonstrated irf to be a major hub connecting ifn-mediated responses in virus-induced asthma exacerbations. in this study, we piloted a new pcr-ldr-based method for respiratory virus detection. twelve of samples were concordant between the two viral detection techniques; in two cases, samples were positive by seeplex and negative by pcr-ldr. the precise cause of the discrepancy between the two tests is not certain, but the seeplex test, which relies on visual detection of bands on agarose electrophoresis gels, may be liable to false positives. we also optimized our pcr-ldr test for maximum specificity, with the goal of avoiding false positives. this may have resulted in a reduction in sensitivity if copy number of viral nucleic acid was low. we measured the effect of natural colds on feno. previous studies have shown feno to be a reliable measure of eosinophilic airway inflammation, steroid responsiveness, and clinical control in children and adults with allergic asthma [ ] [ ] [ ] [ ] . feno levels have also been shown to increase during emergency steroid treatment of acute asthma exacerbations [ , ] . in contrast, we found that despite increases in airway cytokines and respiratory symptoms, there was no increase in feno during natural colds in children relative to their baseline state. these data are consistent with the notion that airway inflammation in the context of viral-induced asthma exacerbations is different in character than that associated with chronic asthma. there are several limitations to our study. first, although nasal sampling has the advantage of increased safety and decreased participant burden, sputum speci-mens more closely reflect the response of the lower airways to viral infection. however, as noted above, levels of ifn-c, cxcl , ccl , ccl , ccl , and ccl significantly correlated with respiratory symptoms, including lower respiratory tract symptoms such as cough, wheeze, chest tightness, and shortness of breath, each of which are indicative of asthma exacerbation. in addition, previous studies have found similar changes in nasal aspirate and sputum cytokine concentrations following viral exacerbations of asthma [ , ] . finally, observations demonstrate consistent effects of nasal and bronchial provocation [ ] . second, we did not measure inflammatory cells in the nasal aspirates. we therefore could not assess the functional effects of chemokine release in our subjects. previous studies have shown recruitment of neutrophils, eosinophils, and macrophages to the respiratory tract following rhinoviral infection of asthmatic subjects [ , , , , [ ] [ ] [ ] [ ] . also, we cannot determine the cellular source of the mrnas measured in our study. pilot analysis showed primarily epithelial cells and neutrophils in the nasal aspirates, suggesting that epithelial cells were the primary source of mrna. third, as noted above, we did not compare responses of asthmatic subjects to controls. a recent study showed that asthmatics and non-atopic subjects experience similar colds, including chemokine responses, following experimental rhinovirus infection [ ] . fourth, although we were able to detect significant differences in many cytokines following upper respiratory tract infection, a fraction of the samples were too dilute to detect mrna. (there may have been other reasons for the loss of rna, for example sample ribonucleases). dilution may also affect the concentration of measured biomarkers. we did not adjust measurements by total protein content, as vascular leakage due to inflammation may alter protein concentrations in children with upper respiratory infections [ ] . in our experience, total protein is difficult to measure in many nasal (and tracheal) aspirate specimens, even when cytokines are readily detectable, and therefore normalization for total protein does not increase the reliability of the concentrations. finally, studying naturally occurring colds in children using home-based methods inherently involves some variability in sample timing and handling that is not present in the controlled environment of the laboratory. yet this approach also offers an opportunity to observe 'real world' scenarios. our ability to detect statistically significant changes in specific mrnas and proteins using the current study design suggests that this is a viable approach for use in future investigations, for example, studies examining whether an early adjustment of asthma medications could abort or temper the impact of respiratory viral infections. we conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, ifns, and ifn-responsive genes. cytokine responses are sustained the first week following viral infection. future analyses of controls and asthmatics which combine the determination of cytokine responses and asthma health outcomes will provide further insight into the cellular pathways responsible for virus-induced asthma exacerbations. respiratory viruses and exacerbations of asthma in adults community study of role of viral infections in exacerbations of asthma in - 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hhi wps authors: sarid, ronit title: investigating an emerging virus during a sudden pandemic outbreak date: - - journal: rambam maimonides med j doi: . /rmmj. sha: doc_id: cord_uid: hhi wps at the time of writing, in july , the recently emerging sars-cov- pandemic has attracted major attention to viral diseases, in particular coronaviruses. in spite of alarming molecular evidence, documentation of interspecies transmission in livestock, and the emergence of two new and relatively virulent human coronaviruses within a -year period, many gaps remain in the study and understanding of this family of viruses. this paper provides an overview of our knowledge regarding the coronavirus family, while highlighting their key biological properties in the context of our overall understanding of viral diseases. in march , bill gates, the cofounder of microsoft, gave a ted presentation in which he stated that while many countries are investing in developing nuclear defense measures, they should be investing similar efforts in developing protection measures against deadly virus pandemics. he referred to the ebola virus outbreak, which killed more than , people in west africa, and warned that the usa and other countries were not ready for the future epidemic that could be even more devastating. "if anything kills over million people in the next few decades, it's most likely to be a highly infectious virus rather than a war," gates said. gates repeatedly urged governments to prepare for pandemics in a manner similar to their preparations for war. five years later, in , when the world health organization declared the coronavirus disease (covid- )-caused by the newly emerging sars-cov- virus-to be a pandemic, this talk was widely acknowledged to be almost prophetic. unfortunately, society, governments, public health officials, pharmaceutical companies, and researchers were not properly prepared for this outbreak. concerns about the potential emergence of an influenza virus pandemic have been raised and extensively discussed over many years. these concerns were based on the destructive spanish pandemic flu, as well as on subsequent influenza virus outbreaks, the most recent of which emerged in mexico in . little attention was paid to coronaviruses, which were temporarily in the spotlight when the severe acute respiratory syndrome (sars) disease emerged in china in , [ ] [ ] [ ] and later when the middle east respiratory syndrome (mers) appeared in saudi arabia in . , these outbreaks attracted some research interest to coronaviruses, yet large pharmaceutical companies did not devote substantial resources to these diseases, because therapies and vaccines for these diseases did not appear likely to be profitable. nevertheless, a clear potential threat by coronaviruses was recognized by a number of investigators. [ ] [ ] [ ] [ ] this was based on the wide range of viral reservoirs and previous documentation of cross-species spread in veterinary medicine, as well as knowledge of their replication mechanism. our understanding of other viruses, in particular regarding coronaviruses, can provide guidance for understanding and control of the sars-cov- new outbreak. yet, many gaps remain in the study and understanding of these viruses. this paper will discuss recent emerging evidence on the sars-cov- virus in the context of our general understanding of viral diseases. covid- is caused by a newly identified virus, sars-cov- , belonging to the coronavirinae subfamily in the coronaviridae family. based on phylogenetic relationship and genomic organization, this subfamily has four different genera-alpha, beta, gamma, and delta-of which alpha-and betacoronaviruses have mammalian hosts, while the reservoir of the gamma-and deltacoronaviruses is mainly birds. sequencing of the sars-cov- genome was accomplished soon after its discovery, promoting its classification to the betacoronavirus and subsequent attribution of certain basic properties that can permit some basic assumptions. coronaviruses (covs) are the largest known rna viruses, encoding - kb positive-sense singlestranded rna with a ′-terminal cap structure and a ′-polyadenylated tail. virions of coronaviruses have a mean diameter of nm, while their most notable feature is a halo of spikes that project from the envelope and give the viral particle the shape of a solar corona, inspiring the name of this family of viruses. four major structural proteins form the coronavirus particles: the nucleocapsid (n) phosphoprotein, which forms a helical ribonucleoprotein core together with the viral genome; the trimeric spike (s) glycoprotein, which mediates virion attachment to host cell receptor and membrane fusion; the membrane (m) protein, which is highly abundant and shapes the virion envelope; and the low-abundance integral envelope (e) protein, which plays an important role in viral envelope assembly. certain coronaviruses also include the hemagglutinin-esterase (he) protein, which forms short protrusions beneath the s protein spikes. all structural genes, except the he, are encoded in the ′-most third of the genome, while the other two-thirds encode the viral non-structural proteins (nsps), such as replication enzymes and additional accessory genes. two partially overlapping open reading frames, orf a and orf b, are encoded in this region, producing the orf a polypeptide or being continuously translated via ribosome frameshifting to produce a large orf ab polyprotein. these polyproteins are cleaved by virus-encoded proteinases into a number of mature nsps. as cleavage of the polypeptide is essential for production of functional viral proteins, drugs that target these proteinases have potential therapeutic applications. the assembled nsp replicase-transcriptase complex (rtc) is responsible for rna replication and transcription. of note, the coronaviruses encode several viral proteins with rna-modifying or replication activities, including rna-dependent rna-polymerase (rdrp), a helicase, an exonuclease (exon), and ′-o-ribosemethyltransferase. replication of the cov rna genome takes place on cellular membranes while generating dramatic changes in their organization. replication involves the synthesis of a full-length negative-strand rna, which serves as a template for the synthesis of additional positive-sense rna genomes. the 'most open reading frames, orf a and orf ab, are translated from full-length genomic rna, while a series of subgenomic (sg) mrnas, which commonly include the - -nucleotide untranslated leader sequence from the ′ end, and extend to the ′ end of the rna genome, are produced to enable translation of downstream genes. production of these mrnas involves discontinuous transcription of the full-length positive-strand rna genome. this process may encompass switching between templates and can therefore result in homologous or hetero-logous recombination when more than one cov virion infects a given cell. this capacity, along with the high theoretical mutation rate of the rna genome by rdrp ( nucleotide substitutions/site/ year) can potentially enable rapid genetic changes in coronaviruses. accordingly, long-term coronavirus infection in some animals, combined with high mutation and recombination rates, increases the probability that a virus mutant with an extended host range might arise. , , nevertheless, a proofreading function during cov replication is provided by the viral protein exon and may balance between fidelity and misincorporation rate that could result in lethal mutagenesis. exon inhibitors are expected to reduce the fidelity of cov replication and thus promote high-level mutagenesis of the viral genome that could drive virus extinction. [ ] [ ] [ ] before the sars epidemics, covs were predominantly associated with devastating outbreaks of severe diseases in livestock, mostly gastroenteritis and bronchitis, affecting domestic animals including cattle, pigs, and chickens. coronaviruses also infect a variety of wild animals including birds, snakes, mice, and bats. human cov (hcov) infection (table ) was first described in the s and was primarily associated with mild upper respiratory diseases (the "common cold") caused by hcov- e and hcov-oc . [ ] [ ] [ ] two other hcovs were identified almost years later: hcov-hku , which was linked with chronic pulmonary disease, and hcov-nl , which was associated with both upper and lower respiratory tract disease in children and adults. in addition, hcov-nl was associated with croup in infants and young children. interestingly, association of hcov-nl with kawasaki disease, an inflammation of blood vessels that mostly occurs in children, has been reported, yet subsequently failed to be confirmed. , all four reportedly mild pathogenic coronaviruses are associated with %- % of cases of the common cold, - yet they have the potential to cause severe lower respiratory tract infection in infants, in the elderly, and in patients with other underlying illness, while hcov-oc , like sars-cov- , has been associated with neurologic dysfunction as well. interestingly, sequence and phylogenetic analysis of hcov-oc dates its transmission from cattle into the human population to around , concurrently with a pandemic which killed approximately million people worldwide and was traditionally ascribed to influenza virus. the three other hcovs can infect the lower respiratory tract and cause a severe acute respiratory syndrome (sars). the sars-cov was identified with the emergence in of an atypical pneumonia that resulted in a % mortality rate, and mers-cov subsequently emerged in and caused % mortality. both hcov- e and hcov-nl are alphacoronaviruses, while all other hcovs belong to the betacoronavirus genus. all known hcovs originated from animals, with rodents or bats being the most likely ancestors, and passed to humans either directly or through an intermediate host. , other examples of transmission across species barriers occurred with hcov-oc , which crossed species to infect dogs, and were suggested for sars-cov- , which can infect ferrets and cats. the occurrence of pathogenic hcov outbreaks with a zoonotic origin has led to the search for animal reservoirs and the identification of a multitude of previously unknown coronaviruses, mostly in birds and bats, that have the potential to cross species. this reinforces the need to monitor animals to gain control over the emergence of these viruses. an important parameter towards understanding any newly emerging virus concerns virus particle attachment to host cells. attachment determines virus host range and may help in understanding its pathogenesis. attachment of covs to the host cell receptor is mediated by the surface transmembrane s glycoprotein, which may also induce cell-to-cell fusion. the s protein is synthesized as a single polypeptide that is cleaved by cellular proteases into two polypeptides, s and s . this cleavage may take place during virion assembly and release from the infected cell or during initiation of infection. the s protein, composed of the s protein amino terminal domain, forms the tip of the spike, and contains the receptor-binding domain (rbd) of most covs. of note, s was suggested to be a modular protein that can exchange domains and thus provides a low barrier for selection of new coronaviruses that were either generated by mutations or recombination. the host protein, angiotensin-converting enzyme (ace ) was found to function as a critical determinant for hcov-nl , sars-cov, mers-cov- , and sars-cov- cellular tropism. like other viral receptors ace is a cellular molecule which has an important physiologic function. decoding the molecular and structural bases for receptor usage is crucial for understanding crossspecies transmission but may also suggest potential therapeutic strategies. development of animal models for sars-cov- infection is vital in providing comprehensive understanding of the pathogenic mechanisms involved but may also serve for screening anti-viral drugs and vaccines. accurate large-scale diagnostic testing is critically important in the control of a pandemic outbreak. it is especially important for a novel virus with no specific treatment and no vaccine. identification of infection among hospitalized patients is of high priority to prevent nosocomial infection and subsequent community transmission. ideal diagnostic tests are highly specific and sensitive, avoiding false-positive and false-negative outcomes, respectively. viral infections can be diagnosed through the detection of viral antigens or genomes by various methods, of which the most widely used is based on amplification of viral genomic sequences. indeed, sars-cov- testing is based on respiratory specimens, either oropharyngeal, nasopharyngeal, or both, that undergo rna extraction followed by reversetranscriptase polymerase chain reaction (rt-pcr). several assays have been optimized to have high sensitivity and minimal cross-reactivity with circulating viruses present in the community. these tests can diagnose or rule out covid- in suspected patients and may also track the infection in those who were in contact with infected individuals. scaling up these tests to expand testing capacity has been a major goal during the covid- outbreak. other nucleic acid-based diagnostics, such as nucleic acid sequencing, were established and are commercially available. of note, inadequate sample collection may yield false negative tests. early implementation of sars-cov- diagnostics for the identification and subsequent isolation of infected individuals has been helpful in control of the outbreak. however, different implementation strategies and indications for diagnostic testing were chosen in different countries depending on public health policies, testing capacity, and the morbidity rate. an alternative to nucleic acid detection is to identify viral antigens, in particular those that are highly abundant, such as the sars-cov- nucleocapsid protein. such assays, which are generally rapid and based on immunoassay, are under development for sars-cov- . finally, serologic tests to identify a virus-specific immune response, including igm and igg antibodies, have potential diagnostic implications. the antibody response to infection takes days to weeks to be detectable. therefore, these tests cannot be used to detect an acute infection and will generally also be negative during the first days post infection. however, accurate serologic assays enable rapid screening and evaluation of the proportion of infected individuals within a given population, herd immunity, and epidemiologic assessments. furthermore, quantitative serologic assays combined with virus neutralization assays could help define the long-term effectiveness of the anti-viral immune response. several immunoassays have been developed for sars-cov- , though their accuracy remains to be fully validated. epidemiological investigations have played major roles in mitigating covid- . in the context of an emerging virus, epidemiologists gather data regarding new cases, including their timing, location, origin, and outcome. the data are based on diagnosed cases that present symptoms, as well as on laboratory-confirmed cases of infection, which could also be asymptomatic. potential animal reservoirs and sources of environmental contamination are also being evaluated. the collected data are incorporated into models that illustrate modes of transmission, such as droplet infection, and predict the infection spread. populations at high risk for infection and severe outcome, such as the elderly or individuals with chronic disease, can also be identified via epidemiologic investigation. among other parameters, the prediction of disease spread is based on the basic reproductive number (r ) representing the average number of people who will become infected directly by a single infected individual. one of the goals of epidemic containment is to restrain the transmission of the virus and reduce r , which is modulated by the time interval of viral shedding, viral infectiousness, and the interface that mediates transmission between infected and susceptible individuals. understanding how a virus spreads in a population, together with knowledge on the virus stability in the environment (e.g. aerosol, water, or different surfaces), helps develop guidelines for prevention and containment. sequence analysis of viral genomes may also help tracking virus dissemination within a population. epidemiology also allows determination of case fatality rate (cfr), although it is very difficult to determine this value during an ongoing outbreak, in particular when the number of asymptomatic infections is high. finally, research has shown that people become infectious before any symptoms of illness appear, during the pre-symptomatic incubation period, while others do not develop a disease and remain asymptomatic. asymptomatic individuals may be those in whom the virus replication is low and therefore their ability to spread the virus is limited. however, because disease symptoms are affected by the immune response, lack of symptoms may reflect a well-balanced immune response and does not necessarily correlate with high or low viral loads. either way, it is interesting to examine the relationship between the severity of the disease and the response of the innate, humoral and cellular immune systems over time. knowing the proportion of asymptomatic infections and whether asymptomatic individuals are contagious and can spread the virus are top public health objectives which may help define ways to avoid infections, such as wearing masks and social distancing. drug treatment for most viral diseases is limited, and relies primarily on symptomatic treatment and supportive care with no attempt to impair virus propagation. this approach rests on the notion that viruses are cellular parasites that employ cellular processes to reproduce, and hence any attempt to target their propagation is likely to be toxic to the host cells as well. therefore, few anti-viral drugs are available with a sufficient level of safety. in addition, anti-viral drugs must be highly effective, as incomplete inhibition of the virus infection may enable selection of drug-resistant virus strains that can cause an even greater threat. accordingly, most antiviral compounds target viral enzymes, such as proteases , and replication enzymes. , new anti-viral drugs, some of which are still under development, focus on blocking attachment of the virus to the host cell, preventing its entry into cells and virion uncoating, inhibiting regulatory functions, or targeting the viral rna. potential drugs with such activities can be identified by using purified viral proteins in high-throughput mechanism-based experimental screens. computational approaches, based on the atomic structure of the target molecule, enable virtual screening and design of new drugs. in addition, a comprehensive understanding of the molecular interactions between the virus and the host cell, together with whole-genome sequencing and methods to block gene expression, enables identification of cellular gene products that are essential for virus replication, and which can be used as targets for treatment without damaging the host. obviously, any new compound has to reach appropriate bioavailability and pharmacokinetics with no toxic effects. the success of developing a number of agents to treat or inhibit human immunodeficiency virus (hiv) infection, as well as anti-hepatitis c virus (hcv) medications to bring about complete cure, reinforces the belief that any viral disease can be effectively treated by specific, safe, and effective anti-viral drugs. however, unfortunately, at present there is no effective drug to treat sars-cov- infection or any other cov. the emergence of covid- highlights the need for novel drug discovery for the treatment cov infections, in particular sars-cov- . the use of a variety of strategies to detect potential inhibitors of sars-cov- infection is expected to identify a large panel of potential anti-viral compounds. for example, the rna-dependent rnapolymerase (rdrp) encoded in the genome of all coronaviruses is a promising candidate for targeting sars-cov- . given the conserved nature of rdrp among different coronaviruses, drugs that target this protein could constitute a pan-cov anti-viral drug. however, like with any new drug, development and evaluation processes take time. therefore, screening of previously approved drugs for anti-viral effects and repurposing them to treat the current pandemic may save time in the development process. finally, evaluation of the effectiveness of type i interferon combinations, which is known to induce cellular anti-viral responses and to enhance host immunity, in combination with other already known and new drugs, is warranted. vaccines against viral diseases have been in use for hundreds of years and have proven powerful in preventing infection. the objective of the vaccines is to generate an effective and safe immune response that will then protect from natural infection. vaccination may be mediated through passive immunization, which involves injection of components of the immune response, or through active immunization, which triggers an active host response. passive immunization provides immediate protection as it does not require a response by the patient, but it does not induce immune memory. in the context of an acute viral infection, such treatment involves administration of anti-viral antibodies that were either derived from the plasma of individuals who recovered from infection or synthetically designed. treatment can be provided during infection or prophylactically to protect individuals at high risk such as healthcare personnel. it can also be used for prevention following suspected exposure. transfusion of convalescent plasma was proven effective in treating several viral infections including the h n and h n strains of influenza virus and sars-cov, and treatment protocols were established for ebola virus , and mers-cov infections. accordingly, transfusion of convalescent plasma is likely to be beneficial to sars-cov- , , yet its effect on virus shedding and disease outcome must be evaluated when given to healthy individuals and patients at different stages and severity of the disease. importantly, diverse antibodies are produced following viral infection, yet only a fraction of these antibodies is capable of neutralizing the infection. accordingly, while convalescent plasma could be useful in the short term, it is preferable to identify the most potent antibodies to allow large-scale manufacturing and standardization. as the main target of coronaviruses neutralizing antibodies is the spike protein, samples from patients are screened to identify b cells that produce sars-cov- antibodies specifically directed at spike. the genes encoding these antibodies are then cloned, and the antigenbinding motif may be used for therapy. active vaccines employ attenuated live viruses, inactivated viruses, recombinant viruses, recombinant or purified viral proteins, or virion-like particles that mimic infectious virions. in all cases, the vaccine induces an immune response that should protect against future exposure to the virulent pathogen. when developing a vaccine, it is important to ensure that the vaccine is safe and does not cause major side effects or other unwanted effects that may, in some cases, even exacerbate infection. after the safety tests, vaccine efficacy, which measures how well it protects from disease, is tested. usually the first test experiments are done in animal models and only then in humans. they involve vaccination and subsequent evaluation of neutralizing antibody production and other potential immune responses. in certain cases, they involve challenge with the virus and long-term follow-up of vaccinated individuals as compared to control groups. the response to immunization must be evaluated in various populations including the elderly who are more vulnerable to infection but unfortunately mount lower responses to vaccines. an effective vaccine is one that induces an immune response that provides protection against infection and pathogen-related disease. ideally, a vaccine should provide life-long protection, yet short-term vaccines may also be useful, at least during an intermediate period until alternatives are available. a race to generate a sars-cov- vaccine is ongoing, involving dozens of candidates in the development pipeline. among others, new approaches involving rna and dna vaccines that enable production of viral antigens upon injection are also being evaluated. a universal coronavirus vaccine designed to target conserved viral epitopes, most likely in the spike proteins, may enable protection from a wide range of related viruses, including cov types that could emerge in the future. realistically, vaccination is a long-term goal that could take more than a year. in addition, development of a sars-cov- vaccine may prove difficult or even unfeasible as is the case for hiv and hcv, which, even after many years of research and huge development budget, are yet to have an effective and safe vaccine. furthermore, even once a safe and effective vaccine is devel-oped, billions of doses have to be manufactured and distributed worldwide. the urgent need to control the spread of the sars-cov- outbreak has brought together a large number of scientists and entrepreneurs to look for solutions that would delay and prevent infection. these solutions include active face masks embedded with anti-viral molecules or particles, fabrics containing anti-viral compounds, anti-viral coatings for surfaces, and various low-toxicity environmental disinfectants. at the same time, ventilators and supportive equipment for patients have been recently developed. artificial intelligence, big data, and machine learning have also been instrumental in tracking virus spread, understanding the epidemiology of the disease, monitoring patients, and preventing the spread of the virus, as well as identifying other clues that may help understand viral pathogenesis towards mitigating and combating covid- . finally, by combining large-scale knowledge, literature, transcriptome data including single-cell rna sequencing, data learning can help finding potential drug and vaccine targets. current management of covid- has been mainly dependent on the prevention of infection, including case diagnosis followed by epidemiologic investigation and quarantine, as well as supportive care. collaborations are key for fighting this new pandemic; it is important both for knowledge acquisition and innovative initiatives for prevention and treatment, but also essential in the modern, globally interconnected world. lessons learned from other viruses as well as newly collected data and its rapid dissemination, describing virus transmission, genetic evolution, receptor binding, immune response, and pathogenesis, can be combined towards solving the puzzling outbreak and disease. in the battle against the covid- outbreak, virology serves as a major axis around which a variety of other fields of expertise, such as epidemiology, medicine, immunology, mathematics, chemistry, computer and data science, move together. a key question is whether sars-cov- will stay with us and become endemic, like the influenza virus and other hcovs such as hcov-oc and hcov- e, or will eventually disappear like sars-cov. endemic viruses may acquire mutations that reduce their virulence, yet the opposite scenario, the emergence of a more dangerous strain, is also possible. therefore, constant monitoring of the population to examine the incidence of infection as well as the genetic shift of the virus is essential. furthermore, given the history of coronaviruses, they should be regarded as an ongoing threat to human health, as additional animal viruses of this family may undergo genetic changes enabling them to infect humans. whether infection with one virus family member confers a certain level of immunity to others, and how long the immunity to a given virus persists, remains to be defined. scientists need to know how the virus functions upon infection. this involves virus cultivation and propagation in cell culture, and development of appropriate cell/organ culture and animal models. laboratory studies should also describe the virus host range, which is primarily determined by the susceptibility or permissiveness of different cells. further basic study of the biology of these viruses is warranted as it may identify new targets for therapy. the next outbreak? we're not ready. ted presentation the influenza pandemic: years of questions answered and unanswered a novel coronavirus associated with severe acute respiratory syndrome the severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia crossref . decaro n, 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strain detected in italy susceptibility of ferrets, cats, dogs, and other domesticated animals to sarscoronavirus angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target diagnostic testing for severe acute respiratory syndromerelated coronavirus : a narrative review structure-based design of antiviral drug candidates targeting the sars-cov- main protease ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study sars-cov- rna dependent rna polymerase (rdrp) targeting: an in silico perspective coronaviruses -drug discovery and therapeutic options treatment with convalescent plasma for influenza a (h n ) infection convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h n ) virus infection evaluation of convalescent plasma for ebola virus disease in guinea administration of brincidofovir and convalescent plasma in a patient with ebola virus disease feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol treatment of critically ill patients with covid- with convalescent plasma convalescent plasma transfusion for the treatment of covid- : systematic review key: cord- -k x z h authors: abu-farha, mohamed; thanaraj, thangavel alphonse; qaddoumi, mohammad g.; hashem, anwar; abubaker, jehad; al-mulla, fahd title: the role of lipid metabolism in covid- virus infection and as a drug target date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: k x z h the current coronavirus disease or covid- pandemic has infected over two million people and resulted in the death of over one hundred thousand people at the time of writing this review. the disease is caused by severe acute respiratory syndrome coronavirus (sars-cov- ). even though multiple vaccines and treatments are under development so far, the disease is only slowing down under extreme social distancing measures that are difficult to maintain. sars-cov- is an enveloped virus that is surrounded by a lipid bilayer. lipids are fundamental cell components that play various biological roles ranging from being a structural building block to a signaling molecule as well as a central energy store. the role lipids play in viral infection involves the fusion of the viral membrane to the host cell, viral replication, and viral endocytosis and exocytosis. since lipids play a crucial function in the viral life cycle, we asked whether drugs targeting lipid metabolism, such as statins, can be utilized against sars-cov- and other viruses. in this review, we discuss the role of lipid metabolism in viral infection as well as the possibility of targeting lipid metabolism to interfere with the viral life cycle. the rapidly growing coronavirus disease (covid- ) pandemic represents a serious global challenge [ ] . the emergence of sars-cov- has been manifested as the third revelation of a highly pathogenic coronavirus into the human population after the severe acute respiratory syndrome coronavirus (sars-cov) in and the middle east respiratory syndrome coronavirus (mers-cov) in [ , ] . coronaviruses are a family of enveloped viruses with a large single-stranded positive-sense rna genome, named for their crown-like appearance under the electron microscope [ ] . the factors that influence the survival of such viruses on various surfaces depend on several factors such as the viral load, type of surface, suspension medium, humidity, temperature, and others [ ] . following covid- infection, sars-cov- can penetrate the mucous membranes of the nose, eye, and/or mouth and move the host lipid biogenesis pathways play crucial roles in controlling virus replication. lipids can act as direct receptors or entry co-factors for all types of viruses at the cell surface or the endosomes [ , ] . they also play an important role in the formation and function of the viral replication complex, as well as the generation of the energy required for efficient viral replication [ ] [ ] [ ] . moreover, lipids can regulate the appropriate cellular distribution of viral proteins, in addition to the assembly, trafficking, and release of viral particles [ , ] . coronaviruses firstly seize host cell intracellular membranes to create new compartments known as double-membrane vesicles (dmvs) needed for viral genome amplification. a specific phospholipid composition is required by different viruses to form the perfect replicative organelles that are suitable for their replication [ ] . dmvs are membranous structures that contain viral proteins and an array of confiscated host factors, which jointly orchestrate an exclusive lipid micro-environment ideal for coronavirus replication [ ] . a recent study indicated that cytosolic phospholipase a α enzyme (cpla α), a crucial lipid processing enzyme belonging to the phospholipase a superfamily, is critical for dmvs' formation and coronaviruses' replication [ ] . accumulation of viral proteins and rna, as well as creation of infectious virus progeny, were significantly reduced in the presence of cpla α inhibitor [ ] . similarly, increased expression of age-dependent phospholipase a group iid (pla g d), an enzyme that usually contributes to anti-inflammatory/pro-resolving lipid mediator expression, resulted in worsened outcomes in aged mice infected with sars-cov, suggesting that inhibition of such factor could represent a potential therapeutic option [ ] . however, to date, the change and tempering effects of specific lipids involved in lipid remodeling upon coronavirus infection stay largely unexplored. earlier reports have shown that most rna viruses such as rhinoviruses show an enhancement of glucose uptake and dependence on both extracellular glucose and glutamine for optimal viral replication [ ] . this glucose uptake enhancement and phosphoinositide -kinase (pi k)-regulated glucose transporter (glut ) upregulation were found to be pi k driven and reversible by pi k inhibitor. moreover, metabolomic studies revealed increased levels of metabolites associated with glycogenolysis, a process that has not been described so far in the context of viral infections. lipogenesis and nucleotide synthesis were found to be elevated as well. lack of both glutamine and glucose impaired high-titer rhinoviruses replication in cells, and the glycolysis inhibitor -deoxy-d-glucose ( -dg) effectively repressed viral replication and reversed the rhinoviruses-induced alterations of the host cell metabolome. these findings underscore the potential of metabolic pathways as a target for host-directed antiviral therapy [ ] . citrate, which is the main carbon source for fatty acid (f.a.) or cholesterol synthesis, can usually pass across the mitochondrial membrane and is cleaved into acetyl-coa that gets carboxylated by acetyl-coa carboxylase (acc) to yield malonyl-coa [ ] [ ] [ ] . on the other hand, f.a. synthase (fasn) catalyzes the production of palmitic acid (c : ) from cytosolic acetyl-coa and malonyl-coa. the palmitic acid can then be further processed and used in the synthesis of cell membranes, storage in lipid droplets, or the palmitoylation of host and viral proteins. as for sterol biosynthesis, two units of acetyl-coa are processed to make acetoacetyl-coa, which then enters the metabolic -hydroxy- -methyl-glutaryl-coa reductase (hmg-coa reductase) pathway for cholesterol production. moreover, f.a.s can be metabolized by catabolic beta-oxidation to yield high amounts of atp [ ] [ ] [ ] . cholesterol and f.a. are essential for viral replication as they constitute the main components of viral membranes. hence, the metabolism of these lipids has been shown to be required for the replication of various viruses [ ] . fasn enzyme is an important player in this process as it controls f.a. synthesis and can regulate the viral replication. some viruses can even harness this process by increasing the expression of fasn and enhancing their activity [ ] . as previously shown, the impact of viral infection on various lipid species has been dramatic. wu et al. showed that the effect of treatment on sars patients was long-lived and could significantly impact their serum metabolites. in their study, wu et al., recruited sars survivors years after their infection and compared them to healthy controls [ ] . they demonstrated that sars survivors were more susceptible to lung infections, tumors, cardiovascular disorders, and abnormal glucose metabolism compared to controls. more importantly, to this review, sars survivors had a higher level of phosphatidylinositol and lysophosphatidylinositol compared to uninfected controls. this increase in metabolites was believed to be caused by the high dose of steroid treatment using methylprednisolone [ ] . in another study, nguyen et al. explored changes in the lipidome of primary human epithelial cells infected with rhinoviruses [ ] . post-infection untargeted lipidomics analysis of infected cells showed time-dependent changes in multiple lipid pathways and in the length of fatty acid saturation and class. these pathways implicate multiple lipid modifying enzymes, which were increased upon infection including lysocardiolipin acyltransferase (lclat), phosphoinositide phosphatase (pip), and d.g. kinase amongst others that present good antiviral targets [ ] . other studies have also looked at changes in the lipidome profile after infection with viruses such as enterovirus a and coxsackievirus and found that lipid species/classes such as arachidonic acid, docosahexaenoic acid, and eicosapentaenoic acid were consistently upregulated after infection [ ] . these data highlight the increase in lipid metabolism that occurs after viral infection where the virus highjacks and utilizes the host lipid metabolism for their own propagation. endocytosis is the process of internalization of various materials into the cell. it is used to internalize fluids, cellular components as well as various solutes. this happens through the invagination of the plasma membrane and the internalization of various components into cells through membrane vesicles. viral entry is dependent on the attachment and fusion of viral membrane with plasma membrane through an endocytosis-mediated process [ , ] . of high relevance to this review and the ongoing covid- pandemic is the role of lipid rafts in viral entry into the host cells. lipid rafts are sphingolipid-, cholesterol-, and protein-rich microdomains of the cell membrane. lipid rafts have been found to be important to multiple viruses including sars-cov, especially during the early replication stage, although there was no increased angiotensin-converting enzyme (ace ) localization in such rafts [ ] . this could be compensated by the presence of caveolins, clathrins, and dynamin that are important for the endocytosis process and viral entry. this process facilitates the fusion as well as the release of the viral genome into the host cell, where many enveloped viruses, including coronaviruses, take advantage of the low ph inside the endosomes to facilitate the fusion and viral genome release [ ] . sars-cov- belongs to the betacoronavirus genus in the subgenus sarbecovirus, which includes sars-cov but not mers-cov, which belongs to merbecovirus subgenus [ ] . the genome structure of sars-cov- has a gene order of -replicase orf ab-s-envelope(e)-membrane(m)-n- , which is shared by other betacoronaviruses [ ] . the spike (s) protein is made of two functional subunits (s and s ) both of which are necessary for virus entry into host cells. while s contains the receptor binding domain (rbd) required for viral attachment to host cell receptors, s facilitates the fusion of the cell and viral membranes. the rbd of the s protein from sars-cov- is well-suited for binding to the human ace receptor, which is also used by the sars-cov albeit more efficiently in the former virus [ ] . for the fusion to take place, the s protein needs to be cleaved by cellular proteases to expose the fusion sequences [ , ] . the amino acid sequence of sars-cov- s protein, unlike other betacoronaviruses, contains a characteristic insertion of polybasic cleavage site (residues prra) for furin (alias pcsk ) at the junction of s and s subunits. furin is a proprotein convertase, which is a serine proteinase. proprotein convertases are involved in posttranslational processing of the precursors of a vast number of cellular proteins [ ] . it was recently reported that sars-cov- uses the serine protease tmprss for s protein priming and it is speculated that furin-mediated precleavage at the s /s site in infected cells might promote subsequent tmprss -dependent entry into target cells, as reported for mers-cov [ ] . the continuous emergence of new strains of coronaviruses such as sars-cov- constitutes a major challenge as global efforts are in a race with time to identify treatments and develop new vaccines. these are generally slow processes that require extensive research and development and ultimately delay the responses to such emerging epidemics. on the other hand, the development of broad-spectrum antiviral drugs could constitute an important first response to such epidemics and enhance the impact of such responses. pathways that are fundamental to viral attachment and fusion as well as pathways involved in the viral replication and assembly are the foundations for drug discovery. please see figure for a summary of current anti-viral drugs that are mentioned in this article [ ] . vaccines. these are generally slow processes that require extensive research and development and ultimately delay the responses to such emerging epidemics. on the other hand, the development of broad-spectrum antiviral drugs could constitute an important first response to such epidemics and enhance the impact of such responses. pathways that are fundamental to viral attachment and fusion as well as pathways involved in the viral replication and assembly are the foundations for drug discovery. please see figure for a summary of current anti-viral drugs that are mentioned in this article [ ] . one important pathway in the viral replication is lipid metabolism that is hijacked by viruses and upregulated to meet the increased demand for viral structural elements such as the viral cell membrane [ ] . cholesterol is an important component of the viral cellular membrane. statins reduce cholesterol synthesis by inhibiting the activity of hmg-coa reductase. multiple studies have eluted to the role of statins in treating different infections [ ] . tleyjeh et al. have performed a meta-analysis to investigate the role of statins in infections. they examined nine cohorts that looked at the role of statins in treating infections like bacteremia, pneumonia, and sepsis [ ] . in conclusion, they showed that the adjusted effect estimate was . ( % confidence interval, . - . ; i ( ) = . %) in favor of statins [ ] . the authors concluded that statins were effective in reducing the infection; however, due to the heterogeneity in the analyzed data, they highlighted the need for randomized clinical trials [ ] . in another study, douglas et. al., have shown that statins can protect against all caused mortality within six months of diagnosis with pneumonia with an adjusted hazard ratio of . ( . to . ) in favor of statins therapy [ ] . in a randomized clinical trial, makris et al. studied the impact of using pravastatin on reducing the frequency of ventilator-associated pneumonia. moreover, they studied if the treatment had favorable outcomes in patients with acute physiology and chronic health one important pathway in the viral replication is lipid metabolism that is hijacked by viruses and upregulated to meet the increased demand for viral structural elements such as the viral cell membrane [ ] . cholesterol is an important component of the viral cellular membrane. statins reduce cholesterol synthesis by inhibiting the activity of hmg-coa reductase. multiple studies have eluted to the role of statins in treating different infections [ ] . tleyjeh et al. have performed a meta-analysis to investigate the role of statins in infections. they examined nine cohorts that looked at the role of statins in treating infections like bacteremia, pneumonia, and sepsis [ ] . in conclusion, they showed that the adjusted effect estimate was . ( % confidence interval, . - . ; i ( ) = . %) in favor of statins [ ] . the authors concluded that statins were effective in reducing the infection; however, due to the heterogeneity in the analyzed data, they highlighted the need for randomized clinical trials [ ] . in another study, douglas et. al., have shown that statins can protect against all caused mortality within six months of diagnosis with pneumonia with an adjusted hazard ratio of . ( . to . ) in favor of statins therapy [ ] . in a randomized clinical trial, makris et al. studied the impact of using pravastatin on reducing the frequency of ventilator-associated pneumonia. moreover, they studied if the treatment had favorable outcomes in patients with acute physiology and chronic health evaluation ii score (icu severity scoring classification) increasing the probability of the survival of the treated group when compared to the untreated group [ ] . however, another randomized clinical trial showed no benefit for statin therapy [ ] . moreover, other studies looked at the effect of a combination therapy using atorvastatin ( mg/day) and irbesartan ( mg/day). statin's beneficial impact on viral infection could be due to its lipid lowering effects that can potentially suppress coronavirus infection, as shown in other viruses where the cholesterol-lowering abilities disturb lipid rafts. even though lipid-lowering capabilities might impact viral replication, statins can also help in mitigating the impact of viral infection through their immunomodulatory and anti-inflammatory properties [ ] . this is particularly important since many cardiovascular patients have been shown to have an elevated covid- infection risk [ ] . additionally, statins can also exert beneficial effects on covid- patients through their stabilization of atherosclerotic plaques [ ] . another lipid-lowering drug that showed potential antiviral properties in vitro is fibrates [ ] . fibrates are drugs that target fatty acid synthesis and increase lipoprotein lipase activity. they have been shown to increase survival of mice infected with influenza virus [ ] . taken together, these studies suggest a beneficial impact for statins and potentially other lipid-lowering drugs such as pcsk inhibitors for treatment of covid- , especially that of the most severely infected people which are suffering from cardiovascular disease and diabetes [ ] . targeting viruses with drugs can utilize the fact that viruses lack basic metabolic processes and rather exploit their host's metabolic machinery. lipid metabolism is an important component of the virus life cycle as demonstrated previously; as a result, targeting the lipid metabolism pathways could constitute an early-intervention and exciting host-directed drug target. yuan et al. utilized such a pathway and tested a lipid drug library and showed that am , a retinoid acid receptor alpha (rar-a) agonist could interrupt the life cycle of mers-cov as well as influenza a virus [ ] . it was also shown that am binds to the sterol regulatory element binding protein (srebp) in host cells. srebps are basic helix loop helix transcription factors that play an important role in regulating lipid metabolism which was highlighted as the mechanism of action for the antiviral activity of am [ ] . sphingolipids are important lipids that regulate membrane properties such as viscosity and tension, which might also make them suitable as novel targets for therapeutic intervention [ ] [ ] [ ] . sphingolipid biosynthesis is modulated in so many diseases, and mutations impacting their metabolism have been shown to cause disease [ ] [ ] [ ] [ ] [ ] [ ] . the sphingolipid biosynthesis pathway starts with the formation of -ketosphinganine from serine and palmitoyl-coa by serine palmitoyltransferase (spt). spt is composed of two major subunits and several small subunits that are most likely involved in its regulation and specificity [ , ] . as sphingolipids, especially ceramides and glucosylceramides, accumulate in a wide variety of diseases, sphingolipid biosynthesis is widely viewed as a therapeutic target for many different indications [ ] [ ] [ ] . in the past few years, it has been established that sphingolipids play a vital protective role in lungs from pulmonary leak and lung injury, and the modulation of their pathways may offer effective therapeutic intervention strategies [ ] . modulation of sphingolipids can be very beneficial and can offer an anti-inflammatory, neuroprotective, and anti-coagulant effects [ , ] . all of these exciting features could likely be exploited to counteract the associated complications of covid- infection [ ] . on this basis, the fda-approved immunomodulator fty (fingolimod), one of the best characterized "sphingomimetic" drugs [ ] , is currently on an ongoing clinical trial (nct and nct -clinicaltrials.gov) for the management of the covid- pandemic [ , ] . fingolimod acts as a nonspecific agonist of sphingosine -phosphate receptors (s pr) and as a selective functional antagonist of the s p subtype by promoting receptor internalization and degradation [ ] . since s p is critical in controlling lymphocyte trafficking, its downregulation causes relocation of the immune cells to secondary lymphoid tissues, causing a reduction from the circulation and henceforth immunosuppression [ ] . this will reduce the central inflammation response and may help since elevated levels of inflammatory cytokines, particularly il- , are thought to be associated with respiratory failure in covid- patients. however, it has been shown that the degree of lymphopenia is not associated with risk of infection, and generally, infectious complications were relatively low on s p modulators [ , ] . it has also been well documented that patients taking these drugs do not suffer from an increased risk of community-acquired viral infections [ , ] . we have recently shown that a higher level of angptl was associated with slow multiple sclerosis progression and good response to fingolimod treatment [ ] . recently, more specific functionally related compounds have been developed, that better differentiate between the various s pr subtypes such as selective compounds krp , ponesimod, and cenerimod [ ] . due to their higher specificity, these new treatments hold great potential for clinical advancements in a broad range of autoimmune and inflammatory diseases. in addition to the immunomodulatory property of fingolimod, its anti-thrombotic and anti-coagulant actions [ ] would make it more suitable and promising for the treatment of covid- patients [ ] . nevertheless, other "sphingomimetic" and "sphingomodulating" compounds, some of which are already in clinical trials for other clinical diseases, can be easily reused and possibly provide further therapeutic options for the management of this global pandemic crisis [ ] . inhibitors of virus cell entry and endocytosis as well as inhibitors of certain cellular pathways involved in the viral cycle constitute an important reservoir of antiviral drugs, such as methyl-β-cyclodextrin (mβcd), which works by depleting cholesterol [ ] . in vitro cell models expressing ace showed that cholesterol depletion by mβcd dramatically decreased the number of bonds with the s protein and reduced ace expression in a dose-dependent manner leading to reduced sars-cov replication [ ] . similarly, phytosterols, which are lipophilic molecules that can interact with the lipid raft and reduce membrane cholesterol and destabilize the membrane structure, impact viral infectivity significantly. other direct inhibitors of the endocytosis process, such as chlorpromazine, chloroquine, and umifenovir (arbidol), have also shown promising results [ , , ] . another example of a broad-spectrum antiviral drug is lj , which is a membrane-binding compound [ , ] . lj possesss a very promising antiviral mechanism that can selectively impact viral membranes but not host cellular membranes at very low antiviral doses (ic ≤ . µm) [ ] [ ] [ ] . this compound is astoundingly capable of inhibiting a wide range of enveloped viruses including hiv, influenza, and ebola, amongst many others [ ] . vigant et al. showed that lj targets unsaturated phospholipids in a manner that was dependent on light and molecular oxygen [ ] . it was postulated that lj leads to increased unsaturated fatty acid hydroxylation that primes the formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer, thus impacting the viral membrane properties [ ] . this drug is viral membrane-specific due to the intrinsic mechanisms in host cellular membranes that are protective against phospholipid hydroperoxides. while the effects of lj are limited to in vitro activity, since it is a light activated compound, newer compounds such as oxazolidine- , -dithiones with membrane-intercalating photosensitizers were designed. these examples clearly highlight the importance of the viral membrane as antiviral targets [ ] . lipid metabolism plays an important role in the viral infection cycle. this role can be harnessed as anti-viral drugs that can constitute a broad-spectrum drug that can be utilized in a makeshift fashion that offers a first response drug. the best example of such drugs are statins that has shown promising beneficial effect in people with various viral infections alongside their main role as a lipid lowering therapy. as a result, more work is needed to focus on the role played by lipid species at the structure and signaling pathway level in the viral life cycle. this will allow us to establish new drug targets as well as enhance existing drugs. such lipid-based therapies can be used alone or in combination with other drugs, but they will have the advantage of targeting multiple viruses. structure, function, and antigenicity of the sars-cov- spike glycoprotein middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease a novel coronavirus outbreak of global health concern covid- : preparedness, decentralisation, and the hunt for patient zero coronavirus infections and immune responses covid- pandemic, corona viruses, and diabetes mellitus characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases 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signaling methyl-beta-cyclodextrin inhibits ev-d virus entry by perturbing the accumulation of virus particles and icam- in lipid rafts methyl-beta-cyclodextrins preferentially remove cholesterol from the liquid disordered phase in giant unilamellar vesicles screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture arbidol inhibits viral entry by interfering with clathrin-dependent trafficking broad-spectrum antiviral agent that targets viral membranes a broad-spectrum antiviral targeting entry of enveloped viruses a mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion acknowledgments: the authors are thankful to irina al-khari for editing the manuscript. the authors declare no conflict of interest. int. j. mol. sci. , , key: cord- - cbh u z authors: honce, rebekah; schultz-cherry, stacey title: they are what you eat: shaping of viral populations through nutrition and consequences for virulence date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: cbh u z nan humans have coexisted with viral pathogens for tens of thousands of years, influencing both their emergence and evolution. however, the pervasiveness of the western diet and disparities in food access and security have altered how we as hosts interact with our viral pathogens. malnutrition, the state of having insufficient, excess, or imbalanced sources of energy, is well known to attenuate immune responses. could nutrition also actively shape how viruses evolve? malnourishment is a global, intersectional issue, and it may soon force a revision of our understanding of how viruses evolve within their hosts (fig ) [ ] . first theorized over decades ago [ ] , a quasispecies population structure has been documented in plant, animal, and human pathogens [ ] [ ] [ ] . a viral quasispecies describes the mutant but related genomes that collectively infect, replicate, and spread among hosts. traditionally, the theory has been applied to rna viruses. because of their short generation times, small genomes, and the inherent lack of proofreading in most rna replication, single nucleotide variants (snvs) emerge at a rate of roughly to more mutations per nucleotide copied compared with dna viruses [ ] . nonsynonymous snvs are continuously accrued and purged from the viral genome. this flux generates a related "swarm" of viruses, which have little effect on the consensus sequence but may show phenotypic differences. mutations with phenotypic consequences are generally deleterious; very few mutations have any fitness benefit. however, if beneficial mutations arise, they may relate to host range, drug resistance or vaccine escape, and replicative capacity [ , ] . both beneficial and the common deleterious mutations balance the structure of the viral swarm through complementation, interference, and cooperation [ ] [ ] [ ] . within a single host, tissue-specific subpopulations may vary in virulence without affecting consensus sequence or phenotype [ , ] . importantly, the consensus sequence should not be considered the "fittest sequence," because selection, competition, and genetic drift act upon the entire viral swarm. therefore, fitness of the swarm exceeds clonal sequence fitness, highlighted by work in vesicular stomatitis virus [ ] and bacteriophage systems [ ] . viruses are obligate intracellular parasites that require a host cell to complete their life cycle. barriers to replication exist within and between susceptible hosts, which restrict viral population diversity to quell infections [ ] . in these wide-ranging environments, a a a a a a heterogenous viral swarm containing isolates with differing abilities to infect, transmit, and survive environmental and immunological onslaughts may safeguard viral existence. however, this genetic plasticity has bounds, with an evolutionarily beneficial middle ground between high-and low-fidelity replication [ , ] . the "goldilocks" approach maximizes fitness by avoiding lethal mutagenesis while ensuring amenability to selective pressures [ ] . too low fidelity leads to error catastrophe and collapse of the viral population; conversely, a highly clonal population may be extinguished by host defenses [ ] [ ] [ ] [ ] . numerous theories have questioned the biological relevance of a quasispecies and challenged its significance [ , ] . however, boosting genetic diversity-to a point-is theorized to increase virulence. a viral swarm may be better equipped to face bottlenecks imposed by infecting hosts, environmental persistence, and transmission. even within a single host, blockades due to infection barriers and the immune response diminish sequence variation, leaving a relatively homogenous population until replicative errors replenish the mutant pool [ ] . so, do viruses harboring higher genetic diversity initially fare better in establishing an infection and displaying virulent phenotypes? in studies with classical swine fever virus, higher genetic diversity correlated with virulence [ ]; however, this conclusion has been challenged [ ] . in other animal viruses, diversity increases precede the selection of virulent genomes [ ] . parallel conclusions have been made for human pathogens. in hepatitis c virus (hcv)-positive patients, high viral diversity prior to transplantation correlated with higher liver fibrotic scoring year post-transplantation [ ] . continued genetic evolution of hcv correlated with progressing hepatitis, whereas resolution was associated with genetic stasis of hcv population [ , ] . a model low-fidelity rnadependent rna polymerase (rdrp) poliovirus variant demonstrates that increasing genetic diversity may not always yield fit populations [ , ], yet high-fidelity rdrp mutants producing nearly clonal populations display reduced fitness in vivo [ ]. selection pressures ranging from host antiviral responses to pharmaceutical interventions mold the viral swarm. upon infection, immune responses restrict genetic diversity by limiting spread and replication, eloquently demonstrated using a model poliovirus rdrp [ , ] exogenous control of infections can affect viral swarm composition. as hosts, we have exploited the high mutation rates of viruses by redirecting viral evolution toward error catastrophe via pharmaceutical interventions [ , ] . interestingly, high-fidelity foot-and-mouth disease viral variants possess a higher level of resistance to pharmacologics but are attenuated in vivo, suggesting that the resulting restricted quasispecies hampers adaptability in the presence of drug or host pressures [ ] . also, antiviral treatment can lead genetic diversity gains that may precede selection of drug-resistant genotypes, as has been observed with oseltamivir [ , ] . globally, in people are undernourished and in are overweight or obese, with innumerable others suffering from micronutrient deficiencies [ ] . consequently, it is of utmost importance to understand whether host nutrition actively shapes how viruses evolve because many hosts do not mirror the actively studied "wild-type" condition. previous work has identified micronutrient deficiencies that may increase pathogen virulence through acquisition of minor variants. in mineral-and vitamin-deficient mice, genetic mutations arise in coxsackie b and influenza virus populations that promote virulence even in well-nourished hosts [ ] [ ] [ ] [ ] [ ] . in our work with influenza virus, we determined that nutrient excesses can drive virulence through population diversification [ ] . experimental evolution of ca/ virus through two models of murine obesity resulted in a viral population displaying increased virulence upon inoculation of a wild-type host. this phenotype was not strain specific; an avirulent h n virus was, upon passage in obese hosts, able to productively infect immunocompetent mice. we observed a significant increase in viral diversity and subsequent virulence after a single round of infection, with the phenotype persisting in obese-derived viral populations across passages [ ] . interestingly, arbovirus-infected obese or protein-deficient mice showed higher morbidity but lower viral diversity, and both malnourished models transmitted virus less efficiently, highlighting that the effects of nutrition may vary based on the natural life cycles of viral families [ ] . it is yet to be determined how malnourishment may impact transmission of a respiratory, as compared with a vector-borne, virus. both undernourishment and obesity are two sides of the same coin and are implicated in blunting immune responses and increasing susceptibility to infection [ , ] . in our studies with influenza virus, we linked the emergence of a more diverse and virulent viral population with blunted interferon responses in obese hosts. interferon treatment of obese mice restricted the emergence of a diverse quasispecies and attenuated the virulence of the resulting viral population, strengthening the claim that a robust innate immune response restricts subsequent infection severity, possibly through reduced viral replication and acquisition of a genetically diverse viral population [ , , ] . dietary metabolites also influence cellular metabolism and can push the body to a state of metainflammation; this prooxidant environment may also directly influence the genetic composition of the viral population [ ] . nutritional excess or deficiency may dampen the host immune responses and alter cellular metabolism, indirectly fostering an advantageous environment for viruses to explore the sequence space (fig ) . the dearth of host responses to infection-particularly innate immunity-and the baseline malnourished state facilitates greater viral replication, permits the diversification of the viral swarm, and potentially allows for the emergence of advantageous mutations. other indirect consequences of poor nutrition may also be involved. blunting of immune responses may alter viral tropism and viral-or immune-induced pathology, thus remodeling the microenvironment in which the virus attacks the host. also, nutrition is increasingly appreciated as an influence on the gut microbiome (reviewed in [ ] ). interestingly, perturbations to the microbiome-both respiratory and gut-dampen interferon responses to respiratory virus infection [ ] [ ] [ ] . however, to our knowledge, no empirical studies connect the obese microbiome to modulating enteric or respiratory viral populations. pathogen virulence is a complex interplay of both host and pathogen properties. host nutritional status has long been considered a risk for infection susceptibility and severity and is now implicated in shaping viral evolution. continued studies on the molecular consequences of obesity and malnutrition at the macro-and micronutrient levels will reveal which host defenses are impaired through malnutrition and how they control quasispecies development and viral pathogenesis. similarly, as we gain insight into how hosts influence quasispecies formation and pathogen virulence, we too can exploit these features for host benefit [ , ] . the global ubiquity of malnutrition is shifting our population toward a more susceptible state. this will undoubtedly influence how pathogens behave within and between hosts. continued study of how quasispecies evolution relates to other human, animal, and plant pathogens will indeed usher in a greater understanding of host-pathogen interactions and provide novel insights into how pathogens impact hosts and hosts impact pathogens. global nutrition report: action on equity to end malnutrition selection and self-organization of self-reproducing macromolecules under the constraint of constant flux subclonal components of consensus fitness in an rna virus clone analysis of newcastle disease virus quasispecies and factors affecting the emergence of 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amyocarditic and myocarditic strains of coxsackievirus b in mice the human microbiome and obesity: moving beyond associations the microbial metabolite desaminotyrosine protects from influenza through type i interferon nasally administered lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection microbiota regulates immune defense against respiratory tract influenza a virus infection attenuation of rna viruses by redirecting their evolution in sequence space key: cord- -da ypiy authors: mônica vitalino de almeida, sinara; cleberson santos soares, josé; lima dos santos, keriolaine; emanuel ferreira alves, josival; galdino ribeiro, amélia; trindade tenório jacob, Íris; juliane da silva ferreira, cindy; celerino dos santos, jéssica; ferreira de oliveira, jamerson; bezerra de carvalho junior, luiz; do carmo alves de lima, maria title: covid- therapy: what weapons do we bring into battle? date: - - journal: bioorg med chem doi: . /j.bmc. . sha: doc_id: cord_uid: da ypiy urgent treatments, in any modality, to fight sars-cov- infections are desired by society in general, by health professionals, by estate-leaders and, mainly, by the scientific community, because one thing is certain amidst the numerous uncertainties regarding covid- : knowledge is the means to discover or to produce an effective treatment against this global disease. scientists from several areas in the world are still committed to this mission, as shown by the accelerated scientific production in the first half of with over , published articles related to the new coronavirus. three great lines of publications related to covid- were identified for building this article: the first refers to knowledge production concerning the virus and pathophysiology of covid- ; the second regards efforts to produce vaccines against sars-cov- at a speed without precedent in the history of science; the third comprehends the attempts to find a marketed drug that can be used to treat covid- by drug repurposing. in this review, the drugs that have been repurposed so far are grouped according to their chemical class. their structures will be presented to provide better understanding of their structural similarities and possible correlations with mechanisms of actions. this can help identifying anti-sars-cov- promising therapeutic agents. the world is facing a huge challenge in the coronavirus disease (covid- ) pandemic: how to fight an enemy without weapons in terms of therapy? unfortunately, even before the severe acute respiratory syndrome coronavirus (sars-cov- ) worldwide spread, there were no clinical treatments nor prevention strategies available for any human coronavirus. it is understandable that both society and researchers urge the discovery of new compounds or even of a drug that is commercially available that can be employed by physicians mainly for patients with the extreme presentation of covid- . there is also urgency in the discovery of medicine with prophylactic action to prevent the entry of the virus in host cells after exposure. vaccine research experts already indicate that rescue from sars-cov- will come from a long but effective journey to produce a vaccine. while this is not a reality, the scientific community, including medicinal chemists and doctors who accompany patients, are trying to identify therapeutic alternatives. this is a meritorious attitude: the commitment with the protection of humanity. nevertheless, the rigorous feature of science in the discovery of a new drug cannot be disregarded, even during a pandemic and in the face of the urgent demand for a treatment, to avoid eventual mistakes and spurious hope. the increase in studies related to sars-cov- during the first semester in has allowed the rather speedy identification of promising therapeutic targets for both developing immunotherapies and producing/identifying antiviral drugs. it is noteworthy the increase in outbreaks of sars-cov ( ) and mers-cov ( ), with accelerated production of knowledge on these hcovs, which has been very useful for ongoing investigations on sars-cov- . one example is the availability of technological devices that allowed the fast sequencing of sars-cov- genome and the elucidation of a promising antigen target, the s glycoprotein. nonetheless, the development of a human vaccine can take years, especially because employing emergent technologies requires extensive safety tests and expansion to large scale production in order to assist the world population, as demanded in the case of the covid- pandemic. the development of new medicine also demands many years of research that involve stages of reasonable planning, synthesis, structural characterization, formulation of prototypes, preclinical and clinical trials. therefore, the literature highlights, as alternative treatments for covid- , the repurposing of drugs, which is fast and useful in emergencies such as the one experienced today. the repurposing of drugs means the use of broad-spectrum medicine for a new disease, once its metabolic characteristics, doses, potential efficacy and adverse effects are pre-established due to drug studies extracellular liquid volume and arterial pressure of the human body. it is largely expressed in fifteen human tissues, including ciliated bronchial epithelial cells and type ii pneumocytes form pulmonary alveoli, the main location of lesions caused by sars-cov- . , after ace receptor-binding, a conformational alteration occurs in protein s allowing the fusion between the viral envelope and the host cell membrane via endosomal. then, sars-cov- releases the rna into the cytoplasm to be translated into viral replicase polyproteins pp a and pp ab, which are processed by cl pro and pl pro proteases, respectively. the cleavage products are nsps that form the transcription and replication complex. next, the positive rna strand is translated into a template of negative strand that allows the synthesis of new genomics and sub genomics mrnas. these mrnas are translated and transcribed producing structural and accessory proteins. viral proteins and rna genomic are put together in virions at endoplasmic reticulum and golgi complex, finally transported through vesicles and released from the cell host for infecting new cells. , the covid- symptomatology starts after the virus is installed in host cells. in general, the symptoms include lasting and high fever, dry cough, shortness of breath, muscles aches or tiredness, sputum production, headaches, and a small percentage of individuals presented gastrointestinal symptoms such as diarrhoea and vomit. the incubation period of sars-cov- , from exposure to first symptoms, lasts to days. the pre-symptomatic stage lasts from - days (possibly more) before the beginning of symptoms. the post-symptomatic stage lasts at least days after the beginning of symptoms and days after lowering of fever and improvement of respiratory symptoms. there are many unanswered questions such as the duration of potential immunity of both symptomatic and asymptomatic individuals when infected with sars-cov- . it is noteworthy that efficient strategies to fight the disease should not depend on the symptoms of patients, once asymptomatic or pre-symptomatic subjects can play an important role in the direct and indirect transmission to others, as demonstrated by arons et al. this investigation reports that half the residents in a nursing facility, who tested positive, were asymptomatic when tested and probably contributed to the transmission to other residents. thus, control strategies focused on symptomatic residents were not sufficient to prevent transmission once sars-cov- had been introduced in the facility. laboratory exams of infected patients showed alterations in haematology and biochemistry. it was verified the increase of leukocytes and the reduction of lymphocytes; increased d-dimer and erythrocyte sedimentation rate (esr), prolongation in prothrombin times (pt), followed by increase in bilirubin levels, aspartate transaminase (ast), alanine transaminase (alt), creatinine, lactate dehydrogenase (ldh), protein c reactive (pcr), hypoalbuminemia (low albumin), microcytosis and thrombocytopenia. in addition, inflammatory factors that indicate the immune condition of patients, such as interleukins (il) il- , il- , il- , and il- and the tumoral necrosis factor-α (tnf-α) become elevated. plasma levels of granulocyte-colony stimulating factor (gcsf), protein induced by interferon gamma, monocyte chemoattractant protein- (mcp- ), macrophages inflammatory protein α and tnf-α also display significant increase. potential risk factors or comorbidities that can lead to complications of covid- include elderly individuals (specially above years of age), cardiovascular issues, cerebrovascular, chronic pulmonary diseases, immunocompromising, renal problems, hepatic disease, hypertension, diabetes and obesity. [ ] [ ] [ ] [ ] [ ] [ ] there is a notorious concern regard the medicine administered to fight these comorbidities because some of them can lead to greater expression of ace , such as treatments for diabetes or hypertension. this may favour or even aggravate covid- infection. these facts justify the urgency of research that contemplate alternative therapeutic targets such as calcium channels blockers for hypertensive individuals as suggested by fang et al. however, there is little clinical evidence on the risk of treating covid- patients with therapies that induce greater expression of ace . further investigation is necessary to explore whether these medicines inhibit or trigger the viral entry into the cells of an infected host. a frequent report in epidemiological data regarding the mortality of covid- concerns the sex of individuals, as men are the predominant fatal victims of the disease. therefore, being of the male sex is considered a bad prognostic factor for infection. , a possible explanation lies in the relation between gonadal hormones and the expression of ace enzymes or even an alleged vitamin d deficiency, according to vignera et al. the latter suggest monitoring of serum levels of testosterone and vitamin d in infected patients for a better understanding of the different fatality rates between sexes, including the hypothesis that women's hygiene justify a lesser rate of infection. understanding the pathogenic effects of sars-cov- for the different organs affected by the disease has also been object of investigation, such as gut-lung crosstalk. data from research conducted thus far indicate that the infection caused by sars-cov- is not only capable of causing pneumonia, but it can also damage other organs such as the heart, the liver, the kidneys and organic systems such as the blood and the immune system. , , patients with the extreme form of the disease frequently manifest lymphopenia, , hepatic insufficiency and viral sepsis diagnostic, whose complications can be related to the severity of the cases and the mortality of patients. , there are reports that the eventual death of such patients is due to multiple organ insufficiency, acute respiratory distress syndrome (ards), cardiac insufficiency, arrhythmia and renal insufficiency. , therefore, great attention is necessary to the disease's potential damage to multiple organs and to therapeutic alternatives to fight covid- , given that some of these alternatives can have side effects on organs initially unrelated to the respiratory system, but that may be susceptible to a systemic compromise prompted by the virus once the treatment has begun. hence, it is possible to observe the existence of different forms of aggravating the disease. in this regard, wang et al. recommend the creation of a system to categorize patients with the severe form of covid- . several investigations report that all hcovs, sars-cov, mers-cov and sars-cov- induce exaggerated immune responses in the host, which are associated to the severity of pulmonary pathology and might lead to the development of acute respiratory distress syndrome (ards) or death. the incidence of the extreme form of the infection is associated with cytokine storm syndrome, characterized by high plasma concentration of several interleukin, inflammatory cytokine, inflammatory chemokines, among other factors that cause infiltrated inflammatory in the organs. , , survivors of this excessive response by the immune system can develop long-term fibrosis and pulmonary damages that might culminate in functional injuries to these organs, thus reducing the patient's quality of life. during the development of drugs to fight microorganisms, the adoption of strategies that allow the design of molecules to act against specific biological targets of bacteria, parasites or viruses is preferred. therapies for covs can be divided into several categories, based on specific paths: ( ) covs proteins or functional enzymes that are essential for viral replication; ( ) structural proteins of the virus that prevent its binding to the respective receptors in human cells or its assembly process; ( ) some viral factor that restores the host's inherent immunity and; ( ) host-specific enzymes or receptors, that prevent the entry of the virus in the host cells. , so far, structural proteins and enzymes that participate actively in the process of viral replication are the most investigated targets for the development of molecules for anti-covs therapies (fig. ) . investigations by wu et al. through bioinformatics, analysed possible sars-cov- therapeutic targets. the proteins coded by this virus were verified and compared to proteins coded by other covs. the results enabled the detection of structural similarities to sars-cov, from which it was possible to conduct homology modelling to build proteins for sars-cov- . among the targets were spike (s) glycoprotein, nsp (rna-dependent rna polymerase -rdrp), enzyme helicase ( cl pro and pl pro ), tmprss, orf a factor and ace presents in the host cells. these targets have pivotal roles in the development of the virus and have great influence on its pathogenicity, hence, some details are provided next. molecular modelling showed that spike (s) glycoprotein is a transmembrane protein of approximately to kda type i whose n-terminal turns to the exterior of the virus and its cterminal segment turns to the interior of the virus. the typical structure of covs is given by the assemble of a bulbous projection of a corolla as trimers of protein s and it is cleaved into two important subunits from the pathogenic perspective: s and s . sars-cov and sars-cov- (s) glycoprotein share about % of amino acid identity and enable the entry of the virus in the host cells. therefore, s glycoprotein present in covs has been considered a promising biological target for antiviral mechanisms. , the moment when the virus approximates the target cell prompts the recognition by the receptor-binding domain (rbd) in the s glycoprotein of its receptor, which leads to the binding to subunit s . next, the subunit s allows fusion of viral and cellular membranes, which enables entry in the cell and the release of viral rna genome. , , some investigations suggest that the strong binding affinity between s protein and ace is essential for viral entry, hence, ace is also relevant for the development of drugs. , molecules that bind to the surface of the virus can destabilize the formation of s glycoproteins and interfere both with the trimerization of the protein and with the continuity of the life cycle of covs. several studies have been conducted on s protein to clarify its sars-cov- structure and its binding process as well as to evaluate its relevance as target for in-silico and in-vitro assays on molecules for anti-sars-cov- therapies. one study conducted by hoffman et al. investigated how the sars-cov- s protein facilitates viral entry in the target cells and how this process could be blocked. results showed that ace is used as receptor for the entry of sars-cov- in host cells and that the spread of this cov in the infected host depends on the activity of tmprss (a cellular serine protease responsible for initiating the binding process between s protein and ace ). this process can be blocked with clinically approved tmprss inhibitor. prior to this, the relevance of tmprss was highlighted in the dissemination of several types of viruses such as influenza a and other covs, which also makes it a relevant target for covid- therapeutic intervention. [ ] [ ] [ ] [ ] [ ] [ ] [ ] binding between s proteins and ace receptors was corroborated through x-ray crystallography conducted by lan et al. to elucidate the interaction between the sars-cov- rbd and ace at a higher resolution. in spite of different interactions with ace , the sars-cov- rbd /ace and sars-cov rbd /ace interfaces share a substantial similarity regarding the surface area, the number of interacting residues and the networks of hydrophilic interactions. such similarity strongly points to a convergent evolution of both sars-cov- and sars-cov rbd structure which improves the binding affinity for the same receptor, the ace . the non-conserved rbd regions in s protein, such as subunit s , could be potential targets for cross-reactive antibodies. considering rbd as a critical region for receptor binding, antibodies that target the conserved epitopes in the rbd are also good candidates for the development of highly potent cross-reactive therapeutic agents against several species of covs, including sars-cov- . investigations on ligands obtained from drugbank . used molecular docking to identify target regions in the pockets of the quaternary structure of sars-cov- s glycoprotein (from protein data bank-pdb). six pockets present in s glycoprotein deserve further investigation in medicinal chemistry due to suitable features for small molecule binding. among the six pockets, the eight best ligand candidates from drugbank were all binding pocket # , which contained residues of amino acids proline, leucine, lysine, asparagine, phenylalanine, glycine, threonine, glutamine, alanine, methionine and tyrosine. one of the best ligands was the drug saquinavir, an antiviral from the class of protease inhibitors, used in anti-hiv therapy. nsps are involved in the rna transcription, translation, protein synthesis, processing and modification, viral replication and infection of the host. significant functional proteins, cl pro , pl pro , helicases and rdrp are important targets for the development of small-molecule inhibitors, due to their biological function and vital enzyme active site. factors nsp , nsp c and orf a are related to assistance to the immune evasion of sars-cov- . interaction between nsp and the host ribosomal subunit induce the degradation of mrna, allowing the virus to develop resistance to the host innate immunity. binding between orf a and bone marrow matrix antigen (bst- ) inhibits activity and blocks bst- glycosylation. these results suggest that all three structures are potential targets for antiviral medicine. proteases pl pro and cl pro mediate the proteolytic cleavage of polypeptides produced by βcoronavirus sars after genome transcription, thus generating other proteins. the cl pro , known as nsp , cleavages several non-structural proteins of importance for viral replication and the maturation of nsps, which is essential in the life cycle of the virus. therefore, it is an attractive biological target for that has been inhibited in-silico by several antiviral, anti-inflammatory and anti-hypertensive drugs from the database zinc (fda). in addition, docking and molecular dynamic studies conducted by qamar et al. showed that non-toxic natural products formed strong bonds with sars-cov- catalytic dyad cis -his of cl pro . moreover, the proteinase pl pro is responsible for cleavages of n-terminus in the replicase polyprotein to release nsp , nsp and nsp , which are essential for correcting virus replication significant to antagonize the host's innate immunity. analysis of the docking model showed that ribavirin formed hydrogen bonds with residues gly , gln , tyr , asp as well as hydrophobic interactions between tyr and the pl pro residue. these results indicate ribavirin as a powerful pl pro enzyme inhibitor, which means it has promising features for anti-covid- therapy given the inhibition of a likely pl pro therapeutic target. helicase (nsp ) has been identified as a promising target for antiviral drug discovery, particularly against sars-cov- . it is a multifunctional protein necessary for a wide range of biological processes, such as genome replication, recombination and dislocation of proteins related to chromatin and nucleic acid remodelling. for covs, helicase is indispensable for viral replication. in studies on molecular modelling, several antibacterial, antifungal and antiviral drugs were analysed and presented elevated affinity to helicase, suggesting it as a good target for sars-cov- therapy. rna-dependent rna polymerase (rdrp -also nominated nsp ) catalyses the viral rna, which performs a key role in the replication/transcription complex of sars-cov- , possibly aided by nsp and nsp complex as cofactor. , nsp has been studied as potential target for several sars-cov and mers-cov inhibitors, due to its importance for viral control. satisfactory results of rdrp inhibition by several ligands were presented in the modelling studies by gao et al. yin et al. those ligands included antiviral analogous to nucleotides, such as remdesivir, which already shows great potential in the treatment of covid- infections. in addition, some nonstructural proteins, including nsp b, nsp e, nsp , nsp , nsp , nsp , nsp , nsp and nsp , also stood out as useful targets due to their significant role in the synthesis and replication of viral rna. cl pro is key enzyme for covs, also called main protease (m pro ), that plays a pivotal role in mediating viral replication and transcription, making it an attractive target for anti-sars-cov- drugs. such claim is reinforced by studies by jin et al. after the virtual screening of n inhibitor. results show that n ( ) is a time-dependent irreversible inhibitor of this enzyme and that a stable covalent bond is formed between n and cl pro . high-throughput screening (hts) was applied to , drugs and drug candidates, demonstrating that ebselen ( ), px- ( ) and carmofur ( ) are all able to covalently bind to cl pro do sars-cov- , with ic that varied from . to . µm (fig. ) . it is likely that a part of the hits identified by hts are bonded to the catalytic cysteine of cl pro through their sulfhydryl groups. in-vitro studies on antiviral activity were performed to corroborate the results. real time quantitative pcr (qrt-pcr) demonstrated that ebselen and n had the strongest antiviral effects at a concentration of μm treatment in sars-cov- infected vero cells. after plaque-reduction assay, the dose-response curves suggested that both could penetrate cellular membrane to access their targets. this result strongly supports the hypothesis that developing a single antiviral agent targeting cl pro or in combination with other therapies could provide an effective first line of defence against all covs related diseases. in relation to sars-cov- therapy, some of the aforementioned targets have been explored for both new drug proposition as well as for sars-cov- drug repurposing. our focus is on this last type, and for each medicine, the putative mechanism of action and viral target will be described trying to find an understandable rational therapy even for an immediate illness situation like covid- pandemic. carmofur ( ). as previously mentioned, sars-cov- is an enveloped virus, whose nucleocapsid consists of a positive rna genome surrounded by multiple copies of nucleocapsid protein. this virus, after entry in the host cell, replicates fast the viral genome with new virion production. the rna replication into the cell host depends on enzymes and substrates for rna synthesis, such as ribonucleotides (adenine, guanine, cytosine or uracil) that have nitrogenous bases in the purine or pyrimidine classes. compounds can mimic these chemical structures and interfere with the formation or use of one of these essential normal organism metabolites. the interference is generally prompted by enzyme inhibition in the biosynthetic pathway of the metabolite or by incorporation, as a false building block, into vital macromolecules such as proteins and polynucleotides. so, this class of therapeutic agents is called antimetabolites. , diverse antimetabolites have been indicated as promising anti-sars-cov- . they are described next. pyrimidine derivatives are aromatic organic compounds necessary for all life forms. examples of pyrimidine derivatives are nitrogenous bases cytosine ( ), uracil ( ) and thymine ( ) (fig. ) . they are found in dna and rna and participate in the metabolic process that involves carbohydrate and lipids. , these heterocyclic rings share two nitrogen atoms at and positions, but display variations between themselves, such as an amine group at -position in the cytosine and a methyl at -position in the thymine. from the pharmacologic perspective, nitrogenous bases are investigated as pharmacophores and are found in the structure of many drugs and experimental substances with various activities, such as antitumoral, antibacterial, antiparasitic, and antiviral. , regarding antiviral activity, there are several approved drugs that are classified as pyrimidine nucleotide biosynthesis inhibitors (pnbi) because, after phosphorylation, they are incorporated either into the dna or into the rna and inhibit hosts or pathogenic enzymes, such as polymerases. therefore, the likely mechanism of action of some pyrimidine derivative drugs has been considered for repurposing. some pyrimidine derivatives with antiviral activity are often formulated as prodrugs. this format solves issues of high polarity in its final structure prompted by the phosphonic acid, which interferes with pharmacological properties and causes low cellular permeability and low oral bioavailability. one compound appointed as potential anti-sars-cov- is the -fluorouracil ( ) ( -fu) (fig. ) , a heterocyclic aromatic amine similar to uracil (u) that presents a fluorine-carbon bond at -position. this compound is used in the treatment of oesophageal cancer, stomach cancer, breast and colon cancer. the similarity between -fu and uracil allows the direct action on nuclei acid as it is incorporated into the genetic material and inhibits replication. tests with -fu as monotherapy confirmed its failure against any coronaviruses. the reason proposed to such failure relied on the fact that coronaviruses rna proofreading activities involve a ' → ' exoribonuclease in the nsp , which removes -fu during replication and metabolism. hence, the combination between -fu and deoxyribonucleoside and deoxyribose was suggested so that, after its insertion in the rna, it escapes rna proofreading and prompt lethality and/or lethal mutagenesis in the virus. despite the proposition of using a widely marketed drug to treat several types of cancer, which means it has well-established efficiency and safety, no other type of test has been made to confirm its efficacy against sars-cov- . therefore, further experiments are necessary to explore -fu potentialities. another antitumoral drug considered for its anti-sars-cov- potential is gemcitabine (gct) ( ) (fig. ) , an analogue of deoxycytidine whose pharmacological action is triggered after the intracellular transformation into triphosphate gemcitabine. the latter competes with endogenous nucleoside triphosphates by incorporation into the genetic material, thus inhibiting dna synthesis. initially, gct was developed for antiviral activity, however, initial results caused it to be redirected for anticancer therapy. it became, then, widely used against non-small cell lung cancer, pancreas, bladder and breast cancers as well. [ ] [ ] [ ] in-vitro analyses of gemcitabine hydrochloride inhibited mers-cov and sars-cov, with a ce of . μm and . μm, respectively, in addition to low cell toxicity for vero e cells. , these data are indicative of a possible activity against sars-cov- , but complementary preclinical investigations are necessary before clinical trials. albeit considered a safe drug under predetermined doses, gct adverse effects are noteworthy and include myelosuppression and disruption of liver functions. in february , the european union (eu) approved baricitinib ( ) as second-line oral treatment for mild to severe active rheumatoid arthritis in adults. a differential feature of baricitinib structure is the azetidine ring bearing an ethylsulfonyl, beyond an acetonitrile group at -position. the same ring binds to the n atom at -position in the pyrazole, which, in its turn, binds to the pyrimidine conjugated to a pyrrole ring. this medicine can modulate human innate and adaptive immune system. based on this property, presumably, one of the important mechanisms of action of baricitinib in the treatment of rheumatoid arthritis is the inhibition of the il- / jak / jak pathway. the promising nature of baricitinib and other small molecule inhibitors against sars-cov- was pointed by richardson et al. through in-silico tests using benevolent ai. the authors evaluated compounds to show that sunitinib ( ) and erlotinib ( ) inhibit ap -associated protein kinase (aak ) interrupting the virus entry to the cells and the intracellular assembly of new viral particles (fig. ) . regarding these two antitumor drugs, it is known that sunitinib is an oral oxindole multitargeted kinase inhibitor that inhibits certain tyrosine kinases including vascular endothelial growth factor receptors (vegfr types and ), platelet-derived growth factor receptors (pdgfr-α and pdgfr-β), stem cell factor receptor (kit), fms-like tyrosine kinase- (flt ), glial cell-line derived neurotrophic factor receptor (ret) and the receptor of macrophage-colony stimulating factor (csf r). concerning erlotinib, it was developed as reversible and highly specific small-molecule tyrosine kinase inhibitor that competitively blocks the binding of adenosine triphosphate to its binding site in the tyrosine kinase domain of epidermal growth factor receptor (egfr), thereby inhibiting autophosphorylation and blocking downstream signalling. however, these oncological drugs have serious adverse effects such as diarrhoea, loss of appetite and skin rashes. in addition, high doses of these medications can aggravate those effects. in relation to baricitinib, its anti-sars-cov- potential was explained in three ways: aak inhibition like sunitinib and erlotinib; the kinase associated to cyclin g, which is another endocytosis regulator; and the janus kinase, that inhibits the action of cytosines that triggers the inflammatory process. because baricitinib can inhibit aak at the therapeutic dose ( or mg/day), the drug is indicated for clinical trials. it is highlighted that baricitinib is not indicated for patients with neutropenia or lymphopenia, once it lowers rates of neutrocytes and lymphocytes, which can lead the disease to progress and increase anaemia. furthermore, treatment with baricitinib can reactivate varicella-zoster, herpes simplex and epstein-barr viruses. this implicates in a conflict between the potential effect and the adverse effects of baricitinib against covid- to prevent aggravating the disease and the mortality of patients. an analogue of adenosine, galidesivir (gsv) ( ) is a broad-spectrum antiviral drug that blocks viral rna polymerase by replacing a natural nucleotide with galidesivir triphosphate. this alteration prompts changes in electrostatic interactions and prevents the formation of the rna elongated strand. , adenosine and gsv differ in that galidesivir has one carbon at -position in the pyrimidine ring and nitrogen in the ribose ring, whereas adenosine has one nitrogen in the former and oxygen in the latter (fig. ) . it is noteworthy that gsv has not been approved for clinical trial and is an experimental drug in advanced stages of development. gsv was first developed against hepatitis c (hcv) but first clinical trials were conducted to ensure its safety (in healthy individuals) and efficacy against yellow fever. furthermore, gsv displayed in-vitro and in-vivo antiviral activity against filoviridae, alphavirus, bunyavirus, arenavirus, paramyxovirus, flavivirus, orthomyxovirus, picornavirus and sars and mers coronaviruses. , , recent in-silico studies have shown the existence of a strong bond between gsv and sars-cov-rdrp to demonstrate the capacity of alterations in rna polymerase, which can eradicate the virus. although, preclinical and clinical trials are necessary to either confirm or deny this hypothesis. it is noteworthy that investigations have pointed the inactivity of gsv against sars-cov- at concentrations lower than mΜ. the existence of antiviral activity against other coronaviruses indicates that more investigations on gsv against sars-cov- are required to elucidate its potential activity in advanced testing. next, sofosbuvir (sbv) ( ) is an example of successful nucleotide prodrug, approved by the food drug administration (fda) since , against chronic hepatitis c infections. sbv is also combined with other antiviral drugs, such as ledipasvir, velpatasvir and voxilaprevir. , the structural similarity between sbv (fig. ) and uridine allows that drug to act on hcv rdrp, incorporate itself into the viral rna and terminate the synthesis of the nucleotide sequence. structural analysis of sbv revealed that its elevated potential is partly due to the presence of the 'phosphate, which terminates the primary enzyme transformation monophosphate inhibitor. the antiviral activity has been explored against other viruses through in-vitro and in-silico studies and shown potential for inhibiting the dengue virus, yellow fever, telbivudine (tbv) ( ) (fig. ) is a thymidine nucleoside analogue used with specific activity against the hepatitis b virus (hbv). it starts acting after phosphorylation by cellular kinases, which results in the active metabolite, telbivudine '-triphosphate, enabling dna polymerase and inhibiting viral replication. the hydroxyl at -position in the sugar β-l- '-desoxirribose provides specificity to hbv polymerase. suggesting repurposing tbv to fight covid- was prompted by virtual screening to find drugs that act on viral m pro . among other results were ribavirin, tbv and two vitamins, cyanocobalamin (b ) and nicotinamide (b ). researchers suggest that these four drugs can be combined and used against covid- , once they are safe, marketed and approved by the authorities. notwithstanding, the suggestion of repurposing these drugs requires more information, including on drug interaction parameters. in spite of well-tolerated and safe for monotherapy, associating tbv and ribavirin, another antiviral drug, can increase hepatotoxic activities of tbv. , it is also important to consider the elevated risk of resistance to tbv, which and pyrimidine derivatives drugs: -fluorouracil ( ); gemcitabine ( ); baricitinib ( ); sunitinib ( ); erlotinib ( ); galidesivir ( ); sofosbuvir ( ) ; telbivudine ( ). purine is a and -membered bicyclic ring. similar to pyrimidines, purine derivatives are essential to life. they are basic constituents of nitrogenous bases adenine (a) ( ) and guanine (g) ( ) (fig. ) . the safety of rdv for humans infected with ebov was evaluated in the democratic republic of congo. results confirmed its safety but did not point rdv as the best therapeutic option, once its mortality rate reached % of treated group. it has been proved that gs- inhibits epidemic and zoonotic hcov. of viral loads and weight loss in murine. therefore, rdv is a potential drug to treat mers-cov infections. regarding covid- , rdv was used to treat the first us case. the patient was years old, had slight cough, low fever and no evidence of pneumonia at day of the disease. when the clinical symptoms became worse, the patient was given vancomycin and cefepime. as the symptoms worsened, intravenous treatment with rdv was administered at day , and vancomycin and cefepime were no longer administered. at day , the patient displayed clinical improvement, unfortunately details on the doses and duration of treatment were not provided. after this first case, a clinical trial with a larger number of covid- patients was conducted. this study of efficacy involved patients infected with sars-cov- who displayed saturation equal or inferior to % while they were breathing ambient air or receiving oxygen support. the treatment lasted days, patients were given mg intravenously on day , followed by mg daily for the remaining days of treatment. follow up of patients treated for days indicated that, after the first dose of rdv, % improved oxygen support whereas % of patients got sicker, % were discharged and mortality rate was %. the most common adverse events ( % of patients) were increased hepatic enzymes, diarrhoea, rash, renal impairment, and hypotension. some limitations were noted in the study, such as the small size of the cohort, the short duration of follow-up and the lack of information on the patients. hence, the efficacy of rdv requires validation by the ongoing randomized, placebo-controlled trials. one advantage of repurposing rdv is the availability of data on safety and pharmacokinetics, which were obtained previously at phase clinical trial. in addition to the promising results shown by rdv, other purine analogues have been investigated for sars-cov . ganciclovir (gcv) ( ) also named, according to its chemical structure, -( , -dihydroxy- -propoxymethyl) guanine (fig. ) , is a guanine analogue, similar to acyclovir, except for the bond between the methyl group and one hydroxyl. gcv inhibits the human herpesvirus and is also indicated in the treatment of cytomegalovirus infections related to acquired immunodeficiency syndrome (aids). gcv is converted into ganciclovir triphosphate by cellular kinase, which inhibits dgtp and disrupts viral dna synthesis due to substitution of various adenosine bases in the dna chain. recently, gcv was used to treat covid- patients in china. the drug was administered with other antivirals, such as oseltamivir and kaletra. as a descriptive study, the relation between gcv as key factor in clinical outcome of the patients ( % of which were discharges) was not possible. therefore, gcv efficacy as monotherapy or part of combined therapy is yet necessary for more robust investigations. valganciclovir ( ) (fig. ) is the antiviral prodrug of gcv taken by mouth. it is indicated for the same treatments as gcv (cytomegalovirus in people who have acquired immunodeficiency syndrome, gastrointestinal disorders related to aids). the drug has great bioavailability and is converted by hydrolysis into ganciclovir. using valganciclovir in its oral form enables clinical treatment and makes patients more comfortable. [ ] [ ] [ ] the mechanism of action is the same of gcv. valganciclovir was computationally evaluated for covid- . the assay with the main proteins coded for sars-cov- allowed the determination of possible binding targets, of which were proteins and host targets. valganciclovir, one of the drugs used in the study, was presented as a possible anti-sars-cov- therapeutic drug due to its high binding affinity to two wellestablished viral targets. the first target was pl pro , indispensable enzyme in viral replication; the second, rdrp, conserved nsp in coronavirus, which is vital for its replication/transcription. therefore, valganciclovir could be a significant antiviral drug to treat sars-cov- . but there are no clinical reports on valganciclovir used to treat covid- in addition to what has been reported about gcv. hence, its efficacy is yet to be confirmed as anti-sars-cov- therapeutic. tenofovir (tfv) ( ) is another adenine analogue pointed as promising covid- therapeutic (fig. ) , it is also called tenofovir disoproxil fumarate or alafenamide tenofovir (taf). approved by the fda in , tfv is a prodrug used to treat hiv and cases of nucleoside resistance. tfv is an analogue reverse-transcriptase inhibitor (ntrti). inside cells, tfv is phosphorylated and competes with deoxyadenosine '-monophosphate (d-amp), thus preventing the formation of dna. once incorporated into a growing dna strand, it causes premature termination of dna transcription and prevents viral replication. , modelling and docking studies evaluated the antiviral effects of tfv and verified a strong bond to sars-cov-rdrp, which can disrupt this polymerase and terminate the viral infection. however, in-vitro tests showed that tfv lacks apparent antiviral effect at concentrations inferior to μm for sars-cov- . in spite of lukewarm in-vitro and in-silico outcomes, an ongoing clinical study on tfv (chictr ), expected to end in june , aims at assessing the effect of the combination tenofovir + emtricitabine (cytidine analogue) related to lpv/r in covid- patients. in addition to the efficacy of treatment, clinical trials can validate the prevalence of adverse effects related to the toxicity of tfv in patients. tfv is also a powerful nephrotoxic drug causing damage to proximal tubular cells. in spite of that, interrupting the treatment is sufficient to improve adverse effects, which makes monitoring of patients essential. heterocyclic compounds with different heteroatoms such as nitrogen, sulphur and oxygen can present different pharmacological properties. one such property is to serve as analogue of nitrogenous bases of nucleic acids, such as triazoles, which have a five-membered ring of two carbon atoms and three nitrogen atoms. this aromatic ring can assume two isometric forms, , , -triazole and , , -triazole. the former is stable under acid and basic conditions and becomes more reactive when binding to electronegative elements. , triazoles are important and stand out for their various biological activities, such as anticancer, antituberculosis, anti-inflammatory, antimicrobial, and antiviral. specifically for the latter action, triazole-based derivatives have shown promising invitro activity against coronavirus, probably by cl pro inhibition. ribavirin ( ) (fig. ) is a powerful triazole-based antiviral analogue to guanosine. it presents a wide range of pharmacological activities related to several viruses, for instance: herpes simplex virus, human immunodeficiency (hvi- ), influenza, respiratory syncytial (rsv) and hepatitis c. , the drug was initially used in to treat syncytial virus in children, generally combined with interferon (inf). however, ribavirin treatment presents undesirable adverse effects, like lowering of haemoglobin, which limit clinical use. its action mechanism relies on the inhibition of enzyme inosine monophosphate dehydrogenase, necessary in the synthesis of guanosine triphosphate, which prevents viral dna and, mainly, rna replication. the necessary concentration for in-vitro inhibition of rsv and influenza ranges from - μg/ml. site similar to other sars-pl pro inhibitors. the formation of hydrogen bonds and π-π stacking were also predicted. these findings suggest that ribavirin as a powerful pl pro inhibitor. nonetheless, investigation on triazole derivatives for anti-sars-cov- therapy are still preliminary. on the other hand, favipiravir (fpv) ( ) (fig. ) is a prodrug, approved in in japan, to treat cases of influenza a and b that displayed resistance to first line drugs. it has provided results that indicate a promising character and is currently undergoing clinical trials against covid- . its antiviral efficacy has also been investigated in different countries to fight ebola and lassa, for example. the molecular structure of the drug consists of a pyrazine heterocyclic ring with fluorine at -position, carboxamide at -position and a double bond between oxygen and carbon , which renders its analogue to guanine ( ). , the metabolization of the prodrug into its active form, favipiravir-ribofuranosyl- '-triphosphate, requires intracellular ribosylation and phosphorylation. fpv therapeutic targets are rdrp enzymes, necessary in viral transcription and replication, and its inhibition blocks synthesis of viral rna for a spectrum of viruses, including human coronavirus. a different investigation compared patients treated with fpv plus interferon inhalation to lpv/rtv. patients under fpv therapy responded better to the progression of the disease with accentuated viral depuration. also, the incidence of nausea and vomit was higher for lpv/rtv. in addition to these clinical trials, the antiviral activity of fpv against sars-cov- was also evaluated but no clear antiviral effect was noted for doses lower than μm. an in-vitro study using molecular docking focused on the binding properties of sars-cov- protein structures to antiviral agents, including oseltamivir. the study showed that molecules form bonds to sars-cov- crystal proteins. however, data did not show oseltamivir as the best structure because lopinavir, asunaprevir and remdesivir interacted with more than two protein structures in the virus. hence, they are likely more promising than oseltamivir. notwithstanding, we suggest further look into oseltamivir against other enzyme targets since different studies achieved positive results regarding its use as anti-sars-cov- . nelfinavir ( ) (fig. ) is a safe anti-retroviral drug largely used for hiv- protease inhibition with strong in-vivo activity. generally, nelfinavir is combined with other anti-retroviral medication as part of a highly active antiretroviral therapy (haart) that reduces significantly the viral load by increasing cell number to mm - cd (+) lymphocytes. the drug is prescribed for children, young individuals, adults and pregnant women. , nelfinavir and its active metabolite m strongly bind to serum proteins, displaying optimal tissue distribution. a frequent adverse effect is light to moderate diarrhoea, reported for to % of patients. the sars-cov outbreak in several countries triggered the search for antiviral drugs active against the disease. among the drugs likely to inhibit sars-cov, nelfinavir stands out in all assays. the mechanism of action suggested for nelfinavir involves preventing sars-cov replication after its entry in the host cell and disrupting virion production. based on results from previous studies as well, nelfinavir was considered a likely therapy for covid- after its indication for clinical trials as a promising anti-sars drug. recently, , drugs were evaluated for their binding affinity to sars-cov- m pro . among the compounds, drugs were selected based on the docking score and three-dimensional atazanavir ( ) (fig. ) is an antiretroviral drug protease inhibitor used to treat hiv infections with in-vitro inhibitory concentration of , - , nmol. compared to other protease inhibitors, atazanavir has the advantage of allowing a daily posology regimen with a favourable metabolic profile and low frequency of adverse effects. , several hiv- resistant to protease inhibitors are still sensitive to in-vitro atazanavir, which is considered safe and well tolerated. the atazanavir acts to inhibit hiv- protease, which is indispensable in the processing of polyproteins precursors of viral structures and prevents the formation of infectious and mature viral particles. the good activities reported for this drug as well as the search for safe and fast therapy for captopril ( ) (fig. ) is an angiotensin-converting enzyme inhibitor (acei). it is a zinc metallopeptidases inhibitor that converts angiotensin-i into angiotensin-ii, an essential function that regulates arterial pressure. it is predominantly indicated as vasodilator in patients with cardiac insufficiency. this drug was suggested as potential antibiotic capable of inhibiting zinc succinyls/dipeptidase by blocking its zinc catalytic center. , tolerance to captopril has been largely investigated; its single dose by mouth is well-established and confirms the pharmacological activity in the short term ( - minutes) at the cellular level. this capacity is related to captopril transport mainly through plasma proteins such as albumins with absorption rate between - %. reported adverse effects are neutropenia, proteinuria, dysgeusia and cough, but less frequent for low doses. , some investigations have suggested captopril as possible covid- treatment. serafin et al. indicated captopril as potential for inhibiting the bond between human sars-cov- and ace- and reduce severe pneumonia symptoms. in-silico studies using molecular docking were conducted with fda-approved drugs capable of binding to the main active site in proteinase cl pro . two drugs were identified as ligands for the enzyme active site: captopril and disulfiram. the former binds to the active site at the same position of n inhibitor (a standard inhibitor that reacts irreversibly in the same site with cl pro cys ). it is, thus, suggested that captopril binds to the same site of n , obstructing the function of cys -his catalytic dyad. captopril probably inhibits the enzyme in two stages. initially, it establishes non-covalent bonds to sites in the enzyme targets, then, a reaction takes place between the critic groups, which results in a more stable inhibitor complex. the hypothesis is that captopril can bind covalently to cl pro cys . although the potential of captopril on the enzyme has been demonstrated, therapeutic use against covid- is controversial, once the drug induces overexpression of ace- -the main receptor used by sars-cov- to entry the cells. therefore, combination with other drugs, such as angiotensin-ii receptor blockers, needs analysis to clarify the effects of captopril in covid- treatment. the cyclosporin a (csa) ( ) (fig. ) is isolated from the fungus beauveria nivea and was approved for use by the fda in . this drug has been used for decades to prevent organ rejection and to treat t cell-associated autoimmune diseases such as behcet's disease, psoriatic arthritis, lupus nephritis, rheumatoid arthritis, systemic lupus erythematosus or interstitial lung disease. such drug exerts its immunosuppressive function and anti-inflammatory effects by inhibiting the transcription of genes required for t cell proliferation, notably interleukin- . [ ] [ ] [ ] due to the severity of covid- , csa can be potential to prevent hyperinflammation-induced lung injury. in this regard, it is known sars-cov nps induces the expression of interleukin- via nuclear factor of activated t cell (nf-at) activation, which can trigger the cytokine storm seen in patients with severe covid- status. another advantage presented by csa in relation to other antiinflammatory drugs is its already known anti-cov action against all genus, including sars-cov, [ ] [ ] [ ] at low and non-cytotoxic micromolar concentrations verified in cell culture assays. this antiviral property is thought to be mediated by the inhibition of cyclophilin-a-dependent viral assembly as well as inhibition of the nf-at pathway or even by genetic or pharmacological specific inhibition of cyclophilin-d, hindering the viral replication. , as already reported, sars-cov and sars-cov- are very similar ( . % sequence identity). teicoplanin ( ) (fig. ) is an antibiotic used against gram-positive bacteria with major compounds at different side chains. it prevents polymerization of peptidoglycans and inhibits the development of the cell-wall, thus prompting cell death. it is a big molecule that has displayed antiviral activity on an early stage of the viral life cycle by inhibiting the low-ph cleavage of the viral spike protein by cathepsin l in the late endosomes thereby preventing release of viral rna and replication. this compound has already shown inhibitory activity against ebola virus, mers-cov and sars-cov. recent investigations have suggested teicoplanin as alternative treatment for covid- after an in-vitro assay achieved ic value of . µm, thus proving its efficacy against sars-cov- . these results need to be confirmed through randomized clinical trials, which are still to be conducted. , , using antibiotics to fight viruses, albeit completely ignored, can become useful to treat covid- . , some studies report the possibility of repurposing drugs like terconazole, which displayed good in-vitro results against mers-cov and sars-cov, dasabuvir ( ) (fig. ) is a drug from the naphthalene class and phenyl-naphthalene subclass due to the bond between its naphthalene ring and a phenyl group. dasabuvir is a first line drug used as combined therapy for chronic hepatitis c. dasabuvir is a non-nucleoside inhibitor that binds to nps b (non-structural protein b -rdrp) and induces conformational change that makes rdrp incapable of elongating the viral rna. repurposing of this drug can be useful as sars-cov- therapy due to its antiviral activity. dasabuvir was subjected to docking studies against sars briefly, dasabuvir forms π-cation interactions with lys a (present in the ace ), π-π interactions with phe b (s protein residue) and hydrogen bonds to ace residues glu a and asp a and gly b and ser b (s protein residue). the authors highlight the importance of repurposing drugs as new therapeutic alternatives not only for the new coronavirus but for the next viral outbreaks. darunavir ( ) (fig. ) is a benzene derivative that has been evaluated for repurposing against covid- . this drug is an antiviral used in the treatment of hiv- infections. it provides a great genetic barrier to resistance and is highly active against resistant strains of hiv- that are not susceptible to other protease inhibitors. darunavir is administered orally as pills or suspension and is often used with low doses of ritonavir as part of a combined art protocol. its mechanism of actions works by protease inhibition. darunavir establishes high affinity bonds to hiv- protease forming a stable complex, thereby selectively inhibiting polyprotein gag-pol coded by the virus. this prevents the formation of mature viral particles. fda-approved drugs against cl pro , rdrp, helicase, exonuclease ′ a ′, endornase e ′-o-ribose methyltransferase. among the best drugs in the assay, darunavir was a surprise because, despite inhibiting viral proteinase, the study showed that it binds to the replication complex components of sars-cov- with inhibitory potency kd < nm. one example is rdrp, whose kd value was . nm and exonuclease ′ to ′ with k d value of . nm. a docking study was conducted by sang et al. as in-silico evaluation of anti-hiv drugs in their interaction capacity to proteinase cl pro . results suggest that all drugs have higher binding affinity to sars-cov- cl pro than to the homolog sars-cov proteinase. among the evaluated drugs, indinavir and darunavir displayed the highest docking scores, therefore, they were subjected to molecular dynamic (md) simulations, free binding energy calculations and molecular mechanics poisson-boltzmann surface area (mm-pbsa) to detail molecular interactions between inhibitors and proteinase. the data suggest darunavir had better binding affinity to sars-cov- cl pro with binding affinity of - , kj / mol. in addition, darunavir bind to sars-cov- cl pro via contact residues and to sars-cov cl pro via residues. this difference explains the lower binding energy values between darunavir and sars-cov cl pro . it was also noted hydrogen bonds between darunavir and sars-cov- cl pro but none for indinavir and this proteinase. because hydrogen bonds are important in the stability of the inhibitor-enzyme complex, darunavir is probably more promising against covid- . finally, a different in-silico assay was conducted by pant at al. to assess a large variety of compounds ( ) from several data banks and potential compounds from fda-approved drugs. the compounds were tested against sars-cov- cl pro . darunavir was among the best fda-approved drugs, with a score of - . . all data collected from in-silico studies still require experimental studies to validate the anti-sars-cov- activity of darunavir. a research conducted by dyall et al. performed a robust in-vitro assay and showed that the drug dasatinib ( ) (fig. ) , a kinase signalling inhibitor developed to treat human cancers, inhibited mers-cov and sars-cov, exhibiting ec values . and . , respectively. this study also revealed that kinase signalling may also be important for replication of this hcovs. nevertheless, the authors reported that dasatinib may be valuable against coronaviruses infections if a dosing regimen that minimizes immunotoxicity while still blocking viral replication can be defined. results indicated this drug as a likely therapeutic alternative against sars-cov- infection. an in-silico study carried out by qiao et al. showed that dasatinib, among others, is one of the most promising drugs for the inhibition of sars-cov- cl pro . more preclinical and clinical studies are required to prove whether dasatinib is really promising for covid- patient treatment. imatinib ( ) (fig. ) is an oral anticancer agent that inhibits the activity of some tyrosine kinases, most prominently the bcr-abl fusion oncoprotein (whose overactivation can lead to chronic myeloid leukaemia, cml), c-kit (involved in gastrointestinal stromal tumours development), platelet-derived growth factor receptor (pdgfr), and the native abl kinase, which has a ubiquitous expression and plays important roles in several biological processes. in addition to this well-known antitumor effect, imatinib has also shown in-vitro antiviral properties against several virus, such as infectious bronchitis virus (a viral model for studying the role of tyrosine kinase activity during cov infection), by interfering with virus-cell fusion, and other rna viruses including coxsackie virus, hepatitis c virus, ebola, among others, mainly by blocking viral entry or egress from the host cell. besides, this drug showed activity against sars-cov and mers-cov, both phylogenetically related to sars-cov- . in this regard, it is reported that imatinib has anti-cov activity in two points of the virus life cycle. in the early phases of infection, it inhibits virion fusion with the endosome and subsequent release into the cytoplasm, thus preventing viral entry and viral replication via abl-mediated cytoskeletal rearrangement. in a later phase of the infection, abl protein expression, which is inhibited by this drug, enables sars-cov and mers-cov replication, which suggests that abl is a new host cell protein required for viral growth. furthermore, evidences suggest that imatinib can modulate the immune response by sundry mechanisms, [ ] [ ] [ ] for several diseases, such as rheumatoid arthritis, asthma, and crohn's disease. this information insinuates that such drug might perform its potentially beneficial immunomodulatory role as a treatment alternative for covid- pneumonia. in addition, the use of imatinib as treatment appears to be reasonable from an economic point of view and its high availability in hospitals, since this drug is well tolerated and the risk of severe adverse effects is relatively low, especially in short-term administration. it is also recognized that adverse effects, mostly mild to moderate in intensity, will be easily controlled by dose reduction or discontinuation. in light of this information, tatar and turhan used the docking methodology to better understand the mechanism of inhibition of the sars-cov- n protein with antiviral compounds. based on this study results, imatinib was one of the highly binding affinities performed against the aforementioned target, with the lowest micromolar ki values among the compounds evaluated. in line with this study, an in-vitro research carried out by weston et al. found fda approved drugs that inhibited sars-cov- at non-cytotoxic concentrations. the authors indicate imatinib as one of the hits, since it exhibited ic value of . μm. they subsequently determined the mechanism of action, demonstrating this drug inhibits fusion of covs with cellular membranes, precluding their entry. this result indicates imatinib use against sars-cov- . however, its efficacy and safety need to be better confirmed in further preclinical and clinical trials in order of elect him as candidate drug in the treatment of covid- . synthesized for the first time by jean francois rossignol in the beginning of the s, the acetyloxy-n-( -nitro- -thiazolyl) benzamide, sold under the name nitazoxanide (ntz) ( ) (fig. ) , is the result of a structural modification in the antiparasitic niclosamide when a benzene ring is replaced with a nitrothiazole. , developed and sold as antiparasitic, ntz is also a first line broad spectrum antiviral with good results against parainfluenza, coronavirus, rotavirus, hepatitis and other respiratory infections. [ ] [ ] [ ] [ ] following oral administration, ntz is absorbed in the intestine, where it is rapidly hydrolysed by plasma esterase into the active metabolite tizoxanide. the mechanism of action of ntz varies according to the pathogen. in relation to antiviral activity, ntz blocks the maturation of the viral hemagglutinin at the post-translation stage in treatments against influenza. in treatments against hcv (hepatitis c), it activates protein kinase r (pkr), which leads to phosphorylation of the eukaryotic initiation factor- α thereby preventing translation. cao et al. conducted an in-vitro evaluation of ntz against recombinant murine coronavirus expressing the firefly luciferase (mhv- afls). the strand was pivotal to triage the drugs with likely anti-cov activity. the first assay resulted in molecules among which was ntz. the antiviral effect of ntz verified for mouse astrocytoma (dbt) and fibroblasts ( cl- ). the dbt cells infected with mhv- afls were treated with ntz at µm for hours, after which the viral titer (tcid ) was determined and viral n protein was subjected to western blot. results show the strong inhibitory effect of ntz on the viral titer. in-vitro studies on ntz or its metabolite tizoxanide were also conducted to verify efficacy against different coronaviruses. the replication of canine coronavirus (strain k ) in a cells, for instance, was blocked by the tizoxanide with ic of µg/ml. on the other hand, ntz inhibited the viral n protein in bovine coronavirus l (βcov-l ) and human enteric coronavirus (hecov- ) with approximate values of . µg/ml. , ntz is also responsible for inhibiting pro-inflammatory cytokines, such as tnf-α, il- , il- nitazoxanide ( ) drugs. more recently, molecules with a quinoline group have been widely investigated as treatment for the new coronavirus (sars-cov- ), such as chloroquine (cq) ( ) and hydroxychloroquine (hcq) ( ) (fig. ) that belong to the quinoline class and aminoquinoline subclass. both are quickabsorption synthetic drugs approved to treat malaria (plasmodium falciparum) by several regulating agencies in the world. cq and hcq are water soluble; the latter is more soluble due to presence of hydroxyl group. they are currently used to treat autoimmune diseases such as lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis as they have immunomodulatory and antithrombosis properties. [ ] [ ] [ ] therefore, these drugs could be useful against covid- due to the elevated levels of cytokine caused by cov infections in humans. the mechanism of action of cq for anti-malarial treatment is not entirely clear, but the interference with the digestion of haemoglobin by the parasite has been suggested. hcq has a similar mechanism, however, in regard to sars-cov- , clinical trials showed it to be safer. [ ] [ ] [ ] therefore, hcq, rather than cq, is used against sars-cov- . recent studies report antiviral activity of cq and hcq as they impair viral entry and release in different in-vitro and in-vivo models. , a factor that can also justify viral mechanisms is the aminoquinoline bioaccumulation in the tissues, as defended by patil, singhal & masand. a factor that facilitates viral replication is the acidic ph of endosomes, lysosomes and golgi complex of the host. thus, cq is promising because it increases the ph of intracellular vacuoles, binds to the cellular receptors, changes the glycosylation and because of its selective and reversible immunomodulator effect on human cd + t cells. hcq exerts similar mechanism of action: a) increases the ph; b) modulation of activated immune cells; c) reduces the number of proinflammatory cytokine and other mediators to control inflammation. it has also been suggested by roldan et al. a likely involvement of hcq in iron homeostasis during sars-cov- infection, which is a similar mechanism to other viral infections in humans. [ ] [ ] [ ] the little difference between the therapeutic and the toxic dose of cq is also known, and poisoning is related to cardiovascular complications that can be fatal. using either cq or hcq, then, requires strict prescription and self-medication is not advised. based on the recovery data, it was concluded that hcq is not effective in patients admitted with covid- . this highlights the importance of randomized clinical trials and the collection of clear data on the efficacy and safety of medications. ivermectin ( ) (fig. ) is an fda-approved broad-spectrum antiparasitic agent used in the treatment of tropical diseases, such as onchocerciasis, lymphatic filariasis, strongyloidiasis and lice. there is also evidence of its effectiveness in the management of myiasis, trichinosis, malaria, leishmaniasis, trypanosomiasis, chagas disease and schistosomiasis as well as bed bugs, inflammatory skin lesions, epilepsy, neurological diseases, tuberculosis and some cancers. it is known that ivermectin is capable of inhibiting the bond between a virus and the nuclear transport mediated by the superfamily of importin proteins (imp α/β ) based on the fact that sars-cov- is a rna virus deeply related to sars-cov, studies on sars-cov proteins have revealed a potential role for imp α/β during infection in signal-dependent nucleocytoplasmic shutting of the sars-cov nucleocapsid protein. furthermore, the sars-cov accessory protein orf has been shown to antagonize the antiviral activity of the stat transcription factor by sequestering imp α/β on the rough er/golgi membrane. considering the ivermectin nuclear transport inhibitory activity, such drug is widely believed as a promising therapeutic approach against sars-cov- . recently, caly et al. a different mechanism of action that consolidates the use of ivermectin against covid- is its immunomodulatory property. the inflammatory response (proinflammatory cytokine) is exaggerated in patients with the extreme case of the disease, which is likely explained by the hypoxiainducible factor (hif- α) that is activated by the virus when no inhibitory medication is administered. this understanding can be explained by the study conducted by kosyna et al. , whose lab tests with and without ivermectin aimed to examine whether the properties of the bond between hif- α and imp α/β and between hif- and nuclear localization signals were affected by the hypoxia mechanism on the cellular level. the authors concluded that ivermectin inhibited both imp α/β and hif- α. regarding this hinders its indication and clinical decisions. thus far, data indicate ivermectin is useful at the early stages of the disease, even the epidemiologic profile of covid- shows significant differences regarding the age of patients for the affected countries and patients with comorbidity. therefore, large scale randomized clinical trials are necessary to standardize clinical, laboratory and image evaluations, as well as combined drug therapy with vitamins and zinc, for example. financially, ivermectin is inexpensive and its doses and protocols are well established for different purposes. in addition, this drug has little side effects. indole, also named benzo [b] pyrrole, is a planar bicyclic heteroaromatic, whose ten π electrons move across its structure making this chemical group behave as a weak base. , the indole ring is the most abundant heterocyclic in nature and is commonly found in biologically active natural products, such as vegetables and seafood. it is also present in the structure of the essential amino acid tryptophan, which interferes with protein synthesis and with the regulation of physiological mechanisms such as precursors for serotonin and vitamin b . and adding the indole ring to spirothiazolidinones conducted to better influenza a/h n inhibition. now, the antiviral activity of indole and its derivatives for covid- therapy is supposed. arbidol ( ) is also a promising drug to fight covid- (fig. ) . this drug is classified as antiviral and has been used for there is high expression of eca , identified as human receptor for virus entry. notwithstanding, a different clinic trial concluded that arbidol monotherapy is best for the patient than lpv/rtv. despite the results, both investigations recognize their own limitations due to small cohort size and lack of placebo-control group. according to the authors, these limitations are inherent to pandemic times when placebo-control groups are difficult to conduct due to life-threatening conditions. most studies with arbidol use mg/ *day , , following the chinese guidelines. previous investigations on the pharmacokinetics of arbidol in healthy chinese patients showed that a single mg oral dose is sufficient for cmax de ~ , μm. this value was obtained through invitro assays that pointed the drug as effective and promising against sars-cov- . hence, clinical trials are still necessary to confirm the efficacy of arbidol at elevated doses to treat covid- . rizatriptan (rzt) ( ) (fig. ) is used to treat migraine and is a selective receptor of serotonin ( -ht) type b and d, structurally and pharmacologically related to other selective antagonists at these receptors. its structure is based on an indole ring replaced with methyltriazole at -position and unsubstituted ethanamine replaced with methyl at -position, the substitution sites are the same of melatonin (mlt). after virtual triage through molecular dock at spike-ace interface, ligations π-cation, interactions π-π and hydrogen bonds were identified between rzt and the sars-cov- protein complex. as one of the outstanding compounds in the analysis, in-vitro tests are still necessary. it is noteworthy that overdosing of the drug can trigger dizziness, fainting, cardiac issues, hypertension, bradycardia and vomiting. despite the safety of the drug in regular doses, there are no reports on either in-vitro or in-vivo tests to support the theoretical data and the antiviral action thus far. one last indole derivative that could be repurposed to treat covid- is melatonin (mlt) ( ) (fig. ) . mlt is classified as a hormone and nutraceutical, as it is naturally produced by the pineal gland and released into the bloodstream. it regulates the sleep-wake cycle as well as our mood, learning and memory, fertility, reproduction and the immune system. from a chemical perspective, it is an indole derivative with a methoxy group at -position and one ethylacetamide at -position. regarding its antiviral potential, mlt acts indirectly through anti-inflammatory, antioxidant and immune modulating activities. an investigation with murine infected with semliki forest virus (sfv) and west nile virus (wnv) showed the efficiency of mlt in reducing mortality rates for these viruses as well as in reducing the levels of pro-inflammatory cytokines. the anti-inflammatory and antioxidant properties of mlt point to its antiviral effects in humans. based on computational data, zhou et al. suggested using combined medication to fight sars-cov- . one such combination involves mlt plus mercaptopurine in synergic action against the following targets: pl pro , ace , c-jun signal and anti-inflammatory vias. therefore, experimental studies on modifications of ace pathways caused by mlt are useful to understand this drug. on the other hand, it has been suggested that using this neuro-hormone can mitigate the extreme form of the disease, the acute respiratory syndrome that has caused most deaths by sars-cov- cases. despite its safety for humans, the lack of data on the relevance of its use for covid- patients emetine ( ) is an approved anti-protozoal drug used against amebae with reported inhibitory activity for enterovirus infections, zika virus and ebola by interfering with the process of viral replication and entry in host cells. emetine is an isoquinoline alkaloid that presents methoxy groups in its structure (fig. ) . studies also confirm emetine has in-vitro activity against coronaviruses, including sars-cov and mers-cov. assays to clarify the activity of these drugs and the mechanism involved in the antiviral action of emetine both in isolation and combined to other drugs. another alkaloid candidate to repurpose against covid- is homoharringtonine (hht) ( ) (fig. ) . hht is an fda-approved drug in semi-synthetic form known as omacetaxine. this drug displays antitumoral activity in the treatment of myeloid chronic leukaemia. the mechanism of action implicates the ribosomal bond to prevent protein translation. in addition to antitumor activity, there are data in the literature that describe antiviral activity of hht against several types of viruses including covs. , a recent in-vitro evaluation of anti-sars-cov- activity displayed ec of . µm. however, the mechanism of action is not yet clear, which demands further investigation on ideal doses of hht to achieve the clinical results expected of a covid- therapeutic drug. the first reports on tetraethylthiuram disulfide, disulfiram (dsf) ( ) (fig. ) , date back to . however, only in the s that dsf would become popular when it was discovered that it could form copper chelates which favoured the death of micro-organisms and enabled treatment of intestinal parasites. [ ] [ ] [ ] in , dsf alcohol sensitivity was discovered accidentally and it was soon used in the clinical treatment of alcohol dependence. , dsf is used to treat alcohol dependence because it irreversibly inhibits the acetaldehyde dehydrogenase enzyme and modifies cysteine residues in its active site. this change prompts the formation of a disulphide bond between two cysteine residues in the active site. dsf effectiveness is based on its similarly to several proteins yielding a range of biological activities, such as antitumoral, , antimicrobial, and anti-sars and mers-cov. adding to the list of drugs to be repurposed against sars-cov- , recent studies indicate that dsf is able to inhibit other enzymes, such as methyltransferase, urease and kinase, all by reacting with important cysteine residues that suppress the natural cycle of the enzymes, suggesting broad-spectrum characteristics. , covs have two viral enzymes, m pro and pl pro , that are cysteine protease involved in the formation of structural and non-structural proteins that constitute the viruses and favour control of host cells. different assays, such as proteolytic and binding synergy assays, were also conducted and described. although outcomes indicate dsf for anti-mers-cov and anti-sars-cov therapy, to the present date ( th june ), no other article was published claiming the availability of the compound as promising anti-sars-cov- . recently, an announcement was published on the oxford university website on the results of one randomized evaluation of covid- therapy. more specifically, the study focused on dexamethasone ( ), a corticosteroid with fluorine at -position (fig. ) . the drug is mostly used as anti-inflammatory, which works by inhibiting vasodilation, reducing leukocyte migration to the inflammation site and increasing vascular permeability. immunotherapy is an effective intervention in viral infections. most attempts at immunotherapy were successful in fighting viruses similar to sars-cov- . the principal methods include vaccine, neutralizing antibodies (nabs) candidates and convalescent plasma. , , , , , , in addition, according to the evidences from viral infections (ebola, influenza, sars and mers), immunotherapeutic interventions can reduce viral load and mortality rate of patients. , development of either monoclonal (mabs) or polyclonal (pabs) neutralizing antibodies is a commonly adopted immunotherapeutic alternative due to its specificity, purity, low contamination by blood-transmitted pathogens and relative safety. however, there are limitations to the use of nabs once its development and large-scale production for clinical use are a complex, expensive and slow process. promising scientific investigations have suggested using mabs or pabs as prophylactic and therapeutic measures against influenza and hcovs, such as mers-cov and sars-cov. targets reported as promising for hcovs immunotherapy were cytokine, s -receptor-binding domain (s -rbd), s n-terminal domain (s -ntd) and some other region of subunit s in order to block the rbds bonds to their respective receptors and to interfere either with s -mediated membrane fusion or with the entry in the host cells, thus inhibiting infection. these researchers have encouraged the development of nabs with cross reactivity potential and/or cross neutralization effect on sars-cov- infections, as shown by tian et al. data suggest that mab cr can be developed as therapeutic candidate, either isolated or combined with neutralizing antibodies to prevent and treat covid- , given that it could potently form bonds with sars-cov- rbd (kd of . nm). a different study by wang et al. reported the discovery of a human mab ( d ) that promoted cross neutralization of sars-cov and sars-cov- in a culture of cells through an independent receptor-binding inhibition mechanism that targets a conserved epitope on the spike hcovs rbd mentioned above. it is also reported the on-going investigation of convalescent plasma or immunoglobulin as last resource to improve the survival rate of patients with several viral infections such as h n avian influenza, a possible explanation for the efficacy of convalescent plasma is that immunoglobulin antibodies in the plasma of recovered patients can suppress viremia. shen et al. reported that five patients with extreme symptoms of covid- received blood transfusion containing convalescent plasma with specific sars-cov- antibodies. after a series of blood transfusions, the improvement in clinical status of patients was observed. viral loads also decreased and became negative within days after the transfusion, and sars-cov- -specific elisa and nabs titers increased following the transfusion. in spite of the limited sample, the authors concluded that convalescent plasma transfusion benefited patients infected with sars-cov- . therefore, testing safety and efficacy of transfusing convalescent plasma in patients infected with sars-cov- can be of value. , among the modalities of immunotherapy, vaccines are expected to be more promising, hence the global engagement in their production. over the last decade, the scientific community and the vaccine industry had to answer urgently to the epidemics of h n , ebola, zika and, more recently, sars-cov- . vaccine development is an expensive and slow process with high risks of failure, which often motivate developers to follow a linear sequence of steps with several breaks for data analysis and fabrication processes. therefore, it is fundamental that vaccines be developed through faithful methods even if it takes longer to move them onto clinical trials or to make a large number of doses available, a challenge during a pandemic. developing efficient vaccines for sars-cov- will be essential to reduce the severity of the disease, viral shedding and transmission to control future outbreaks. prior to the covid- pandemic, multiple strategies were used to generate vaccines for the first hcovs (sars-cov and mers-cov). several studies related to sars-cov vaccine production targeting the protein s, due to its function in the receptor binding and fusion to the host membrane, were successful in animal tests against that coronavirus. - these vaccines employed live-attenuated virus vaccines, killed virus, dna vaccines and viral vector vaccines. theoretically, these techniques could be applied to develop sars-cov- vaccines given their similarities from both the genomic perspective and the mechanisms employed in the invasion and infection of host cells. gao et al. promoted the pilotscale production of a purified inactivated sars-cov- virus vaccine candidate (picovacc), which induced sars-cov- -specific nabs in mice, rats and non-human primates. in addition, three immunizations using two different doses ( μg or μg per dose) in macaques provided partial or complete protection against the sars-cov- challenge, respectively, without observable antibodydependent enhancement of infection. these data reinforce the use of picovacc in the next steps of clinical trials targeting sars-cov- still for the present year. given the magnitude of the covid- pandemic, it has become indispensable to work as fast as possible to develop vaccines for global distribution. however, protocols are necessary to safekeep the population's health. hence, before allowing human testing of covid- vaccines, regulatory organizations must evaluate their safety against a series of virus strains and more than one animal model. they also must demand preclinical evidences that experimental vaccines prevent infectioneven if it means waiting weeks or months for models to become available. this is time well-spent, once testing vaccines without investing the due amount of time to completely understand the risks can lead to setbacks for current and future pandemics. repurposing potential relies on the mechanism for other viruses, such as hepatitis c, by inducing conformational changes that compromise rdrp activity. it was also possible to identify benzene derivatives used to treat cancer (dasatinib and imatinib) and one antiviral (darunavir), but predominant in-silico and some in-vitro outcomes demand more conclusive studies. representative of benzoic derivatives, ntz displayed significant inhibitory activity against pro-inflammatory cytokines, which can benefit control of ards, in spite of an unclear mechanism against sars-cov- . quinoline derivatives, hcq and cq, were some of the first drugs investigated. after several in-silico, in-vitro and in-vivo assays, based on results presented by recovery to the present date, unfortunately, these drugs were proven ineffective in hospitalized covid- patients. ivermectin represents macrolide derivatives and is suggested as promising due to both immunostimulatory activity and inhibitory activity on nuclear transport when administered at the early stages of the disease. among indole derivatives, arbidol was pointed out as useful for reducing viral binding and releasing of intracellular vacuoles that contain the virus. nonetheless, clinical trials are still necessary after dose adjustment for better outcomes. both indole derivatives (emetine and hht), in spite of promising results, were only submitted to in-silico and in-vitro tests, thus demanding further investigation on their toxicity and mechanism of control. hence, the currently available data on the classes of drugs investigated here revealed that drugs were considered promising mainly after in-silico tests only. in fact, the inefficacy of some of these drugs became evident after in-vitro or in-vitro tests, as cq and hcq, whose clinical trials failed to confirm the so-expected anti-sars-cov- activity. it is necessary to highlight the importance of clinical trials with drugs considered promising in theoretical studies, with due calm and openness to question and refute hypotheses, in the absence of scientific evidence to support their use to treat covid- . similarly, the 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de amparo à ciência e tecnologia none. all authors researched data for the article, contributed substantially to discussion of the content, wrote the article and reviewed and edited the manuscript before submission. key: cord- -pjjvfdnq authors: fontana, juan; lópez‐montero, noelia; elliott, richard m.; fernández, josé jesús; risco, cristina title: the unique architecture of bunyamwera virus factories around the golgi complex date: - - journal: cell microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: pjjvfdnq viral factories are novel structures built by viruses in infected cells. during their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. we have studied how a bunyavirus uses the golgi to build the factory. with the help of confocal and d ultrastructural imaging together with molecular mapping in situ and in vitro we have characterized a tubular structure that harbours the viral replication complexes in a globular domain. numerous ribonucleoproteins were released from purified tubes disrupted in vitro. actin and myosin i were identified by peptide mass fingerprinting in isolated tubes while actin and the viral nsm non‐structural protein were detected in the tubes' internal proteinaceous scaffold by immunogold labelling. studies with nsm deletion mutants and drugs affecting actin showed that both nsm and actin are key factors for tube and virus assembly in golgi. three‐dimensional reconstructions based on oriented serial sections of infected cells showed that tubes anchor cell organelles to golgi stacks and make contacts with intracellular viruses. we propose that this new structure, unique among enveloped viruses, assembles in association with the most stable component of golgi stacks, the actin‐containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories. rna viruses replicate their genome in intracellular membranes of infected cells (mackenzie, ; salonen et al., ) . modified membranes harbouring viral replication complexes (rcs) frequently integrate into a complex structure known as the 'viral factory' where the cytoskeleton participates, cell organelles are recruited and the different steps of the virus life cycle are sequentially connected. characterizing how this happens would help to understand how viral factors take control of cells and modify their architecture. there is no description of the mechanisms involved in the formation of most viral factories, although there is evidence that some viruses induce aggresomes and autophagosomes to generate sites of replication (novoa et al., a; wileman, ) . expression of early proteins such as viral polymerases is probably sufficient to program the cell for organelle recruitment and factory formation, as recently observed in cells transfected with rubella virus replicons (fontana et al., ) . the viral replication machinery is usually inserted in single-or double-membrane vesicles that can be associated with a variety of organelles, such as the rough endoplasmic reticulum (rer), mitochondria, the endolysosomal system and chloroplasts or vacuolar membranes in plants (hagiwara et al., ; novoa et al., a; salonen et al., ; kopek et al., ) . some viruses, e.g. polioviruses, induce proliferation of specific membranes creating a 'novel compartment' (cherry et al., ) where the viral rna polymerase molecules can assemble bidimensional arrays (hobson et al., ; lyle et al., ) . although the purpose of this targeted localization has not been elucidated, one possibility is that association of rcs with membranes provides a structural framework for efficient replication and transfer of replicated rna to assembly sites in contiguous membranes. bunyaviruses comprise a large family of rna enveloped viruses that includes serious emergent pathogens for humans, animals and plants, and are responsible for severe episodes of encephalitis and haemorrhagic fevers in humans (elliott, ) . they assemble a large factory involving the golgi complex where virus particles bud and mature (salanueva et al., ; novoa et al., b) . bunyamwera virus (bunv) serves as a model for the many pathogens within this family. it contains three rna segments of negative-sense polarity. the large segment (l) codes for an rna-dependent rna polymerase (l protein), the medium segment (m) codes for a precursor polyprotein (nh -gn-nsm-gc-cooh), which is cotranslationally cleaved to yield the two virion glycoproteins (gn and gc) and a non-structural protein called nsm, and the smallest segment (s) codes for the nucleoprotein n and a second non-structural protein nss in overlapping reading frames (elliott, ) . the nh -terminal domain of nsm is essential for bunv morphogenesis (shi et al., ) while nss is a non-essential protein that contributes to viral pathogenesis (bridgen et al., ) . there is no ultrastructural description of the bunyavirus replication site that has been defined as 'cytoplasmic' (nichol et al., ) . morphogenesis of coronaviruses, arteriviruses, rubiviruses and bunyaviruses is associated to the golgi complex. the choice of the golgi for viral replication or assembly is somehow surprising. the golgi is a highly dynamic organelle whose function requires continual membrane and protein flow (james morre and mollenhauer, ) . on the other hand, the golgi may be an autonomous organelle with a stable framework (seemann et al., ) . its unique architecture is thought to depend on cytoplasmic matrix proteins and the cytoskeleton. actin seems to participate in the preservation of the flattened shape of golgi cisternae (lazaro-dieguez et al., ) and several actin-binding proteins are known to play some role in golgi function. these include myosins and spectrins (beck, ) . we have studied how a bunyavirus modifies cell structure and builds factories around the golgi complex from early to late steps of its life cycle. in a previous study we observed that golgi stacks contain peculiar virus-induced tubular structures in bunv-infected cells (salanueva et al., ) . with the help of three-dimensional reconstructions and molecular mapping in situ and in vitro we have characterized these tubular elements and discovered that they represent a new structure of viral and cellular origin. tubes assemble in golgi stacks where they seem to be involved in multiple functions including viral genome replication, transfer of viral ribonucleoprotein complexes to assembly sites and viral morphogenesis. we propose that this new multifunctional structure associates with the actin-containing matrix of the golgi stacks providing a stable scaffold for viral replication and early morphogenesis. changes in cell organization during assembly of the viral factory were studied by confocal microscopy (fig. ). control monolayers (fig. a) showed round-shaped nuclei (blue), perinuclear golgi elements (red) and welldefined straight actin stress fibres (green). infected cells (fig. b) have a completely different aspect: nuclei are elongated, the golgi is rounded and concentrates on one side of the nucleus, and stress fibres have moved to the cell periphery. no changes in microtubules or vimentin filaments were observed (not shown). in addition to the wga trans-golgi marker shown in fig. a and b, two additional golgi markers (giantin and galactosyltransferase or gal-t) exhibited a similar transition from a perinuclear distribution in non-infected cells ( fig. c and d) to a more compact pattern on one side of the nucleus in infected cells ( fig. e and f). when observing just nuclei-associated staining a depression was frequently detected, corresponding to the location of the viral factory ( fig. g and h). electron microscopy (em) showed factories as groups of organelles near the nucleus (fig. i) . higher magnifications showed round-shaped and tubular structures in golgi stacks ( fig. j and k). we have detected these structures in several mammalian cultured cell types that support bunyavirus replication such as bhk- (this study), vero (salanueva et al., ) and cho cells (not shown). their number was higher early in infection and their viral origin was demonstrated in previous studies (salanueva et al., ) . in the present work we have studied a large number of serial sections and discovered that viral tubes actually contain a tubular domain and a bigger globular domain on one of the extremities (fig. l ). the tubes are open to the cytoplasm (arrows in fig. k -m). bunv generates round-shaped and tubular structures in infected cells whose representative views in thin sections of epoxy resin are summarized in fig. a -h. the golgiassociated morphogenetic pathway of bunv and the structural and biochemical characterization of the three viral forms have been previously described in detail (salanueva et al., ; novoa et al., a,b) . global understanding of all these structures has been possible through a careful analysis of consecutive serial sections (see below). condensation of material in individual golgi sacculi originates arcs opened to the cytosol ( fig. a) , elongated globular structures of low internal electrondensity (fig. b) and complete viral tubes with both globular and cylindrical domains (fig. c) . the two types of cross-sections originated by a viral tube are shown in fig. d and e. cross-sectioned globular heads are always > nm in diameter and most of these structures have a diameter of - nm. the cylindrical domain of viral tubes has a cross-section of - nm in diameter. virus particles have smaller diameters (fig. f-h) . budding profiles in golgi membranes generate immature viruses or vi that are released into the lumen of a golgi sacculus (fig. f ). intermediate intracellular virus or vii exits the golgi in secretory vesicles (fig. g ) and mature extracellular virions or ve are seen in the extracellular environment (fig. h ). all three viral forms have a diameter of around nm in thin sections of epoxy resin for ultrastructural studies. in cryosections for immunogold detection of specific components structural details of viral tubes and viruses change ( cell fractionation and centrifugation in optiprep gradients allowed isolation of viral tubular structures (fig. a) . negative staining confirmed their general morphology: they contain a cylindrical and a globular domain ( fig. b and c) similar to tubes in situ (fig. l ) and a filamentous internal texture (fig. d ). dsrna was detected in isolated tubes by dot-blot (fig. e ). viral proteins involved in genome replication such as the rna pol and the nuc protein were detected by western blot and coomasie blue staining, respectively (fig. f ). controlled disruption of isolated tubes by saponin treatment showed a proteina-ceous internal scaffold (fig. g ) that was labelled with anti-pol and anti-nuc antibodies ( fig. h and i). complete disruption of tubes provoked the release of viral ribonucleoprotein complexes or rnps (fig. j ). similar release of rnps was observed when disrupting isolated viruses in vitro (novoa et al., b) . nsm is a transmembrane non-structural protein involved in bunv morphogenesis (shi et al., ) that accumulates in the golgi region of infected cells (lappin et al., ; shi et al., ) . immunogold and em showed that nsm localizes both in the cylindrical ( fig. a and b) and globular (fig. c ) domains of golgi-associated viral tubes. nsm was detected in golgi membranes where new virus particles are assembling (fig. d ). western blot analysis confirmed the presence of nsm in isolated viral tubes and intracellular viral intermediates i and ii while extracellular mature virions lacked the protein (fig. e ). the same result was obtained by immunogold detection of nsm and em of isolated viruses disrupted in vitro (not shown). this distribution is typical for the behaviour of scaffolding proteins that assist in the assembly of immature viral precursors and are later degraded by proteolysis during maturation (steven et al., ) . to obtain functional information about the role of nsm in viral tubes and viral particles three nsm mutants were characterized at the ultrastructural level ( fig. f -h). these viruses obtained by a reverse genetics approach have deletions in nsm domains iii and iv (shi et al., ) . they grow slowly generating maximum virus yields -to -fold lower than wild-type bunv. when studying ultrathin sections of cells infected with nsmd mutant virus, viral tubes were rarely seen. more than cells infected with wild-type bunv and an equivalent amount of cells infected with nsmd mutant virus at , and h post infection (p.i.) were studied by oriented serial sectioning. while more than viral tubes per cell were detected when infecting with the wild-type virus, a total of complete tubes (longitudinal sections of the cylindrical domain) were seen in~ cells infected with the mutant virus. tubes assembled by the nsm mutant were apparently less rigid than normal, had small heads and exhibited a diameter of - nm (fig. f ). intracellular immature viral intermediates (type i viruses) accumulated in golgi membranes which suggests a blockade in viral particle maturation (fig. g) . a significant amount of viral glycoproteins was visualized at the plasma membrane by confocal microscopy (not shown) and budding profiles were also frequently seen at the cell surface (fig. h ). similar results were obtained with nsmd and nsmd deletion mutants, with small differences in the amount of intracellular virus particles and budding profiles in plasma membrane compared with nsmd virus (not shown). these data confirmed an active participation of nsm protein in tube structure and virus assembly in golgi, and pointed to viral tubes as elements involved in viral morphogenesis. matrix-assisted laser desorption ionization (maldi) peptide mass fingerprinting and database searching (navarro-lerida et al., ) was performed to detect cellular proteins in purified tubes. the highest scores obtained from this study corresponded to cytoskeletonassociated proteins (actin, myosin i and tubulin), several ribosomal proteins, the eukaryotic translation elongation factor and a retinoblastoma-like protein (table ) . considering the filamentous internal structure of the tubes ( fig. d ), we focused our attention on the cytoskeletal proteins. confocal microscopy showed that although actin is mostly removed from the viral factory some spots of actin remained in the modified golgi region (fig. a ). omitting the actin and golgi-associated signals showed a weak to-pro blue staining that most probably corresponds to rna accumulation in the factory (fig. b ). when the golgi signal is omitted, thin filamentous elements of actin are distinguished (fig. c ). the dimensions of these filamentous structures are compatible with those of viral tubes as seen by em. actin was detected by western blot in isolated tubes and intracellular viruses while it was absent in extracellular mature virions (fig. d ). actin was also detected by immunogold labelling in tubes and viruses as shown in cryosections of infected cells ( fig. e and f) and on isolated tubes ( fig. g -i). intact tubes exhibited a very weak or no immunogold signal (fig. g ), while tubes opened with saponin 'on grid' showed an enhanced signal on the released protein aggregates ( fig. h and i). an actinassociated signal was also detected on protein aggregates released from intracellular immature viruses disrupted in vitro (not shown). these results confirmed the presence of actin as a component of the internal fibres of viral tubes and its incorporation into immature viruses. assembly of viral tubes in golgi stacks could involve golgi membrane components or more stable golgi matrix proteins. to test these possibilities we used brefeldin a (bfa), a drug that induces the rapid fusion of golgi membrane with pre-golgi membranes and rer leaving a separated scaffold of matrix components (seemann et al., ) . from previous studies we knew that drugs disrupting cis-and medial-golgi subcompartments block bunv replication when added at - h p.i. (salanueva et al., ; novoa et al., a) . thus, short bfa treatments were applied to already infected bhk- cells. drug was added to the cultures at h p.i., followed by immunofluorescence staining for galt or giantin ( fig. a-f) . at or min after adding bfa the immunofluorescence signal for galt, a golgi membrane enzyme, was rapidly fragmented and dispersed while giantin, a golgi matrix protein, stayed in the perinuclear location for longer times. similar effects were seen after just min of treatment (data not shown). viral tubes deprived of surrounding golgi membranes had normal globular and cylindrical domains ( fig. g -i) and maintained their internal proteinaceous scaffolds and contacts with mitochondria ( fig. g ). tubes with apparent connections with cytoplasmic fibres that remind cytoskeletal intermediate filaments were observed (fig. h ). viral tubes with short bfa treatments then exhibited the same behaviour than giantin and remained as distinguishable structures when golgi stacks have already disappeared. several drugs that affect actin such as latrunculin a (lta), cytochalasin d (cyd) and jasplakinolide (jpk) were also used to target actin associated to golgi membranes. among them, the actin-stabilizing drug jpk provided the clearest results. lta and cyd had very little or no effect, respectively, on viral replication and assembly as determined by measurement of infectious viral particles released to the culture supernatants at and h p.i. complete tubes, virus budding profiles and viral particles assembled in golgi stacks of cells treated with lta, as observed in thin sections of treated cells studied by em (not shown). however, treatment with jpk led to a decrease in infectious virus release by - % in cell cultures that simultaneously lose the actin stress fibres as observed by fluorescence microscopy (fig. j and k) . em showed that in cells treated with jpk cell organelles were displaced to the cell periphery against the plasma membrane (not shown) and golgi fragments were occasionally distinguished (fig. l) . intact viral tubes were not detected although their globular domains were seen in golgi remnants (fig. l ) and attached to mitochondria (not shown). viral assembly was massively displaced to the plasma membrane where budding profiles were frequently seen (fig. m) . intracellular budding and viral particles in golgi remnants were very scarce and observed exclusively in cells less affected by the drug according to characteristic jpk effects in ultrastructure. budding profiles were not distinguished at the plasma membrane of normally infected cells where budding events were exclusively located in golgi stacks, as confirmed by analysis of oriented serial sections covering all planes from each infected cell. at least cells per condition were studied. our results strongly support a functional participation of golgi-associated actin in assembly and function of viral tubes as well as in anchoring virus assembly sites in golgi membranes. as viral tubes could play multiple functions in bunyavirus factories (replication, morphogenesis and organelle recruitment) a three-dimensional view of how they integrate in the factory and the contacts they establish with factory components can help us to understand how they work. we obtained serial sections of factories for a detailed study of the whole, complex structure. analysis of all planes provided a much better characterization of factories compared with random planes with a more accurate quantification of numbers and dimensions of viral structures. most tubes have a total length of . - . mm although tubes longer than mm were occasionally seen. d reconstructions from serial sections were done for early ( fig. a -f) and late (fig. g-i) factories. early factories with few viral particles are round shaped structures near the nucleus and surrounded by mitochondria (fig. a) . a close-up of the central areas of this factory showed viral tubes that connect golgi stacks with rer cisternae (fig. b) . the consecutive sections corresponding to these tubes are shown in fig. c . this frequent contact was never detected after random analysis of factories in d which points to the importance of covering all the planes of very complex structures in an oriented manner. an important contact previously detected in d analysis occurred between tubes and mitochondria ( fig. d and e) . careful study of d maps revealed that early factories are indeed composed of repetitive units of a basic set integrated by a golgi stack with mitochondria, rer and one or more viral tubes (fig. f ). by contrast, late factories are less compact structures that have abandoned the juxtanuclear location and contain many viral particles (fig. g ). mitochondria and rer do not completely surround the factory and virus particles are moving towards the cell periphery. physical contacts between viral tubes and new viral particles were detected as shown in the encircled area of fig. g and in fig. h and i. the existence of these contacts supports a role for the viral tubes as the physical connections between viral replication and morphogenesis inside the golgi stacks of the virus factory as proposed in the model of fig. . viral factories are probably the most extreme examples of how viruses manipulate cell organization. it is remarkable that just a few different classes of viral macromolecular complexes can transform a whole eukaryotic cell in minutes. we are interested in understanding how this happens. at the same time, by studying viruses, we have a very valuable tool to study cell architecture. in the present work we have characterized the bunyavirus factory built around the golgi complex. although the whole factory is large and complex it is in fact composed of repetitive units constituted by one or more golgi stacks, mitochondria, rer elements and a tubular structure that acts as a link between these organelles. further, the tubes could play multiple functions such as transfer of replicated viral genomes to assembly sites. actin is needed both for assembly of these tubes and for virus morphogenesis in golgi membranes. the presence of tubular structures in thin sections of mouse brain infected with bunv was reported in old literature where they were interpreted as elongated virus particles or nucleocapsids (murphy et al., ) . we also detected these structures in a previous study (salanueva et al., ) and estimated a discreet number of tubes, or , per golgi stack. analysis of serial sections has provided us with a much more accurate appreciation of their real numbers, as all planes of the cell are analysed. as a consequence several new data have been obtained: tubes are in fact more abundant (more than in many cells) and frequently connect with mitochondria and rer cisternae. our structural characterization of viral tubes in golgi membranes was completed with a molecular mapping of the structures both in situ and in vitro after their purification from infected cells. identification of viral factors involved in genome replication and morphogenesis as well as cell proteins such as the translation elongation factor or ribosomal proteins suggested that tubes may harbour fig. . d maps of early and late factories obtained by serial sectioning, tem, d-reconstruction and image processing. a. large round-shaped early factory near the nucleus. mitochondria (red) surround a network of membranes (yellow for the rer and beige for golgi stacks) where viral tubes (grey) are embedded. b. d reconstruction of the encircled central region of the factory in (a) elaborated with the central sections of the series to show contacts between viral tubes and cell membranes in more detail. the large tube is on the centre, a smaller one is encircled. c. three original serial thin sections included in the d map shown in (b). the central tube is marked with arrows. d and e. whole volume (d) and one single plane (e) of the same area of a factory showing a clear contact between a viral tube and a mitochondrion (arrows). f. basic factory unit constituted by a golgi stack, rer elements, mitochondria and a viral tube. a viral particle is coloured in purple. g. d map of a late factory. a significant displacement of components (mitochondria in red, rer in yellow) from the nuclear cavity (arrow) to peripheral areas of the cell is observed. golgi-associated density has been omitted in this volume for better visualization of viral particles (purple). a contact between viral tubes (grey) and viral particles (purple) is encircled. h and i. original thin section and d reconstruction, respectively, of a viral tube touching a viral particle (arrow). n, nucleus; ne, nuclear envelope; pm, plasma membrane; v, extracellular virions; c, centrioles. bar: nm in c; nm in h. post-replication events. a functional analysis of how altered nsm sequences or actin integrity affect factory architecture and viral assembly demonstrated that viral nsm protein and golgi actin are essential for the structure and function of the tubes. a sequence database comparison with bunv nsm showed homology with nsm proteins from other bunyaviruses and a low ( %) similarity with human atp p x receptors. immunoprecipitation assays showed that nsm interacts with n protein and the viral glycoproteins in infected cells (shi et al., ) . interestingly, nsm from tospoviruses, the plant-infecting bunyaviruses, is a movement protein associated with transport of rnps through plamodesmata (storms et al., ) . in mammalian cells tube-associated nsm could facilitate the transport of rnps from the rcs to the assembly sites. our data suggest that nsm could act also as a 'matrix' protein, facilitating binding of rnps to the cytosolic domains of viral glycoproteins gc and gn in golgi membranes. in fact its behaviour as a scaffolding protein that assists in viral assembly is supported by its presence in immature viral intermediates and its absence in mature extracellular virions. during tube assembly nsm aggregates seem to grow from the cytosolic side towards the interior of golgi sacculi creating a new space (fig. k) . as in situ labelling experiments showed that actin and giantin are inside viral tubes when they are normally facing the cytosolic side of golgi membranes, interactions of viral proteins with golgi actin and matrix proteins could be essential for tube assembly. despite its highly organized structure the golgi complex is a very dynamic organelle (james morre and mollenhauer, ) . nevertheless, the golgi manages to maintain its high degree of structural organization thanks to a large group of golgi-resident proteins that form a matrix. various cytoskeletal networks together with coiled-coil proteins of the golgin family, such as the cis-golgins p , gm and giantin, are components of this golgi matrix (short et al., ) that has been visualized by em of detergent-extracted golgi stacks (fath and burgess, ) . microtubules and associated motor proteins and the actin cytoskeleton are of particular importance in golgi organization. accordingly, several myosin motors localize to the organelle, where they are thought to contribute to the formation and transport of golgi vesicles. complex molecular machineries regulate actin dynamics involved in transport events in golgi membranes (fath et al., ; beck, ) . viruses manipulate actin in many different ways. they use actin for entry, intracellular transport, or cell-to-cell transmission (pelkmans et al., ; fackler and krausslich, ; radtke et al., ) . actin is involved in replication and transcription of both nuclear and cytoplasmic viruses and the infectious particles of retroviruses, herp-esviruses, and picornaviruses contain actin (grunewald et al., ; radtke et al., ) . when the actin cytoskeleton is destroyed with specific drugs a compaction of the golgi complex is observed (lazaro-dieguez et al., ) . curiously, this altered golgi is very similar to the round-shaped organelle we observed in bunv factory whose assembly occurs simultaneously with relocation of stress fibres to the cell periphery (fig. ) . in an attempt to test the potential function of actin in the structure and function of viral tubes we have used three actin-disrupting drugs. two of them depolymerize filamentous actin (cyd and lta) while the third one stabilizes filamentous actin (jpk). jpk stabilizes actin filaments in vitro, but in vivo it induces polymerization of monomeric actin into amorphous masses enhancing the rate of actin filament nucleation and disordered polymeric actin (bubb et al., ) . we used three drugs with different actin binding sites and different mechanisms of action because we wanted to target molecules associated to a very particular structure, the tubes inserted in golgi stacks. although we knew that viral tubes are open to the cytosol, we were not sure about efficiency and accessibility of drugs to actin molecules integrated in the tubular structure. in fact, while actindepolymerizing drugs had little effect on viral tube structure and virus production, the f-actin-stabilizing drug jpk altered the structure of viral tubes, the location of virus assembly sites and the release of infectious virus particles to the culture supernatants. golgi-associated actin seems to play a structural role in maintaining the structural integrity of the viral tubes in golgi membranes. brefeldin a is a drug that induces redistribution of most golgi-localized proteins to the er. studies on the nature of the golgi matrix scaffold showed that giantin, gm , grasp and grasp end up in membranes called bfa remnants that are distinct from the er (seemann et al., ) . these results supported the idea of a preexisting template for golgi stacks composed by these and other proteins. by using bfa we wanted to study if viral tubes disappear immediately when golgi membranes fuse with the er or if they stay for longer times following a common behaviour with the golgi matrix scaffold. like giantin viral tubes resisted short bfa treatments, which suggests they could be bound to the stable scaffold of golgi stacks. interestingly, integrity of golgi actin and viral nsm protein is also important for viral tube structure and normal production of infectious virions. our working hypothesis is that assembly of these viruses happens in golgi membranes because replication machinery anchors in golgi matrix components. a detailed study with silencing experiments and transfections with mutated components will be necessary to determine if particular golgi matrix components are specifically involved in the organization and activities of viral tubes. also to explore if myosin i molecules detected in isolated tubes are partici-pating in transport of rnps from the rcs to the assembly sites in nearby golgi membranes. according to our data, in particular the information coming from d maps and molecular detection, multiple interactions and movement of molecules must take place in viral tubes, as proposed in the working model of fig. . tubes represent a new structure that, in communication with the cytosol, would host viral rna replication and assembly of rnps in a protective environment, facilitating the posterior transfer of rnps to the assembly sites. the unusual cylindrical shape of this viral rc-containing structure might be related with the organization of golgi sacculi where viral tubes are anchored. actin and other matrix proteins can form the cellular protein scaffold for nsm interactions and tube growth while the actomyosin-based motors might mediate macromolecular movements. contacts with rer and mitochondria most probably provide necessary factors for tube functions. future work aims to locate viral and cellular proteins in tubes after d reconstruction by electron tomography (cyrklaff et al., ; mcintosh et al., ) . although factories are very large and complex structures for electron tomography we plan to use d maps from serial section reconstructions to assist in segmentation and interpretation of tomograms. in fact, our results also demonstrate that methods of d reconstruction from serial sections can benefit from the same principles of segmentation and noise reduction common to electron tomography. these procedures provide three-dimensional maps with enough resolution and details to analyse contacts between small elements such as virus assemblies inside very large and complex structures such as whole eukaryotic cells. this will hopefully help us to understand how viral macromolecular complexes interact with cell components to create the unique architecture of virus factories. bhk- (c ) cells supplied by the american type culture collection (atcc) were grown in dulbecco's modified eagle's medium supplemented with % foetal calf serum from reactiva s.a. (barcelona, spain). bunv (atcc vr- ) was propagated and subjected to titre determination in bhk- cells by a lysisplaque-forming assay, as previously described (salanueva et al., ) . bunvs containing deletions in the gene for nsm protein and designated rbunm-nsmd (aminoacids - were deleted from the m polyprotein), rbunm-nsmd (deleted aminoacids, - ) and rbunm-nsmd (deleted aminoacids, - ) were generated by reverse genetics as described (shi et al., ) . the mab monoclonal antibody against bunyamwera gc glycoprotein, and an anti-nsm rabbit antiserum against the peptide tdqkytldeiadvlqa (residues - of the m segment precursor) were described previously (watret et al., ; nakitare and elliott, ; lappin et al., ) . rabbit antisera against the amino-and the carboxyl-terminal domains of viral l protein were previously characterized (jin and elliott, ) . rabbit anti-n antiserum was obtained by immunization with a synthetic peptide corresponding to the aminoterminal peptide of the protein (mielefhdvaantsst) following standard procedures. the anti-giantin rabbit antiserum was a kind gift of m. renz (institute of immunology and molecular genetics, karlsruhe, germany). anti-b actin (clone ac- ) and a rabbit anti-actin antiserum were from sigma. anti-gal-t and anti-dsrna k mabs were kindly provided by t. suganuma (department anatomy, miyazaki medical college, japan) and n. lukacs (biological research center, institute of plant biology, szeged, hungary), respectively. anti-dsrna j mab was purchased from english and scientific consulting (hungary). anti-dsrna antibodies have been validated as markers of viral replication for different rna viruses (schonborn et al., ; westaway et al., ) . fluorescent secondary antibodies, alexa , phalloidin (green), wga (wheat germ agglutinin, red) and topro (marker for nuclei) were purchased from molecular probes/invitrogen. monolayers of bhk- cells were infected with bunv or nsm deletion mutant viruses at a multiplicity of infection (moi) of or plaque forming units (pfu) per cell. at , , or h p.i. culture supernatants were removed and cell monolayers processed for immunofluorescence or em. monolayers at h p.i. were incubated , or min with culture medium containing mg ml - bfa. for treatment with drugs for actin culture supernatants were removed at h p.i. and substituted by medium with mm cyd, mm lta or . mm jpk. at h p.i. cells were fixed and processed for microscopy. cell monolayers grown on glass coverslips were fixed min at °c with % paraformaldehyde in pbs before processing for immunolabelling of viral and cellular components as previously described (novoa et al., b; fontana et al., ) . a zeiss axiophot fluorescence microscope equipped with a micromax digital camera and a bio-rad radiance confocal laser microscope were used for image collection. cell monolayers were fixed h at room temperature with a mixture of % glutaraldehyde and . % tannic acid in hepes buffer (ph . ) and processed by conventional embedding in the epoxy-resin eml- (taab laboratories) or in lowicryl k m (taab) after cryo-processing by freeze-substitution as described (fontana et al., ) . ultrathin sections ( - nm) were collected on cupper grids, stained with uranyl acetate and lead citrate or processed for immunogold labelling, and studied in a jeol -ex ii electron microscope operating at kv. cryosections were obtained in an ultracryomicrotome (leica em fcs) operating at - °c by the standard tokuyasu method as described (salanueva et al., ) . for immunogold labelling primary antibodies were diluted in saturation buffer (pbs containing % bsa) as follows: : for anti-giantin, anti-l, anti-nsm and rabbit anti-actin, : for anti-n and : for anti-dsrna. secondary antibodies conjugated with nm colloidal gold particles from bbc were diluted : in the same buffer. negative staining of isolated tubes and viruses was performed with uranyl acetate using standard procedures. for immunogold labelling samples adsorbed to em grids were submitted to short incubations with primary and secondary antibodies before negative staining as described (novoa et al., b) . the newly synthesized viral rna in bunv-infected bhk- cells was labelled from to h p.i. with mm brutp (sigma). at h before labelling, mg of actinomycin d (sigma) per ml was added to the medium to block cellular rna synthesis. brutp was introduced into cells using dotap liposomal transfection reagent (roche) as described (westaway et al., ) . incubation with the mixture brutp-dotap-actinomycin d was maintained h at °c. cells were then washed with pbs, fixed with % paraformaldehyde + . % glutaraldehyde in pbs and processed for cryosectioning and immunogold labelling using a monoclonal anti-bromodeoxyuridine antibody (sigma) diluted : in saturation buffer followed by a secondary antibody-colloidal gold conjugate as described above. the established protocol for the isolation of intracellular viruses (novoa et al., b) was slightly modified for the purification of more labile structures such as the viral tubes. bhk- cells were infected at an moi of pfu/cell and maintained h at °c. cells were washed and collected in ten buffer containing protease inhibitors (roche), and frozen at - °c. after two consecutive cycles of freezing and thawing to release the viral tubes, cell lysate was clarified by centrifugation at g, min at °c. supernatant was centrifuged . h at g through a % (w/v) sucrose cushion. pellet was re-suspended in ml of ten with protease inhibitors and applied to a - % (v/v) optiprep iodixanol density gradient (sigma). centrifugation of samples was performed for . h at g. fractions of ml were collected from the top of the gradient and processed for structural and biochemical characterization. complete intact tubes localized in fractions - from the top, well separated from intracellular viral particles (fractions - ). controlled disruption of isolated tubes was done with saponin after adsorbing them on hydrophilic em grids. tubes were incubated with saponin ( . % in pbs) during s, s or min. after washing with pbs grids were processed for negative staining or immunogold labelling. purified viral tubes were processed by polyacrylamide gel electrophoresis (page) in the presence of sodium dodecyl sulfate (sds) with %, % or % acrylamide gels. coomasie blue staining was used to visualize protein bands (novoa et al., b) . for western blot analysis proteins were transferred from gels to nitrocellulose membranes by standard blotting procedures. membranes were saturated overnight at °c with pbs containing non-fat dry milk and . % tween , and incubated h at room temperature with primary antibodies diluted in saturation buffer ( : for anti-l, : for anti-nsm and : for anti-b actin). after washing with this buffer, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody diluted : . immunoreactive bands were detected by chemiluminescence (ecl kit, amersham). for rna dot-blotting samples were processed under rnasefree conditions as described (brown et al., ) . samples were incubated overnight at °c in a mixture of mg ml - proteinase k and % sds in depc water. a positive control (dsrna birnavirus ibdv, e strain) kindly provided by dr. d. luque (cnb, madrid), a negative control (yeast rna from sigma), purified extracellular bunv virions and fractions enriched in intracellular viral tubes were applied to nitrocellulose membranes. membranes were allowed to dry at room temperature during min and processed for immunodetection of total rna. after incubation with saturation buffer ( % dry milk in pbs) membranes were incubated h with a mixture of j and k anti-dsrna antibodies (diluted : each in saturation buffer), washed three times with the same buffer and incubated h with a secondary antibody conjugated with horse radish peroxidase diluted : in saturation buffer. after several washes in saturation buffer signal in membranes was visualized by chemiluminescence (ecl kit, amersham). fractions containing tubes as detected by negative staining and em and fractions without tubes were submitted to sds-page using % and % acrylamide gels to cover a wide molecular weight range. exclusive bands in fractions containing viral tubes were processed by maldi peptide mass fingerprinting and database searching as described (navarro-lerida et al., ) . ultrathin consecutive sections of approximately nm were collected on formvar-coated parallel-bar copper grids and stained with uranyl acetate and lead citrate and studied by em. after selecting an interesting region in the series, photographs were taken at ¥ magnification. using an epson perfection photo scanner the photographs were digitized at dpi as -bit images with a final pixel size of . nm. the images were then normalized using the bsoft software (heymann and belnap, ; http://lsbr.niams.nih.gov/bsoft/). sections were aligned by selected traces between two consecutive sections using the free editor for serial section microscopy 'reconstruct' (fiala ; http://www.synapse-web.org/tools/index.stm/) taking into account the 'tips for aligning sections' of the user's manual. as sections had an average thickness of nm the voxel in the serial section d reconstruction had an anisotropic size of . nm in xy axis and nm in z axis. segmentation and d visualization were done with amira (http://amira.zib.de). for noise-reduction, images were subjected to three iterations of a median filter (van der heide et al., ) . isosurface was used for d visualization, and the optimal threshold for the different materials was determined using a previously implemented algorithm (cyrklaff et al., ) . only elements with unequivocal identity were included in the d representations. a total of factories were studied by d reconstruction of serial sections and - sections per series were included. as individual sections have a thickness of - nm, the total thickness of the d maps in the z axis was around mm. spectrins and the golgi bunyamwera bunyavirus nonstructural protein nss is a nonessential gene product that contributes to viral pathogenesis analysis of rna by northern and slot blot hybridization effects of jasplakinolide on the kinetics of actin polymerization copi activity coupled with fatty acid biosynthesis is required for viral replication cryo-electron tomography of vaccinia virus molecular biology of the bunyaviridae emerging viruses: the bunyaviridae interactions of human retroviruses with the host cell cytoskeleton golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-i as a cytoplasmically oriented peripheral membrane protein molecular motors and a spectrin matrix associate with golgi membranes in vitro reconstruct: a free editor for serial section microscopy novel replication complex architecture in rubella replicon-transfected cells three-dimensional structure of herpes simplex virus from cryo-electron tomography subcellular localization of host and viral proteins associated with tobamovirus rna replication efficient automatic noise reduction of electron tomographic reconstructions based on iterative median filtering bsoft: image processing and molecular modeling for electron microscopy oligomeric structures of poliovirus polymerase are important for function microscopic morphology and the origins of the membrane maturation model of golgi apparatus function mutagenesis of the l protein encoded by bunyamwera virus and production of monospecific antibodies three-dimensional analysis of a viral rna replication complex reveals a virus-induced miniorganelle localization of bunyamwera bunyavirus g glycoprotein to the golgi requires association with g but not with nsm actin filaments are involved in the maintenance of golgi cisternae morphology and intra-golgi ph visualization and functional analysis of rna-dependent rna polymerase lattices wrapping things up about virus rna replication new views of cells in d: an introduction to electron tomography electron microscopic observations of mouse brain infected with bunyamwera group arboviruses expression of the bunyamwera virus m genome segment and intracellular localization of nsm proteomic identification of brain proteins that interact with dynein light chain lc virus taxonomy. fauquet virus factories: associations of cell organelles for viral replication and morphogenesis key golgi factors for structural and functional maturation of bunyamwera virus local actin polymerization and dynamin recruitment in sv -induced internalization of caveolae viral interactions with the cytoskeleton: a hitchhiker's guide to the cell polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments viral rna replication in association with cellular membranes monoclonal antibodies to double-stranded rna as probes of rna structure in crude nucleic acid extracts matrix proteins can generate the higher order architecture of the golgi apparatus requirement of the n-terminal region of orthobunyavirus nonstructural protein nsm for virus assembly and morphogenesis golgins and gtpases, giving identity and structure to the golgi apparatus virus maturation: dynamics and mechanism of a stabilizing structural transition that leads to infectivity the nonstructural nsm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells synthesis of bunyavirus-specific proteins in a continuous cell line (xtc- ) derived from xenopus laevis nascent flavivirus rna colocalized in situ with double-stranded rna in stable replication complexes aggresomes and autophagy generate sites for virus replication this work was supported by grants bmc - and bfu - /bmc from the ministerio de educación y ciencia of spain (to c. risco), ja-p -tic and mec-tin - (to j.j. fernández) and wellcome trust programme grants and (to r.m. elliott). juan fontana and noelia lópez-montero are recipients of contracts from the comunidad de madrid. we thank sylvia gutiérrez erlandsson for expert support with confocal microscopy and silvia juárez for her support with peptide mass fingerprinting studies, reyes novoa and gloria calderita for technical assistance, sonia ruiz and carmen lópez-iglesias for support with cryosections, xiaohong shi for manuscript reading and alberto fraile-ramos for helpful discussions and critically reading the manuscript. key: cord- - ickafd authors: kapil, sanjay; yeary, teresa; johnson, bill title: diagnostic investigation of emerging viruses of companion animals date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: ickafd in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable, because viruses are highly mutagenic. even the immune response dictates the nature of virus that evolves in a host. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with the diagnostic laboratory to solve the diagnostic puzzle. c linicians and laboratorians are usually the first to detect most outbreaks of emerging diseases in animals. much attention is rightfully given to emerging diseases of commercial food animals; however, small animal practitioners also have an obligation to be vigilant to the possibility that new and devastating viral diseases might emerge that infect the companion animals in their charge. canine parvovirus (cpv) type , emerged in and spread worldwide within less than years [ ] . in , a new antigenic type, cpv- c, was reported in italy [ ] , which has since caused outbreaks in western europe, asia, south america, and the united states [ ] because current vaccines offer no protection for this type. in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. the term emerging virus is defined broadly and includes these categories: variants of a known virus that has gained enhanced virulence or that is able to infect completely vaccinated animals a known virus that has reappeared in the population after a decline in incidence novel or previously unidentified viral agents detected for the first time because of improved diagnostic capabilities ''mystery diseases'' with large numbers of naive animals involved that are caused by previously uncharacterized viruses spread of an emerging virus among small companion animals is multifactorial and includes animal health and sanitation practices; migration of a pathogen from a wild reservoir to domestic animals because of changes in populations, trade, climate, land use, and the introduction of invasive species (eg, plant, animal, insect); and, finally, globalization, as was the case with west nile virus (wnv). emerging viral infections may take a heavy toll on the health of cats or dogs whenever they are brought into situations in which groups of animals are housed together, even temporarily, such as at greyhound racetracks, kennels, catteries, animal shelters, animal obedience training classes, dog parks, pet stores, pet day care facilities. this is especially true when pets are allowed by their owners to roam at will, commingling with ownerless feral dogs and cats and wildlife. for example, the rapid spread of cpv- , which is extremely stable in the environment and highly contagious, was caused not only by the movement of dogs by their owners but by the transfer of fecal material on shoes and clothing of travelers and, unintentionally, through national and international mail [ ] . according to the to national pet owners survey conducted by the american pet products manufacturers association, the us pet cat population is estimated to be . million and the pet dog population is estimated to be . million [ ] . municipalities throughout the united states commonly pass animal control ordinances to protect the public health and safety and general welfare of the citizens and animals residing within the city. typically, animal control codes limit the numbers of companion animals that individuals may own or keep on their private property, require that cats and dogs be licensed annually by owners and vaccinated against rabies, prevent animals from running at large, require proper disposal of animal waste, and prevent the feeding of wild or feral cats or dogs. vaccination of dogs and cats by compliant pet owners for rabies prevention has, since , dramatically reduced the occurrence of this disease; currently, most animal cases reported to the centers for disease control and prevention (cdc) now occur in wildlife [ ] . compliance with other animal control ordinances is variable, particularly among pet owners with respect to leash laws for dogs and cats and among well-intentioned individuals who maintain wild or feral colonies of cats and dogs by providing food, water, and shelter. statistics from the humane society of the united states indicate that to million companion animals are admitted to shelters each year and nearly half are adopted or reclaimed by their owners, whereas the remaining animals are euthanized [ ] . no census of ownerless dogs and cats is available. estimates of the feral cat population in the united states range from million to million animals living primarily in or near urban settings with ample opportunity to interact with pets that are allowed to roam and with wildlife [ ] . thus, ownerless, wild, or feral dog and cat populations may transmit infectious and zoonotic diseases between wildlife and companion animals. from a public health standpoint, this is of particular importance because emerging viral infections from wildlife are often transmitted to human beings by means of a pet that is allowed to stray. it is widely believed by virologists and public health epidemiologists that most viruses emerging from wildlife have an rna or single-strand dna genome [ ] because they have a high propensity for mutation. two significant canine viruses have emerged recently and meet this hypothesis: cpv and canine distemper virus (cdv). canine distemper has re-emerged in the past decade [ , ] because of antigenic and genetic drift in the surface protein (h glycoprotein). in a multicontinent study, variant cdv strains, (but not the vaccine strain of cdv virus) were the cause of illness within weeks after vaccination. in and , large outbreaks of cpv variants (cpv- c and cpv- b*) in kennels occurred in oklahoma and other states [ ] . diagnostic and molecular studies detected mutations in the parvovirus isolates that explained the failures of current commercial cpv vaccines from conferring protection and of approved commercial diagnostic kits from detecting these new viral isolates. another recent example is outbreaks of hemorrhagic symptoms associated with virulent feline calicivirus (fcv) in the united states [ ] ; however, molecular basis of gain of virulence in fcv is not yet understood. in addition to virus evolution, in some cases, the virus can be reintroduced back after the population immunity has declined after a period of disease-free status. thus, diseases that have been eradicated from developed countries but are still circulating in developing countries [ ] may re-emerge by reintroduction from trade or movement of animals. there is a major commitment by the us department of agriculture (usda) in this country and in cooperation with foreign governments and international agencies worldwide to monitor the health of food animals and certain wildlife but not of companion animals [ ] . the primary mission of the cdc is to promote and protect human health. to this end, the cdc performs surveillance for noninfectious and infectious diseases, including zoonoses [ ] ; however, the only chosen reportable viral diseases of animals that are collected by the cdc are rabies and avian influenza (h n ), and those that are reported to the cdc arbonet system are avian, animal, or mosquito wnv infections. largely, surveillance of companion animal diseases, many of which have zoonotic potential, has not been considered to be a priority until recently [ , ] . in , the cdc partnered with the purdue university school of veterinary medicine to establish a pilot surveillance system to monitor clinical syndromes and diseases of small animals [ ] to determine whether animals can serve as sentinels of health hazards to human beings. the national companion animal surveillance program (ncasp) initially drew exclusively on the database of the privately owned organization, banfield, the pet hospital, which provides medical care to approximately . million pet dogs and cats in states, and it now integrates data from antech diagnostics to detect potential emerging and zoonotic infections. a long-term goal of the ncasp is to become a national resource in veterinary public health. in the meantime, the front line of companion animal surveillance for emerging diseases is at the home front, with astute small animal clinicians playing a major role. it can be a challenge for busy and isolated veterinary practices to receive the information on emerging viruses. linking to a health-related network for companion animals might fill the gap. recently, a space-time permutation scan statistic, which was applied in the anthrax terrorist attacks in [ ] , wnv outbreaks [ ] , and enzootic raccoon rabies [ ] , has been applied to veterinary diagnostic data in the unite states and europe [ ] . this analysis provides important information about potential clusters of medical conditions and issues medical alerts about the developing situations based on mortality and confirmed diagnosis of important disease conditions. earlier and more timely notifications should lead to more thorough investigations and reduce losses, especially from emerging viral diseases. it is important to keep in mind that clinical syndromes tend to be multifactorial, and it is essential to review the entire history, including environmental factors, with the specialist in a small animal specialty practice and also with a small animal teaching hospital before arriving at a conclusion about the case. the purpose of this article is to encourage companion animal veterinarians to think outside the routine diagnostic plan when atypical cases of infectious disease are presented at their practices. detecting emerging viral diseases of companion animals requires interaction and discussion among clinicians, pathologists, and virologists, and practicing small animal veterinarians must stay engaged in communication with these specialists through their state diagnostic laboratories or nearby colleges of veterinary medicine. veterinary diagnostic medicine is rapidly progressing, and it is critical for the successful practitioner to stay abreast of new developments in small animal infectious diseases and their diagnosis through continuing education [ ] [ ] [ ] . the development of monoclonal antibody technology in the s and the advent of the polymerase chain reaction (pcr) assay in the s have reshaped veterinary diagnostic strategies, especially in the subspecialty of virology. now, these molecular techniques, which are becoming mainstream applications in routine viral diagnoses, are proving their merit in facilitating the diagnosis of emerging animal viruses. the authors offer practical information on the applications of diagnostic techniques for investigating viral disease outbreaks in companion animals. the authors provide this brief overview of diagnostic techniques in the modern virology laboratory that are used for routine diagnosis and in identifying novel and emerging viruses. every step of diagnostic investigation-history, specimen collection, transportation, and laboratory examination-has to be carefully aligned for optimal outcome. small animal clinicians are familiar with symptoms of common infectious diseases and are often the first to recognize the emergence of new disease problems. in some cases, there may be a history of vaccination compliance, yet some animals develop disease [ , ] . it is important to record the complete history, including the body system involved (eg, respiratory, gastrointestinal, reproductive tract, nervous system), clinical symptoms and their duration, the presence of lesions, and vaccination history. particularly when the case is confounding, the client must be carefully and thoroughly interviewed as to how he or she manages the pet (ie, is the pet free to roam; has the pet traveled recently and where; if this is a new pet, where and how was it obtained; are there other pets in the household). consulting a book on differential diagnoses can be useful to list the potential causes [ , ] . when a history of unusual symptoms is presented, clinicians, recognizing that these cases may be important to individual and universal animal health, should refer these cases to an accredited veterinary diagnostic laboratory. it is convenient to attach copies of all relevant hospital records to the laboratory submission form to aid the diagnostician. correct diagnosis depends on a thorough case history of the affected animal and submission of appropriate specimens that are collected and transported in a manner to preserve the integrity of the viral agent. submitting a comprehensive collection of specimens in a timely manner to the diagnostic laboratory from affected animals when the disease does not fit a familiar clinical picture, as is the case with emerging viral diseases, is of paramount importance. all the system(s) that are potentially involved and all the tissues with gross lesions should be sent to the diagnostic laboratory. it is important to check for concurrent infections. viral diagnosis depends on the quality and type of specimen collected [ ] . the best time for collection of specimens is immediately after symptoms of disease are first noticed. samples from all body systems involved in the acute stage of the disease of affected animals should be submitted to the diagnostic laboratory in a timely manner by overnight delivery. at least to g or ml of each sample should be collected. recovery of virus in cell culture depends on the condition of the specimen received by the diagnostic laboratory. freezing specimens can be detrimental to virus isolation efforts (and also to electron microscopic identification) and should only be done (À c) if it is not possible to deliver the specimen to the laboratory within hours. use wet ice for shipping virology samples, because dry ice (solid carbon dioxide gas) can inactivate many viruses, preventing isolation in cell culture. tissues intended for virus isolation should always be shipped in separate packages from specimens that are immersed in formalin to prevent fumes of formaldehyde from reaching the fresh tissues. it is imperative that tissues and organs from animals that have died be harvested as soon as possible after death. postmortem tissues should be placed in sterile containers with a small amount of transport medium ( - ml), if possible. when the clinician is unsure as to what specific organs and fluids should be retrieved, the entire carcass of the dog or cat may be delivered to the laboratory for examination. to obtain more specific details regarding specimen collection, packaging, and submission, contact the diagnostic laboratory of your choice by telephone or consult its specimen submission and fee schedule guidelines, which are often available on an internet web site. individuals who ship biologic substances for diagnostic testing are required by federal law to be in compliance with all regulations governing packaging and labeling of interstate shipments of causative agents. failure to follow the regulations results in heavy fines (fig. ) . complete instructions on appropriate packaging for laboratory specimens to be mailed or shipped by a common carrier may be accessed in several sections of the code of federal regulations (cfr). health and human service regulations define such terms as diagnostic specimen and etiologic agent and describe requirements for packaging and labeling viruses have a simple structure with a protein coat enclosed with only one type of nucleic acid (dna or rna) rather than both. thus, methods for viral diagnosis target one of the components of the virus structure. for a definitive viral disease diagnosis, four basic approaches are used: direct detection by virus isolation or direct identification, viral serology for detection of a specific antibody, viral antigen detection, and molecular-based detection of genetic material. a brief discussion of the principles of diagnostic assays representative of each approach follows. gross pathologic and histopathologic findings histologic (fig. ) and cytologic examination (fig. ) of tissues and fluids by a board-certified veterinary pathologist contributes valuable information about the pathologic signs, gross and microscopic, that distinguish infections caused by viral or bacterial pathogens and other possible etiologies. tissue tropism, mononuclear infiltrates, development of inclusion bodies (intranuclear, cytoplasmic, or both), and the formation of syncytia are some of the characteristics that differ among viruses and can sometimes distinguish different viral infections. for example, most dna viruses replicate in the nucleus, and thus tend to produce intranuclear inclusions, whereas most rna viruses form cytoplasmic inclusions, although there are exceptions. as part of the pathologist's examination, immunohistochemistry testing (figs. and ), fluorescent antibody testing, and possibly in situ hybridization (ish) studies on tissues may be ordered; these methods are considered elsewhere in this article. a complete histopathology report should include possible differentials for the lesions. the pathologist might note that some findings do not exactly fit the routine lesions he or she has observed in previously. in cases in which there are deviations in lesion type or distribution or when gross lesions and histopathologic findings suggest the involvement of a viral disease but routine virology tests do not detect the expected conventional viral agents, variant or ''emerging'' viruses or even iatrogenic infections may be suspected. in early , blue tongue virus serotype was introduced in canine populations from a commercial modified-live multivalent canine vaccine that was associated with high mortality in dogs [ , ] . in some situations, second or even third opinions from pathologists at other laboratories who have special expertise should be solicited [ ] . with the application of telepathology to veterinary case materials, networks of specialists, including veterinary pathologists, small animal clinicians, infectious disease specialists, and laboratory diagnosticians, are able to exchange patient histories, clinical data, and images (gross and microscopic) through the internet for consultation, diagnosis, and education. this allows timely access to expert opinions at other locations throughout the world [ , ] . the use of telepathology can facilitate rapid intervention through the synergy of computer technology and special pathology expertise (eg, system-and speciesspecific pathologic findings) to understand the lesions in difficult cases better. conventional virus isolation techniques are often the backbone of investigation of novel viral diseases, provided that the virus is cultivable in available cell lines or primary cell cultures. virus isolation may be relatively slow depending on the growth characteristics of the virus; however, roller culturing or centrifugation of samples onto cell monolayer(s) can enhance viral replication and recovery. in many of the recent emerging viruses from wildlife (eg, bats), the virus was first cultivated, allowing further characterization of the virus. it is important to keep in mind that virus isolation, even if the effort is successful, may have a slow turn-around time, approximately to weeks. definitive identification of virus in cell culture can only be accomplished with specific antibody nucleic acid testing, and in the case of an ''emerging'' virus, existing reagents may not be reactive with the ''new'' virus. if culture is successful, however, the viral material may be studied by electron microscopy (em) and by molecular techniques, as described in this article, to characterize the new isolate. virus isolation requires fresh tissues and cannot be done on formalin-fixed tissues. em is often used in veterinary diagnostic laboratories to detect enteric viruses in fecal samples retrieved during the course of viral diarrheal disease. additionally, em is indispensable for identification of emerging and previously unidentified viruses in clinical samples [ ] , and this method has helped in the identification of many new viruses, including, most recently, bat lyssavirus [ ] . viruses can be classified up to the virus family based on size, shape, and distinctive structural features, such as envelopes or protein spikes, particularly for parvovirus, rotavirus (fig. ) , coronavirus, astrovirus, herpesvirus, poxvirus, and picornavirus. em allows detection of multiple viruses simultaneously. application of antibodies to supplement the em diagnosis provides higher sensitivity and further confirmation of the viral diagnosis. sensitivity is the major limitation of em, and at least to virus particles per milliliter must be present in the sample being examined. because the electron microscope is an expensive piece of equipment that requires special technical skills and a high level of expertise, it is not available in many laboratories. viral components can also be determined by several basic biochemistry experiments. acridine orange (ao) staining can determine the nature of the nucleic acid of purified viral particles [ ] . differentiation as to whether the nucleic acid is single-or double-stranded in nature is based on the color developed on ao staining; double-stranded dna or rna nucleic acids stain yellow green, whereas single-stranded dna or rna acids stain flame red. nuclease susceptibility of the purified virions differentiates dna from rna. the presence of envelope on viruses can be determined by susceptibility to the virus to heat, ether, or other lipid solvents [ ] . the titrated virus preparation is treated with ether or chloroform. a decrease in virus titer of greater than log is considered to be significant to indicate the presence of envelope on the virus. the presence of envelope indicates that virus is susceptible to common disinfectants. lack of envelope indicates that the virus is resistant to the use of common disinfectants. classic serology tests indirectly determine the viral etiology of disease by detecting the presence of antibody in serum (red-topped tube) to a specific test viral antigen, and thus provide retrospective evidence of an immune response or exposure to a virus. serologic methods still provide powerful tools in the virology laboratory of today for diagnosing viral diseases that are seen routinely and for discovering and characterizing novel viral diseases. serologic tests are now used to detect antibody or antigen in serum and body fluids. typically, methods used in the virology laboratory are serum neutralization (sn), hemagglutination-inhibition (hai) test, indirect fluorescent antibody test (ifat), and elisa. serologic results require interpretation by an expert diagnostician based on critical clinical observations, confirmation by pathology examination, virus isolation, and mass screening of the populations by serology. if animals in populations that have never been exposed to or vaccinated against a given virus have specific antibodies detected in their serum, it is expected that this is most likely attributable to recent exposure to the emerging virus. paired serum samples are important to demonstrate a fourfold significant increase in antibody titers, which indicates that the diagnosis of recent exposure may be attributable to infection as opposed to previous exposure or vaccination depending on the vaccination history. serology is also useful to study the antigenic distance of the emerging virus and provides clues as to whether the newly emerged agent is or is not likely to be protected by an available vaccine(s), such as heterologous virus in another species of animal. viral hemagglutination (ha) occurs between the viral protein; hemagglutinin (hn), which is present on the viral capsid or envelope of only certain families of viruses; and specific receptors on red blood cells (rbcs) that bind to hn, causing their agglutination and precipitation from solution. this phenomenon is the basis for a powerful and sensitive assay, the hai test. when a hemagglutinating virus is mixed with serum containing antibodies specific to that virus, rbcs that are added to the mixture do not agglutinate and precipitate from solution. feline panleukopenia, cpv, influenza a, and parainfluenza antibodies may be detected by hai testing. the hai method may also be used to identify unknown virus utilizing antibodies of known specificity; however, most often, this test is applied to detect the presence of antibodies in a serum sample against specific hemagglutinating viruses. variants of cpv and feline parvovirus can differ in the hemagglutinating activity of swine erythrocytes [ , ] . sn measures the inhibitory activity of a hyperimmune serum against viral isolates in cell culture. commonly performed in a cell culture microwell format, this is a long-standing method for quantifying virus-specific antibodies, and it is usually performed to test for antibodies to viruses that typically cause cell damage (cytopathic effect [cpe] ) to the host cell culture they infect. when a virus is mixed with hyperimmune serum containing antibodies specific to that virus, the antibodies bind the virus, preventing infection of the cell culture. the sn test can diagnose current infection using acute and convalescent serum samples from individual animals. it may also be used to determine immune status conferred on vaccinated animals. vaccination antibody titers often differ from antibody titers developed in response to natural infection. usually, vaccination titers are lower relative to infection titers, and maximal titers occur approximately to days after vaccination. sn assays are commonly performed to detect antibodies to fcv, herpesvirus, enteric coronavirus, and syncytial viruses and to canine herpesvirus, cdv, coronavirus, parainfluenza virus, and adenovirus. elisa this is useful for screening large numbers of samples for the presence of antibodies against viruses. the elisa format is flexible, and it may be used to detect antibody or antigen in clinical specimens. in either case, the detection system is an antibody conjugated to an enzyme. when the enzyme-linked antibody binds to the analyte being measured, the enzyme reacts with a chromogenic substrate, causing a color change to occur that may be measured spectrophotometrically or evaluated visually. several elisa kits are available to detect antiviral antibodies in companion animals, including cpv and cdv, feline leukemia virus (felv), feline immunodeficiency virus (fiv), and feline coronavirus. the immunoglobulin m (igm) elisa is a method used to distinguish current infection from past infection. during acute disease or immediately after vaccination with modified-live viruses, igm is the first class of immunoglobulin produced in response to infection, appearing to weeks before there are detectable levels of igg in the serum. because it is short-lived, igm levels typically disappear months after infection. a single acute-phase serum test sample is sufficient to diagnose current infection with an igm elisa. testing of igm titers is available for several viral agents, including cdv and cpv among others. elisa is useful for screening naive animal populations for the presence of antibodies against viruses to track the origin and spread of emerging infections. antibodies to wnv have recently been detected in dogs and cats by igm-capture elisa [ ] . a related method known as virus neutralization can be used to identify the serotype of a newly discovered virus. western blot (wb) may be used as a supplementary test to confirm antibody elisa results for fiv testing [ ] . to perform the assay, purified virus is disrupted using detergent; the constituent proteins are then separated on the basis of molecular weight by electrophoresis in a polyacrylamide gel. the proteins are transferred (blotted) from the gel to a nitrocellulose or polytetrafluoroethylene (ptfe) membrane for stabilization. the electrophoretically separated proteins are the antigen substrates for analyzing the test sera for the presence of specific antibodies. as with the elisa format, the western immunoblot uses an enzyme-labeled antispecies antibody that binds to the test serum antibodies that have bound to the separated viral antigens. substrate reacting with the enzyme-labeled antibody in the presence of a colorless soluble benzidine derivative results in conversion to colored insoluble precipitate at the protein bands where test serum antibodies are bound. the molecular weight of the protein detected is characteristic for a particular viral component. immunoblot results of the unknown test antisera are compared with positive control test sera for interpretation. a major advantage of the immunoblot technique is that a full antibody profile of a single serum sample is made simultaneously, identifying each of the individual particulate viral antigens that patient antibodies bind. as an epidemiologic tool, wb analysis may be used to detect currently circulating viral subtypes within a population and to characterize new emerging viral subtypes. immunoblotting is also a valuable research technique for antigen detection that is often used to characterize novel viruses by comparing them with known related viral family members using standard antisera or monoclonal antibodies. immunofluorescence assays on cells from clinical samples can be applied for rapid diagnostic investigations ( - minutes), provided that the fluorescent microscope and expertise are available in a laboratory. with the pooling of primary monoclonal antibodies against potential viral agents, the assay can be used as a screening tool and the sample tested again with individual conjugates to obtain specific virus diagnosis (fig. ) . the elisa is also a means for detecting viral antigens present in clinical specimens, and it offers a relatively quick turn-around time. antigen test elisa kits are available to detect antiviral antigens in companion animals, including cpv, felv, and fiv. additionally, it is a common practice by many veterinary diagnostic laboratories to appropriate the use of some rapid antigen test kits intended for the human diagnostic market, specifically, rotavirus test kits. when monoclonal antibodies are used as capture antibodies in elisa test kits, however, they fail if there is a mutation in the epitope of the viral surface protein present in the specimen that is being tested. lateral flow immunoassay is a special application of the elisa that provides a rapid, economic, portable, sensitive, and specific technique that is convenient for performing testing outside of the laboratory. it is the technique of choice for emerging viral infections [ , ] , and it has gained attention for use in diagnosing foreign animal diseases and zoonotic and emerging viral infections of animals, such as influenza virus and wnv, in the field. the test kits are small in size (size of credit cards), extremely stable at ambient temperature ( c), and take minutes to perform. an advantage of nucleic acid-based testing is that specimens submitted for analysis do not have to have viable viral particles present to be detected by this means. there is a trend toward application of molecular or gene sequence-based techniques to routine virology testing in diagnostic laboratories, which is justified under several circumstances. first, a molecular technique may be the test of choice if conventional methods of diagnosis are technically weak, such as when a viral agent is noncultivable or there are biocontainment concerns with culturing the virus, the virus has amorphous morphology by em, antibodies are unavailable or not specific to the virus, and serologic tests result in a confounding diagnosis. second, molecular techniques may be essential to detect and classify the sequence type or genotype of a virus. third, a viral agent may be characteristically slow to replicate, such as c-herpes virus; thus, a molecular method might provide a better turn-around time for diagnosis. in this instance, a rapid diagnosis might be achieved by pan-herpesvirus pcr. finally, a novel viral isolate that cannot be definitively identified by the routine diagnostic methods described previously may merit investigation and characterization by molecular-based techniques, which are indispensable in the classification of new and emerging viruses. these advanced techniques may confirm a diagnosis of viral etiology when other tests have failed; however, they are, unfortunately, relatively expensive. furthermore, the presence of nucleic acid does not equate to infection, and infections are attributable to subclinical, latency-associated nucleic acids or defective interfering virus particles, such as in paramyxoviruses, produced in nonproductive infections in genetically resistant hosts. clients, who bear the financial burden, should be counseled as to the benefit and shortfalls of this testing before ordering molecular-based tests. an excellent review of molecular-based techniques for diagnostic testing of infectious diseases has appeared in a previous issue in this series [ ] . the most familiar nucleic acid testing technique, pcr, has been used for more than a decade; however, over the past few years, real-time pcr has taken its place, revolutionizing diagnostic virology. in this procedure, the pcr chemistry may be combined with detection using a single-stranded dna probe with a fluorescent label [ ] . moreover, the procedure may be completed within an hour, and it allows for quantitation of results. because the hands-on steps are reduced and the pcr reactions are not opened, it eliminates the chances of cross-contamination in the laboratory. real-time pcr protocols are gaining more acceptance in routine veterinary diagnosis. in situ hybridization ish involves using nucleotide probes with an attached label. non-isotopelabeled probes (digoxigenin or fluochrome) can be applied in veterinary diagnostic laboratories. diagnostic applications of ish involve identification of virus-specific sequences (dna or rna) in the tissues or cells [ ] . although uncommon in veterinary diagnostic laboratories, ish is in routine use in human diagnostic laboratories for detection of the genotype of human papilloma viruses in cervical samples. for ish, smears and tissues (fresh, unfrozen, and fixed tissues) are suitable. in electropherotyping and restriction fragment length polymorphism (rflp), double-stranded dna (rflp) or rna (electropherotypes) is purified and size-separated on agarose or acrylamide gel electrophoresis. because nucleic acids are charged and double-stranded molecules bind more ethidium bromide compared with single-stranded nucleic acids, under the electric field, the nucleic acids migrate and larger sized molecules separate out higher than smaller sized molecules. for dna molecules to be tested, the double-stranded viral dna-or pcr-amplified fragments are digested with restriction enzymes. these techniques allow quick differentiation of viral genomes (dna or rna). both techniques have applications in molecular epidemiology of rotaviruses [ ] . new generation molecular techniques viral genome sequencing technologies viral genome or mrna sequencing is a powerful molecular epidemiologic tool and has been applied for epidemiology of rabies virus [ ] . sequences of novel or emerging viruses may be derived based on known conserved sequences of previously characterized viruses within the same family. although virus sequencing is gaining more routine application in veterinary laboratories, it does add cost, and thus should be used judiciously. when these methods fail to identify a newly discovered virus, which is truly novel, metagenomic analysis, which is largely used in research laboratories, may be applied. pyrosequencing is a recent variation on sequencing short stretches of pcrgenerated dna without the need for labeled primers, labeled nucleotides, and gel electrophoresis [ ] . although this variation on pcr and nucleic acid sequencing is currently used exclusively as a research tool, it is likely to be adapted for clinical diagnostic work in future years because it has been demonstrated to detect many different unrelated viruses simultaneously in a single reaction and to identify viral serotypes and detect viral isolates that could not previously be typed by classic procedures [ , ] . a biochip or microarray is small solid support, such as a nylon membrane, silicon chip, or glass slide, on which nucleic acid fragments, antibodies, or proteins are immobilized in an orderly arrangement. thousands of different molecules, referred to as probes, may be machine-printed as spots on the support, allowing for high throughput of samples using lower volumes of analyte in less time than conventional laboratory techniques take to complete. microarrays are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions that are most commonly detected through the use of fluorophores. the fluorescent signal patterns formed by each analyte are then compared by the computer software using complex algorithms to make an identification of its contents. biochips enable researchers to screen large numbers of biologic analytes quickly for a variety of purposes, ranging from disease diagnosis to detection of bioterrorism agents. biochip technology is still relatively new and has not yet entered the mainstream of clinical diagnostics techniques, although it is widely used in research institutions. as an epidemiologic tool, the use of nucleic acid microarrays was instrumental in the rapid identification of the first severe acute respiratory syndrome (sars) coronavirus outbreak in china [ ] . coronavirus protein microarrays have been used to screen canadian sera [ ] for specific antibodies to sars and to other coronaviruses in a comparative study with the traditional elisa. scientists around the world are assessing the feasibility of using microarrays as tools for surveillance and diagnosis of influenza viruses [ , ] . once issues of sensitivity and assay validation have been addressed satisfactorily and the cost of the technology has become more affordable, microarray technology may find a place in clinical diagnosis. pathogenic virus or ''orphan'' virus or ''vaccine-source'' virus molecular methods for detecting and identifying viral pathogens are powerful. it is possible to detect a virus in a specimen, but it may have no association with the clinical condition. these types of viruses are called ''orphan viruses.'' minute virus of canine is a parvovirus, and it causes no clinical disease [ ] . as a result of the advent of sensitive molecular techniques, it is quite common to detect viral sequences of agents that may be present in a sample but not associated with the disease (orphan viral agents). it is possible to study the association of the viral agent with the pathologic findings observed to support the diagnosis. moreover, the pcr protocols targeting structural genes that are expressed only during active infection are useful and avoid the potential false-positive results attributable to latency or persistent viral infections. moreover, the sense and antisense probes offer the opportunity for resident and replication intermediates of viruses. obviously, the history of recent vaccination should be known, and the vaccine virus from the same lot of vaccine should be simultaneously included in the testing run and sequenced over critical regions to ensure that the virus in the sample is the same or different from the vaccine. when fluorescent antibody testing or immunohistochemistry testing is performed, false-negative findings result even when a related virus is present. because of changes in the sequence of the target protein epitopes, antibodybased detection methods may fail to provide the diagnosis; monoclonal antibodies used may fail to react and polyclonal antibodies may cross-react weakly when a variant strain of virus is present. thus, a sudden trend in lack of correlation between tests may signal an emerging variant of the virus. if a new variant of the virus arises, it may be associated with a change in the clinical profile and we may or may not understand the molecular basis of this shift. it is possible that the polyclonal antibodies may react weakly with the new variant of the virus. in many cases, the pcr primers may fail to amplify the new variant if the mutation occurs in the hypervariable region of the target gene amplified. for example, in the recent emergence of cpv variants, many practitioners noted clinical symptoms compatible with cpv but the commercial field tests were not working. if a new variant of virus emerges, a polyclonal antibody antiserum prepared in a heterologous species (rabbit or goat) can be used as a primary antibody against the whole virus, because it is possible that the monoclonal antibody might fail. the molecular techniques are more likely to fail compared with the antibody-based techniques because of the degeneracy of codons. it is important to keep in mind that factors other than emerging viruses can also affect the performance of usda-approved tests. for example, local anesthetic can also affect the outcome of antibody tests. in one study, the use of lidocaine was recommended over oxybuprocaine to avoid false-positive results [ ] . it should be clear to the readers that veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable because viruses are ''perfect'' obligate parasites. even the immune response dictates the nature of virus that evolves in a host. thus, vaccines are to be viewed as preventive tools rather than as a cure for emerging viruses. in some situations, the best vaccine is bound to fail. similarly, the diagnostic methods have to be tailorfitted to keep up with the emerging viruses. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with diagnostic laboratory to solve the diagnostic puzzle. your state veterinary diagnostic laboratory may be the first place that issues an alert to veterinary professionals and the public at large to possible emerging viral diseases. newsletters from your state diagnostic laboratory can be a good source of information about emerging viral diseases in your area. additional sources that are dedicated to dog and cat health issues and public health are available on the internet [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . an annotated historical account of canine parvovirus evidence for evolution of canine parvovirus type in italy canine parvovirus types c and b circulating in north american dogs in and american pet products manufacturers association. industry statistics and trends: - national pet owners survey rabies surveillance in the united states during feral cat population statistic the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia canine distemper viruses circulating in north american dogs an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus future risks from infectious diseases of animals. presented at the world association of veterinary laboratory diagnosticians linking human and animal health surveillance for emerging diseases in the united states-achievements and challenges united states department of health and human services centers for disease control and prevention animals as sentinels of environmental health hazards world health organization. future trends in veterinary public health. who technical report series purdue university-banfield national companion animal surveillance program for emerging and zoonotic diseases early statistical detection of anthrax outbreaks by tracking over-the-counter medication sales dead bird clustering: a potential early warning system for west nile virus activity spatial and temporal patterns of enzootic raccoon rabies adjusted for multiple covariates a space-time cluster investigation of an outbreak of acute respiratory disease in norwegian cattle herds methodology in diagnostic virology a primer on diagnostic virology: specimen selection and serology. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian a primer on diagnostic virology: direct and molecular-based detection of viral pathogens. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian antemortem diagnosis of cdv infection by rt-pcr in distemper dogs with neurological deficits without the typical clinical presentation clinicopathological findings in dogs with distemper encephalomyelitis presented without characteristic signs of the disease differential diagnosis in small animal medicine small animal medical differential diagnosis diagnostic virology bluetongue disease in dogs associated with contaminated vaccine iatrogenic infection of a pregnant dog with bluetongue virus, serotype interobserver variation among histopathologic evaluations of intestinal tissues from dogs and cats telepathology: its role in disease diagnosis in meat hygiene. presented at the world association of veterinary laboratory diagnosticians th international symposium static image telepathology in perspective overview of electron microscopy and its role in infectious disease diagnosis. presented at the world association of veterinary laboratory diagnosticians th international symposium diagnostic electron microscopy: historical review and future. presented at the world association of veterinary laboratory diagnosticians th international symposium acridine orange staining of purified rat virus strain x recovery and characterization of a minute virus of canines characterization of a nonhemagglutinating mutant of canine parvovirus characterization of a canine parvovirus strain isolated from an adult dog serologic survey of cats and dogs during an epidemic of west nile virus infection in humans use of western blot and radioimmunoprecipitation for diagnosis of feline leukemia and feline immunodeficiency virus infections a comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats, and mink simple rapid, on-site detection for diagnosis of animal disease update on molecular techniques for diagnostic testing of infectious disease real-time pcr in clinical microbiology: applications for routine laboratory testing in situ hybridization and its diagnostic applications in pathology the isolation of rotavirus from calves, foals, dogs and cats in new zealand clinical laboratory advances in the detection of rabies virus pyrosequencing sheds light on dna sequencing type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology identification of enterovirus serotypes by pyrosequencing using multiple sequencing primers micro-array-based detection and genotyping of viral pathogens severe acute respiratory syndrome diagnostics using a coronavirus protein microarray comparison of the mchip to viral culture, reverse transcription-pcr, and the quickvue influenza aþb test for rapid diagnosis of influenza detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate oxybuprocaine induces a false-positive response in immunochromatographic sas adeno test american association of public health veterinarians cdc: healthy pets healthy people available at national association of state public health veterinarians united states animal health association code of federal regulations (search engine) key: cord- -gw mgg m authors: junter, guy-alain; lebrun, laurent title: cellulose-based virus-retentive filters: a review date: - - journal: rev environ sci biotechnol doi: . /s - - - sha: doc_id: cord_uid: gw mgg m viral filtration is a critical step in the purification of biologics and in the monitoring of microbiological water quality. viral filters are also essential protection elements against airborne viral particles. the present review first focuses on cellulose-based filter media currently used for size-exclusion and/or adsorptive filtration of viruses from biopharmaceutical and environmental water samples. data from spiking studies quantifying the viral filtration performance of cellulosic filters are detailed, i.e., first, the virus reduction capacity of regenerated cellulose hollow fiber filters in the manufacturing process of blood products and, second, the efficiency of virus recovery/concentration from water samples by the viradel (virus adsorption–elution) method using charge modified, electropositive cellulosic filters or conventional electronegative cellulose ester microfilters. viral analysis of field water samples by the viradel technique is also surveyed. this review then describes cellulose-based filter media used in individual protection equipment against airborne viral pathogens, presenting innovative filtration media with virucidal properties. some pros and cons of cellulosic viral filters and perspectives for cellulose-based materials in viral filtration are underlined in the review. abstract viral filtration is a critical step in the purification of biologics and in the monitoring of microbiological water quality. viral filters are also essential protection elements against airborne viral particles. the present review first focuses on cellulosebased filter media currently used for size-exclusion and/or adsorptive filtration of viruses from biopharmaceutical and environmental water samples. data from spiking studies quantifying the viral filtration performance of cellulosic filters are detailed, i.e., first, the virus reduction capacity of regenerated cellulose hollow fiber filters in the manufacturing process of blood products and, second, the efficiency of virus recovery/concentration from water samples by the viradel (virus adsorption-elution) method using charge modified, electropositive cellulosic filters or conventional electronegative cellulose ester microfilters. viral analysis of field water samples by the viradel technique is also surveyed. this review then describes cellulose-based filter media used in individual protection equipment against airborne viral pathogens, presenting innovative filtration media with virucidal properties. some pros and cons of cellulosic viral filters and perspectives for cellulose-based materials in viral filtration are underlined in the review. virus capture/purification/concentration is critical in a number of biopharmaceutical and clinical applications. validation of virus clearance is essential in the manufacture of therapeutic proteins, in particular blood products (bryant and klein ; klamroth et al. ; de mendoza et al. ; radosevich and burnouf ; shukla et al. ). on the other hand, large-scale, efficient purification schemes of viruses/ virus-like particles are required for the production of prophylactic vaccines and gene therapy vectors (rodrigues et al. ; segura et al. segura et al. , vicente et al. a, b) . virus inactivation technologies are commonly used to fulfil viral safety. they include physical (e.g., heat application, ultraviolet-and gamma irradiation) and chemical methods (e.g., solvent/detergent treatments) or their combination (e.g., exposure to photosensitizer plus uv light) (bryant and klein ; klamroth et al. ; klein and bryant ; pelletier et al. ; prowse ; radosevich and burnouf ; solheim ) . common methods for virus capture include filtration (charcosset ; grein et al. ; liu et al. ; van reis and zydney ) and chromatography in column or membrane configurations (charcosset ; gottschalk ; liu et al. ; orr et al. ; van reis and zydney ; segura et al. ). viral filtration is usually the final purification step in the downstream processing of biopharmaceutical products, e.g., monoclonal antibodies ( fig. ) , following one or more chromatography ''polishing'' steps that contribute to the overall virus removal efficiency of the process before concentration of the purified product. filtration technologies are also extensively used for the capture and concentration of waterborne viral pathogens (gibson ) from drinking, environmental, recreational or waste water samples (cashdollar and wymer ; ikner et al. ). in addition, particulate air filters are used in personal respiratory protective equipment (i.e., face masks and respirators) cohen and birkner ; rengasamy et al. ) to ensure shortrange protection of wearers against airborne pathogens-which include a number of viruses (tang et al. )-and in air purifiers/cleaners to limit longrange aerosol transmission of infection in healthcare settings (hyttinen et al. ; tang et al. ) . cellulosic membrane microfilters have been used routinely for ages in laboratories to perform the socalled ''sterile filtration'' (cold sterilization), i.e., the absolute removal of bacteria, yeasts and molds but not viruses (walsh and denyer ) from heat-sensitive liquid media. however, many virus filtration devices currently implemented are made from cellulose and its derivatives. some eighty years ago, gradocol, graded collodion (cellulose nitrate) membrane filters (elford ; bauer and hughes ) have been extensively used in ultrafiltration to estimate the size of several medically important viruses such as foot-and-mouth disease (galloway and elford ) , vaccinia (elford and andrewes ) , herpes (elford et al. ) , poliomyelitis (elford et al. ) and influenza (elford et al. ) (see also ferry ( ) for an exhaustive review). later on, commercial mixed cellulose ester filters with very low pore size, namely vf (virus fine), vm (virus medium) and vc (virus coarse) grade filters produced by millipore (billerica, ma, usa-a subsidiary of merck kgaa, darmstadt, germany), have also been applied to virus ultrafiltration for grouping by size assessment (hsiung ). as part of a series of papers surveying the antiviral applications of polysaccharide-based materials, the present review focuses on viral filtration using cellulosic filter media-most of which are commercially available products that have been extensively tested over the past twenty years for the removal/concentration/purification of viral particles from liquid samples, i.e., biopharmaceutical (namely blood products) and environmental (raw or treated) water samples. the application of cellulose-based filters in individual respiratory protective devices protecting the wearer against airborne viral pathogens is also detailed. polysaccharide-based chromatographic adsorbents for viral clearance or recovery/purification of viruses or virus-like particles will be presented elsewhere. in the processing of biological products, virus removal from the product stream while providing maximum product recovery is a critical task. it is more particularly difficult to eliminate small viral particles such as parvoviruses, which may contaminate blood products and mammalian cell cultures used in the production of recombinant proteins (charcosset ) . viral filters developed to answer this challenge are typically membrane (screen) filters ensuring a size-based rejection of viral particles via a sieving mechanism. given the size of viruses, ranging roughly from (parvoviridae) to nm (poxviridae) (segura et al. ) , viral filtration stands between microfiltration and ultrafiltration among pressure-driven filtration processes (fig. ) , though it is frequently but incorrectly (van reis and zydney ) classified as nanofiltration (burnouf et al. ). . regenerated cellulose hollow fiber (hf) membrane filters common virus filtration membranes are made from poly(ethersulfone) (pes), poly(vinylidene fluoride) (pvdf), and regenerated cellulose (burnouf and radosevich ; carter and lutz ; liu et al. ; van reis and zydney ) . among the latter, the commercial planova tm filters (asahi kasei medical, tokyo, japan) have been widely used to clear viruses from biologically produced pharmaceuticals, in particular blood products. planova filters are composed of cuprammonium regenerated cellulose hf (bemberg tm cupro fibers), prepared from cellulose cuprammonium spinning solution via microphase separation under precise spinning conditions (tsurumi et al. b, c) . the wall of each hf has a threedimensional web structure of pores consisting of large, bulky void pores interconnected by fine capillaries (tsurumi et al. a, b, c) (fig. ). during filtration, as the feed solution containing the product of interest is circulated through the hf bore, viruses accumulate in the large, bulky void pores of the fiber network while the product solution passes through the capillary pores. since the hf wall is several tens of micrometers thick (fig. ) , viruses are captured gradually inside the porous structure that can be considered multi-layered ( - layers) (hongo-hirasaki et al. ) . hence, planova filters behave like ''membrane depth filters'' (walsh and denyer ) operating on the basis of size exclusion. they offer a choice of nominal mean pore sizes, namely nm ( n), nm ( n), nm ( n) and nm ( n)-the large pore n model being essentially used as a prefilter to remove impurities or aggregates prior to final virus filtration-and can be operated in normal (dead-end/flow through) or tangential flow (cross flow) filtration mode (phillips et al. ). planova filters have emerged to answer a publichealth problem of worldwide magnitude, i.e., blood contamination by hiv (hamamoto et al. ; manabe et al. ) and hepatitis viruses (yuasa et al. ; sekiguchi et al. sekiguchi et al. , . table gives examples of viral clearance studies assessing the capacity of planova filters to remove viruses from blood products. in these viral clearance assays, product streams resulting from successive, scaleddown purification steps representative of the manufacturing process of the product (kundu and reindel ) , were artificially contaminated (''spiked'') with model viruses of different sizes (table ) (at much higher concentrations than what might be commonly found in the product intermediate) before being submitted to filtration. the virus removal efficiency of the filtration step was expressed as lrv (log reduction value, i.e., log ratio of the viral load in the spiked product feed stream to that recovered in the product filtrate), which implies that residual virus infectivity can never be reduced to zero but may be greatly reduced mathematically (ich ) . minimum lrv values were most frequently given. they were estimated when no viruses could be detected in filtered samples taking into account the detection limit of the assay (viral titer estimated as infectious unit per sample volume). from the measured lrvs, the filtration operation is usually classified as effective (lrv [ ), moderately effective ( \ lrv \ ) or makino et al. ( ) rev environ sci biotechnol ( ) ineffective (lrv \ ) (phillips et al. ). since filtration complements other viral clearance methods in the downstream processing of biotherapeutics (brorson ; klamroth et al. ; shukla et al. ) , several studies quoted in table also report lrvs for common virus reduction steps such as inactivation by pasteurization (gröner ; gröner et al. ; terpstra et al. ) or solvent/detergent treatment (dichtelmüller et al. ; terpstra et al. ) , and chromatographic capture (gröner ; gröner et al. ) . virus removal during the plasma fractionation process (bryant and klein ) leading from the crude plasma pool to the product (immunoglobulin here) stream has also been quantified (dichtelmüller et al. ; koenderman et al. ; terpstra et al. ) . overall virus reduction factors obtained by addition of successive lrvs are given in these works, as illustrated by table . in the virus spiking studies quoted in table , the actual viral filtration step was preceded by prefiltration to eliminate viral and/or protein aggregates that might be present in the spiked product solution. the presence of aggregated viruses may lead to overestimation of size exclusion effectiveness while contributing with protein aggregates to the fouling of viral filters, which reduces the membrane hydraulic permeability and may affect virus retention (phillips et al. ). pre-filtration was performed on the spikevirus stock suspension (caballero et al. ; gröner et al. ; roberts et al. ) on the product intermediate before virus inoculation (gröner et al. ; koenderman et al. ) , and/or on the virus furuya et al. ( ) and terpstra et al. ( ) cpv canine parvovirus b v gröner ( and terpstra et al. ( terpstra et al. ( , spiked material (chtourou et al. ; dichtelmüller et al. ; koenderman et al. ; terpstra et al. terpstra et al. , . for pre-filtration of the virus preparation, micro filters with a pore size adapted to the spike virus size, i.e., slightly lower than the virus size, were used to remove cell aggregates but not single viral particles-usual pore sizes ranging between . and . lm. micro filters with . - . lm pore size (koenderman et al. ; terpstra et al. ; , but also n (dichtelmüller et al. ) and n (chtourou et al. ) . planova viral filters were used to pre-filtrate the spiked product solution. in the latter work, the association in series of n and n filters led to efficient removal of small viruses such as ppv (chtourou et al. ). this was not the case when n was used as the main viral filter after pre-filtration with n (dichtelmüller et al. ) , which confirms previous date showing that the nm pore size was too large to retain the smallest viral particles (furuya et al. ; hongo-hirasaki et al. ). both n (caballero et al. ; furuya et al. ; gröner ) and n (caballero et al. ; roberts et al. ; terpstra et al. ; planova filters and their combination (gröner et al. ; koenderman et al. ) showed effective virus removal over a wide range of viral particle sizes (table ) . as a general rule, the removal efficiency of the filters increased with the ratio of pore size to virus size, as illustrated by gröner ( ) and terpstra et al. ( ) . this was also demonstrated by roberts et al. ( ) by filtrating poliovirus type (a picorvanirus) through planova filters manufactured with different pore sizes, ranging between (lrv = . ) and nm (lrv = . ). the two-filter combinations were globally more efficient than single filters at removing small viruses from spiked protein intermediates, though two n filters connected in parallel for tangential filtration (koenderman et al. ) yielded lrvs quite similar to those obtained using n alone (terpstra et al. ). care should be taken when comparing vertically the data collected in table , however. in addition to the sizes of viral particles and filter pores, the results of such virus-spiking studies are dependent on a number of process parameters such as the filtration operating conditions (filtration mode, transmembrane pressure, volume per filter area, tempera-ture…), the characteristics of the product feed stream (ph, conductivity, nature and concentration of the product…), or else the purity level and concentration of the virus spike. moreover, several studies include pre-filtration (dichtelmüller et al. ) or viral inactivation (koenderman et al. ; terpstra et al. ) in the calculation of lrvs. this variability in test conditions for filtration-based virus removal makes difficult any comparison between lrvs collected from multiple sources as in table , even though some results (caballero et al. ; hongo-hirasaki et al. ; roberts et al. ) contradict its influence on filtration performance, underlining the robustness of the filtration process. also, some variations in virus removal capacity may occur between commercial filter modules owing to their manufacture from different batches of hf, as illustrated by roberts et al. ( ) for n filters. like membrane microfiltration, depth filtration is widely used as a clarification step in biopharmaceutical purification to ensure removal of cell debris, large aggregates and contaminants from the product stream prior to purification processes such as column/membrane chromatography and viral filtration/inactivation steps (liu et al. ; van reis and zydney ; vicente et al. vicente et al. a, b) . commercially available depth filters currently employed in bioprocessing are composed of cellulose fibers with a porous inorganic filter aid and a resin binder, generally cationic. for instance, the zeta plus tm filter media ( m purification inc.-formerly cuno, meriden, ct, usa) is composed of a cellulose fiber depth matrix containing silica-based filter-aid material and positively charged by chemical bounding of a cationic charge modifier (ostreicher ) . varying retention ratings are available ( . - . lm). the triple layered a hc filter media from emd millipore include two depth filtration layers (cellulose ? diatomaceous earth) of different grades and a . lm-pore-size microporous membrane pre-filter made of mixed cellulose esters (van reis and zydney ). hence, these cellulosic depth filters rely on both size exclusion and electrostatic adsorptive binding to effect separation (liu et al. ) . in viral spiking studies (barnette et al. ; dichtelmüller et al. ; zhou et al. ) , they were found to retain viral particles whose surface is negatively charged over a wide range of ph as their isoelectric points (pi) range most frequently between . and . , with a mean value of . ± . (michen and graule ) . viruses are recognized as a major cause of waterrelated disease (bosch et al. ; fong and lipp ; gibson ; hamza et al. ) . enteric viruses, more particularly, which are associated primarily with gastrointestinal illness (bosch et al. ; fong and lipp ; gibson ; hamza et al. ) , are implied in most waterborne viral outbreaks (gibson ; hamza et al. ). all types of water, including waters used for drinking (surface or ground supplies), recreational waters (fresh, marine, and swimming pool), agricultural waters for irrigation (rivers and groundwater) and waste waters (sewage or industrial effluents), have been shown to be potential vehicles for virus transmission (bosch et al. ) . contamination is most frequently of fecal origin (bosch et al. ; wong et al. ) . although viral levels may be high at the contamination source, e.g., concentrations ranging between and virus particles per gram of feces are referred to in the literature (michen and graule ) , viral concentrations as low as - viral particles per liter may be found in environmental water (julian and schwab ) . since enteric viruses display high infectivity (fong and lipp ; julian and schwab ) , such titers may constitute a health risk and should be detected for reliable surveillance of viral pathogens in water. as a consequence, efficient concentration methods are needed to capture viruses in large-volume water samples and release the retained viral material in concentrated form. most common methods are based on filtration processes (cashdollar and wymer ; ikner et al. ). in particular, virus adsorptionelution (viradel) filter methods have been widely implemented since the s (cashdollar and wymer ) . briefly, in a viradel method, viruses from aqueous samples are reversibly adsorbed to microporous filters and then eluted from the filters in a small liquid volume (apha, awwa and wef ). adsorbent filters carry electrical charges and virus retention occurs via electrostatic interaction rather than by size exclusion. in other words, viradel filters act as depth filters rather than sieves. contrary to viral filters that retain viruses by a sieving mechanism to achieve complete viral clearance, their pore size lies in the microporous range, which allows the high flow filtration necessary for virus capture in large water samples. positively or negatively charged filters can be used in a viradel procedure, among which those made of cellulosic materials have been dominant over the last few decades. since their pi is below the ph of natural water (i.e., around neutrality) ( huang et al. ( ) table continued filter type a water nature no. [ tested earlier for concentration of viruses in water samples of different origins (table ). more recent studies made use of zeta plus tm mds microfilters, however (table ). in these works, pure (e.g., distilled), tap, environmental or sewage water samples were seeded with varying enteric viruses that are common waterborne pathogens, i.e., members of the families picornaviridae (polioviruses, coxsakieviruses, teschoviruses), adenoviridae (adenoviruses), caliciviridae (noroviruses, caliciviruses) and reoviridae (rotaviruses) (fong and lipp ) , or bacteriophages (enterobacteria phages) that are considered as alternative indicators of fecal contamination and as index organisms for the presence of enteric viruses in waters (goodridge and steiner ). naturally contaminated waters containing sufficient levels of indigenous viruses, more particularly bacteriophages, were also used in addition to spiking studies sensu stricto (table ) . following sample filtration, viral particles adsorbed to the filter were eluted in concentrated form using a variety of eluting solutions, the most common eluent consisting of a slightly alkaline (ph . - . ) protein solution (i.e., beef extract), frequently buffered with glycine-naoh or another amino acid solution (sometimes supplemented with salt to aid disruption of electrostatic interactions between viruses and filters (shields and farrah ) , a chaotropic agent (e.g., urea) or a surfactant (e.g., tween ) to affect virus-filter hydrophobic interactions . eluted viruses were quantified-using plaque titer assays, % tissue culture infectious dose (tcid ), quantitative realtime pcr (qpcr) and reverse-transcription pcr (rt-qpcr) (hamza et al. )-to assess the recovery efficiency of the filtration step by comparing to input titers. this efficiency depends on a number of process parameters including the filter type and filtration conditions (e.g., filtration rate and pressure), the elution buffer composition and eluting conditions, the nature, input titer and titration method of the tested virus-in addition to the water matrix characteristics that may affect virus quantification and filter performance (borchardt et al. ) . hence, tables and display a wide range of recovery yields. in practical tests aimed at the detection of naturallyoccurring viruses in field, large-volume water samples, the filtration (adsorption-elution) step is usually a primary concentration step which is followed by a secondary one to reach the virus detection threshold, e.g., organic flocculation or peg precipitation (lewis and metcalf ) . the recovery yield of such two-step concentration processes was evaluated in several studies using seeded or naturally contaminated water samples (tables , ) . as a general rule, the viral loss due to secondary concentration was balanced by a huge increase in concentration factor allowing virus detection in water concentrates from large-volume samples, as reported by chang et al. ( ) and raphael et al. ( ) for indigenous enteroviruses in wastewater and rotaviruses in sewage-polluted surface water, respectively. after determining the optimal elution conditions for mds disk-adsorbed noroviruses following filtration of spiked distilled water samples, lee et al. ( ) (table ) applied the optimized procedure to the detection of noroviruses in environmental water. they combined adsorptive filtration using mds cartridge with organic flocculation to enrich viruses from largevolume surface ( l) and ground ( l) water samples. this integrated two-step process led to high volume reduction factors, i.e., , and , for surface and ground water samples, respectively ( - for filtration, for organic flocculation), but its recovery efficiency was not evaluated by spiking studies. in the same way, beside spiking woo et al. ( ) . a ratio of the virus survival factor in the treated filter to that in the untreated filter, where the virus survival factor in a filter is the number of viruses recovered by elution from the filter divided by the number of viruses removed by the filtration process. b pf pp filter from commercial surgical mask (dupont tm n), ccf coarse pore cellulose filter paper (whatman tm grade , lm pore size), fcf fine pore cellulose filter paper (whatman tm grade , . lm pore size) studies like those detailed in table , mds filter cartridges have been widely applied to concentrate enteric viruses in water samples of different origins (table ) , ranging from large-volume samples of weakly contaminated water intended for drinking or drinking water production (borchardt et al. (borchardt et al. , lee et al. ; sedmak et al. ; verheyen et al. ) to wastewater samples with high viral content-to assess the virus removal efficiency of wastewater treatment plants (kuo et al. ; simmons et al. ) . in these tests of field water samples for enteric viruses, mds filtration was mainly combined with second-stage concentration by organic flocculation as detailed by fout et al. ( ) in the usepa (united states environmental protection agency) manual of methods for virology. the elution of filter-adsorbed viral particles was mostly performed with an alkaline beef extract solution supplemented or not with glycine (buffers eb and eb in table ). the work by verheyen et al. ( ) is an exception to these common procedures. to concentrate viruses in small-volume samples from drinking water sources, these authors used two filter cartridges in series with no additional concentration step and an alkaline powdered milk solution as elution buffer. they found that % ( / ) and % ( / ) of surface and ground (well) water sources, respectively, were contaminated with adenoviruses, with very few samples positive to rotavirus ( / surface water samples, / well water sources). despite the differences in virus concentration methods, these results compare with those obtained by xagoraraki et al. ( ) and cheong et al. ( ) for adenovirus detection in surface ( / samples, %) and ground water ( / samples, %), respectively. they also agree with data reported by borchardt et al. ( ) and cheong et al. ( ) showing the low contamination level of ground water by rotavirus. it is worth noting here, however, that the number of positive samples reported in these viral analyses of water (and the others quoted in table ) is dependent on the sensitivity of the detection techniques used to assess the presence of viruses, i.e., nucleic acid-based amplification methods such as conventional pcr, reverse-transcription pcr (rt-pcr), nested pcr, real-time pcr/rt-pcr or integrated cell culture pcr (icc/pcr) to determine virus infectivity (fong and lipp ; hamza et al. , mattison and bidawid ; watzinger et al. ) with different detection thresholds. it emerges from this glance at literature that cellulose-based electropositive filters are still commonly used for viral monitoring of water. these filters are expensive, however, and face competition with cheaper products, in particular nanoalumina fiber filters and glass wool filters (cashdollar and wymer ; wong et al. ) . the former, nanoceram tm filters manufactured by argonide corporation (sanford, fla., usa), are composed of nanosized ( nm diameter), alumina-based {mainly boehmite, c-alo(oh)} fibrilles dispersed in a microglass fiber matrix, resulting in an electropositive filter media with - lm average pore size kaledin , ) . the latter consist of commercial sodocalcic glass wool coated with mineral oil (type bourre qn/tech loose wool, isover saint-gobain, courbevois, france), hand packed into columns or filter holders in the laboratory (vilaginès et al. ) . these glass wool filters harbor electropositive sites while presenting hydrophobic surface characteristics. efficient enrichment of (seeded or/ and) indigenous viruses from various water samples using nanoceram (gibbons et al. ; ikner et al. ; pang et al. ) , and glass wool (deboosere et al. ; lambertini et al. ; wyn-jones et al. ) filters has been reported in recent years (see also cashdollar and wymer ; wong et al. ) . compared with mds filters (table ) , nanoceram filters showed similar , slightly lower (mcminn ) or higher (karim et al. ) virus concentration efficiency. commercially available under different pore sizes ( . - . lm), mixed cellulose ester membrane filters are negatively charged over a wide range of ph values, their overall negative charge increasing with ph (kessick and wagner ) . microporous filters in the . - . lm pore size range have been used for ages in laboratory and industry for size-based filtration of bacterial particles and cell debris (surface filters). these and larger pore size filters have been shown to retain enteroviruses, however, despite the much smaller size of viral particles compared to the nominal mean diameter of filter pores (cliver ). the presence of salts enhanced virus adsorption to cellulose ester membranes, this effect increasing with the cation valence (i.e., al ? [ mg ? [ na ? ) (wallis et al. ) . acidification of the viral suspension also improved virus adsorption efficiency, even in the absence of exogenously added salts (sobsey et al. ). these early results were later confirmed by lukasik et al. ( ) , who investigated the influence of mono-, di-, and trivalent salts (nacl, mgcl , and alcl ) on the adsorption of poliovirus and enterobacteria phages to the mf-millipore tm membrane filter type ha ( . lm pore size) and the electropositive m tm zeta plus tm mds microfilter at neutral or acidic ph. at ph , salts promoted virus adsorption to ha filter while affecting adsorption to mds filter. at ph . , more than % of the viruses tested adsorbed to ha filter with or without salt added to the viral suspension-the salts interfering again with viral adsorption to mds filter. furthermore, the addition of urea or tween to the salt solution affected virus adsorption to both filters at ph . and ha filter at ph . in agreement with previous studies by the same group (haramoto et al. (haramoto et al. , lukasik et al. ( ) explained these results by the antichaotropic effect of salts that increased hydrophobic interactions between filters and viruses and was impaired by the chaotropic agent or the detergent. at neutral ph, charge screeening by salt addition reduced electrostatic attractive and repulsive forces between viruses and mds or ha filter, respectively. cation-mediated cross-complexation between negative groups on virus and filter surfaces (kessick and wagner ) and, more particularly, strengthened hydrophobic virusfilter interactions contributed to improving the adsorption efficiency of ha filter. at acidic ph, viruses displayed a positive surface charge and their electrostatic interactions with ha filter switched from repulsive to attractive-and inversely for electropositive mds filter. the presence of salt probably affected these interactions but was balanced by the promoting effect of salt on hydrophobic interactions. hence, most tests reported so far for virus concentration from water samples using cellulose ester filters (essentially ha filters) have been performed after addition of multivalent cations (mainly mg ? ) to the water samples with or without ph adjustment to an acidic level. according to katayama et al. ( ) , the virus-loaded filters were rinsed with an acidic solution to eliminate remaining cations before elution with naoh or other alkaline buffers. haramoto et al. ( haramoto et al. ( , haramoto et al. ( , a proposed a variation in the method that consisted in pre-conditioning the filter with al ? ions. aluminum chloride was passed through the filter, making an electropositive ion coating, which avoided cation addition to the water sample. the recovery efficiency of both protocols has been evaluated by seeding water samples with enteric viruses and bacteriophages and quantifying eluted viruses (table ) . similarly to spiking studies (tables , ) and viral analyses of field water samples (table ) with electropositive filters, the ha-based filtration/elution process was frequently associated with a secondary concentration step to increase virus concentration factors. here, centrifugal ultrafiltration (cu) using commercial millipore (centriprep Ò , centricon Ò or amicon Ò ) concentrators was the elective concentration method. these cu units contain a low adsorptive regenerated cellulose membrane (ultracel Ò ) whose nominal molecular-weight cutoff ranges between and kda-a mwco value of kda being most frequently selected for secondary concentration of eluted viruses. they are routinely used in laboratories to purify and concentrate biomacromolecules such as peptides, proteins, and nucleic acids from small-volume biological samples (e.g., - ml for the centriprep filter unit). tested or not for virus recovery by spiking experiments, the combined concentration procedures have been extended to the detection of enteric viruses in field water samples including tap (haramoto et al. ), sea (katayama et al. ) , river (surface) (fong et al. ; hamza et al. ; haramoto et al. ) and waste (fong et al. ; katayama et al. ) water (table ) . samples with different volumes were collected according to the water source, i.e., sample volumes increased as the expected contamination level decreased. for instance, katayama et al. ( ) applied ha filtration, followed by acid rinse of the filter, viral elution with naoh and secondary concentration of the eluate by cu, to concentrate naturally occurring viruses (noroviruses, enteroviruses and hav-hepatitis a virus) in seawater samples. based on the volume reduction factor ( -l sample/ ml final concentrate, i.e., ) and the recovery yield of the two-step process (table ) , a virus concentration factor of was reached. only hav virus was not detected in any sample tested. later on, katayama et al. ( ) followed the same protocol to concentrate enteric viruses in the raw influent of a wastewater treatment plant. samples of -ml volume were collected and submitted to the two-step concentration process, yielding a volume reduction factor of c. . the four tested kinds of enteric viruses were detected in all wastewater samples but one lacking norovirus gi (nov gi). using peg precipitation as the secondary concentration step, hamza et al. ( ) tested river water samples for contamination by enteric viruses and bacteriophages. a volume reduction factor of ( -l sample/ ml final concentrate) and virus concentration factors ranging between (norovirus) and (adenovirus) (see table for recovery yields) were obtained. all analyzed samples were found positive for enteric viruses. human adenovirus and norovirus were detected in . and % of the samples, respectively. haramoto et al. ( ) illustrated the ability of al ? -coated filters to concentrate viruses from large-volume freshwater samples without salt addition by detecting noroviruses in tap water from tokyo university. tap water samples ( -l average volume) underwent two successive filtration/elution steps using -mm and -mm diameter ha filters prior to concentration by cu (final volume: . ml), which ensured a volume reduction factor higher than , . however, the virus recovery efficiency of the -step concentration process was not evaluated. ten of the tested samples were found positive for noroviruses. these results compare to those reported by lee et al. ( ) ( table ) using mds cartridge with organic flocculation to enrich noroviruses from large surface and ground water samples. despite these promising data, the two viradel methods based on electronegative filters (i.e., addition of mgcl to water samples or filter coating with al ? ions before filtration) have been essentially implemented for viral analysis of waters containing high amounts of indigenous viruses, requiring limited sample volumes (table )-electropositive filter cartridges being more adapted to virus concentration from large volumes of weakly contaminated water (table ). beside the above-mentioned work by haramoto et al. ( ) , only two studies among those detailed in table describe virus detection in environmental water samples with low viral content, namely drinking water sources after chlorination or without treatment (rigotto et al. ) and groundwater from artesian wells (chironna et al. ) . both studies used small-volume samples and cu as secondary concentration step, yielding a modest volume reduction factor of . very few from artesian well water samples were found positive to the tested enteric viruses: none for hav and enterovirus, for rotavirus and for norovirus (chironna et al. ) . a large proportion ( %) of samples from drinkingwater supplies was positive to adenovirus, however (rigotto et al. ) . these data are compatible with viral analyses of groundwater and water intended for drinking performed using mds filtration as the first virus concentration step (table ). it should be noted, however, that the same remark applies to data collected in table as to those in table concerning the numbers of virus-positive samples, i.e., their dependence on the virus detection method. this can be illustrated by the two following examples. using icc-pcr (measuring infectious viruses), katayama et al. ( ) detected enteroviruses in of seawater samples from bathing beach, but no sample was found positive by direct rt-pcr. de paula et al. ( ) found / river water samples positive for hav by quantitative real-time rt-pcr, but only / by nested rt-pcr. as attested by the large body of literature data quoted above, virus adsorption-elution methods using electropositive or electronegative filters are routinely applied to the primary concentration of waterborne viruses, each type of filter possessing its own advantages and drawbacks. ultrafiltration is considered another filtration-based option to concentrate viruses from water samples (cashdollar and wymer ; ikner et al. ). it has been indicated earlier that cu with microconcentrators based on cellulose ultrafiltration discs is commonly used as a secondary concentration step following the viradel process. hollow fiber ultrafiltration (hfuf), however, is considered a potential technique for primary concentration of viruses from large-volume water samples, yielding better virus recoveries than the viradel method performed with either electropositive or electronegative filters (cashdollar and wymer ) . commercial hfuf devices operated in cross-flow mode have been applied to the simultaneous concentration of biological particles, including viruses, spiked in tap (polaczyk et al. ) or reclaimed (liu et al. ) water samples, to virus recovery from seeded tap (rhodes et al. ) and estuarine (hernandez-morga et al. ) water samples, and also, more rarely, to virus recovery from field water samples (grassi et al. ; hernandez-morga et al. ). most of these devices are dialyzers that contain synthetic hf made from polysulfone. following the early work by belfort et al. ( ) showing that polysulfone hf membranes were superior to cellulose acetate ones for virus concentration, virus concentration experiments using hfuf dialyzers equipped with hf manufactured from cellulose are scarce. an example is given by liu et al. ( ) . they showed that the exeltra plus cellulose triacetate hf dialyzer (baxter healthcare corp., deerfield, il, usa) and the optiflux Ò f nr polysulfone dialyzer (fresenius medical care, walthamm, ma, usa) provided similar recovery efficiencies for ms and ux bacteriophages, escherichia coli, clostridium perfringens spores, and cryptosporidium parvum oocysts from spiked -l and -l samples of reclaimed water. an array of viral infections can be transmitted by the airborne route, in particular via aerosols (droplet nuclei) tang et al. ; tellier ; verreault et al. ). even though virus inactivation rates in the atmosphere are generally higher than those of bacterial and fungal contaminants, virus-containing aerosols can spread worldwide (després et al. ) . the threat of viral outbreaks and pandemics such as those caused by sars coronavirus (yu et al. ) and highly pathogenic strains of influenza a virus (tellier ) , allied with the fear of bioterrorism using viruses (e.g., smallpox and hemorrhagic fever viruses) (barras and greub ) as biological weapons, has encouraged the search for efficient protection equipment against aerosolized virus-containing particles. individual respiratory protective devices consist in respirators, i.e., air-filtering face masks designed to protect the wearer against inhalation of a hazardous atmosphere (here airborne infectious aerosols)-opposing to surgical masks designed to protect the environment from contaminants generated by the wearer's exhaled breath (i.e., prevention of surgical infections) . currently, most ''filtering facepiece'' (ffp) respirators have a multilayer composite structure with a central filtering layer displaying electret properties (gralton and mclaws ) . these electret filters (thakur et al. ) are produced by imparting an electrostatic charge to a nonwoven fibrous mat composed of synthetic polymer fibers such as poly(propylene) (pp) (mainly), poly(butylene terephthalate), poly(tetrafluoroethylene) and poly(carbonate) fibers. electrostatic charging of the filter media is commonly obtained by corona discharge, triboelectrification and electrostatic spinning (tsai et al. ) . electret filters collect particles through the combined action of mechanical and electrostatic forces (podgórski ; wang (cen ) should retain respectively at least and % of influent particles (nacl particles are used as nonbiological surrogate particles) (rengasamy et al. ). first worn by surgical teams a hundred years ago to prevent bacterial contamination of patient's open wounds, early surgical masks were constructed from layers of cellulose materials, more particularly cotton cellulose (gauze) (haller and colwell ) and derivatives (arnold ) (see also belkin ) . much more recently, face masks made from cotton fabrics have been tested as alternative respiratory protective equipment against pandemic outbreaks such as influenza (dato et al. ; davies et al. ; rengasamy et al. ) . like respirators, current commercially available surgical face masks include several layers of non-woven fabrics with, frequently, a cellulose inner layer in contact with the wearer's face to improve wearer's comfort. however, the filtering layer, usually made of meltblown fibers (ghosh ) , is devoid of electret properties. since particulate filtration is only mechanical, i.e., less efficient than that of respirators, most surgical masks are not certified for use as respiratory protective devices, standard tests to evaluate their filtration efficacy being less stringent (oberg and brosseau ) . while a number of studies have confirmed that surgical masks logically offer lower protection than respirators against aerosol particles, the effectiveness of both types of face masks at preventing viral respiratory infection, in particular influenza, is still a matter of controversy (bin-reza et al. ; cowling et al. ; gralton and mclaws ) . the improper facial fit of respirators coia et al. ; weiss et al. ) may affect their protection efficiency against infectious aerosols. it allows particulate flows outside the filtration area of the mask, resulting in face-seal leaks, i.e., the leakage of infectious particles around the edges of the mask (grinshpun et al. ; lei et al. ) . mask seal leakage can be minimized by applying existing guidelines for correct donning and fit checking of respirators . moreover, human face and head form models have been proposed as a tool for designing respirators with improved protection efficiency, simulating interactions between faces and facemasks and describing their fit (golshahi et al. ; lei et al. ) . the accumulation of viral particles at the surface and within the filtration media of respirators, where they can remain viable and infectious for extended periods of time (coulliette et al. ; sakaguchi et al. ) , is another significant problem with which respirator wearers are confronted in the prevention of viral transmission and spread. incorrect mask handling by the wearer may lead to accidental self-inoculation, cross-contaminations affecting both other healthcare workers and patients, and contamination of fomites (casanova et al. ) . furthermore, the risk of virus reaerosolization from respirators during extended use, if limited, cannot be ruled out (fisher et al. ) . recommendations for respirator doffing do exist but have proven insufficient to prevent virus transfer from respirator to healthcare employees' hands and clothing (casanova et al. ) . adding antiviral properties to the filtration process may contribute to limit the risk of viral transmission by improper handling of used respirators. a number of virucidal facemasks have been developed and patented over the past years (tiliket et al. ) , some of which are commercially available. in the spectrashield tm plus respirator masks designed by nexera medical inc. (fort lauderdale, fla.) (haas ), for instance, two layers of filter media are sandwiched between (inner and outer) antimicrobial layers where a silver-copper zeolite (carrier) antimicrobial agent (agion Ò antimicrobial, sciessent llc, wakefield, mass.) is embedded around the core of synthetic fibers (fosshield Ò , foss manufacturing, hampton, nh). the biofriend tm biomask tm ffp respirator, manufactured by filligent ltd. (hong kong) and distributed by medline industries, inc. (mundelein, ill.) , is another four-layered device in which the melt-blown pp filtration layer is inserted between an inner layer of spunbond pp and two antiviral layers, namely a cellulose/polyester layer containing copper and zinc ions and an outer layer of spunbond pp treated with a low ph (citric acidacidified) hydrophilic plastic coating (davison ; stewart et al. ) . the outer layer absorbs infectious aerosol droplets and viruses are denaturated by exposure to citric acid. in the second layer, inactivation of viruses with damaged structures is completed by the virucidal effect of divalent metal cations. contributing to the overall antiviral efficiency of the structure, the cellulosic component of this layer is a sulphated or sulfonated rayon fabric (stewart et al. (stewart et al. , to which a variety of viral human pathogens bind via the cationic sites of viral envelopes/capsids. beside the commercial biomask tm respirator, various composite structures designed for filtrationinactivation of airborne microbial contaminants, where cellulosic materials play an active antimicrobial role, have been patented in recent years bernard ; nakamura and nakamura ; tsutsumi ; zhang et al. ; zhong ) . several of them are based on bacterial cellulose layers/coatings provided with an antimicrobial component, i.e., silver (zhong ) or zeolite-supported silver (nakamura and nakamura ) nanoparticles, and chitosan (zhang et al. ) . the respiratory protective mask described by zhong ( ) comprises a three-layer bacterial cellulose membrane whose middle layer contains silver nanoparticles. the antimicrobial facemask invented by nakamura and nakamura ( ) includes two base cloth elements both of which are made of a woven textile (gauze), a nonwoven cellulose fabric (e.g., rayon), or a porous sheet (e.g., a . - . mm sliced sheet of urethane sponge). the first base cloth is filled with bacterial cellulose nanofibers that retain silver zeolite and a humectant (e.g., trehalose or , -butylene glycol) in their network structure. additional antimicrobial properties are provided to the mask by impregnating the second base cloth with a carboxylic acid (e.g., citric acid). zhang et al. ( ) also used bacterial cellulose in a composite antimicrobial material suitable for respiratory protective masks. a nonwoven polymer fabric rev environ sci biotechnol ( ) : - was coated with a film of bacterial cellulose mixed with poly(vinyl alcohol)-to improve the film-forming properties, mechanical strength and air permeability of the coating-and chitosan, a non-sulfated polysaccharide produced commercially by deacetylation of chitin that displays strong antibacterial potency (kong et al. ; rabea et al. ) and, to a lesser extent, antiviral activity (rabea et al. ; wang et al. ). in the antimicrobial air filtration device proposed by bernard ( ) , cellulose derivatives-more particularly, cellulose acetate phthalate (cap), a common pharmaceutical excipient for enteric coating of tablets and capsules with antiviral activity (pirrone et al. )-were used as ''biocidal prophylactic compounds'' to provide the classical filter media (e.g., a nonwoven pp web, electrostatically charged or not) with antimicrobial properties. cellulose derivatives could be incorporated as fibers or particles (micronized fibers) into the air filtration device either in a separate layer positioned before or after the filter media, or deposited onto the outer surface of the filter media relative to the air flow direction. to illustrate this invention, handsheets prepared from cap fibers intermixed or not with cap particles were successfully tested for virucidal efficacy by exposure to an aerosol challenge of enterobacteria phage Øx . by exploiting the antimicrobial properties of dialdehyde polysaccharides such as starch and cellulose dialdehydes, baney et al. ( ) proposed a new method for producing low-cost virucidal filters suitable for respiratory protective masks. the treatment of a standard cellulose filter paper (whatman tm grade filter paper) with sodium periodate improved its antiviral potency due to oxidation of some cellulose to dialdehyde cellulose inside the filter. challenged with aerosols of ms enterobacteria phage at high relative humidity ( % at °c), the treated filter showed higher filtration efficiency than a control, untreated one, i.e., better removal of viable viral particles (plaque forming units) joined to a lower resistance to air flow (pressure drop)-due to increased pore size distribution (woo et al. ) . higher inactivation of ms virus by the treated filter was also highlighted, confirming an improvement of its disinfection capability (woo et al. ). however, the filtration and inactivation of airborne viruses were less effective at lower moisture content of the filter, i.e., at air humidity levels commonly encountered in hospital settings, in particular operating rooms (balaras et al. ). furthermore, the inhalation resistance of the treated filter remained too high for application to respiratory protective masks. the same pros and cons apply to cellulose paper filters treated by immersion into aqueous suspensions of dialdehyde starch (das) (woo et al. ) . while this treatment did not affect the removal efficiency of viable ms viruses from aerosols at high relative humidity, virus survival on the treated filters was reduced (fig. ) and the pressure drop decreased as das concentration increased. the drop in air flow resistance remained insufficient for practical application to respirators. das treatment of a pp filter from a commercial ffp mask did not modify the filtration parameters (virus removal efficiency and pressure drop) of the filter but significantly improved its biocidal efficacy (fig. )-an opening of this treatment towards the development of virucidal facemasks. another example of cellulose filter material treated with cellulose derivatives to yield antiviral properties is given by tsurumi (tsutsumi ). this inventor devised a low-cost virucidal mask filter made of a standard cellulose nonwoven fabric impregnated with water extracts of sulfonated or aminated styrenegraft cellulose. to reinforce the water retention properties of the filter media, a superabsorbent resin such as poly(acrylic acid) was added to the graft cellulose extract before impregnation of the fabric or incorporated into a separate layer covering the impregnated fabric. low (hydrogen ions from sulfonic acid groups) or high (hydroxyl ions generated by aminated groups) ph conditions inside the hydrated material were claimed to make it virucidal against influenza a virus and caliciviruses. the patent gives no illustration of such antiviral properties, however. studies published by tiliket et al. ( ) and catel-ferreira et al. ( ) present another type of cellulosebased material for airborne virus filtration in which a low-cost nonwoven cellulose material, i.e., commercial kimwipes Ò (kw) wipes (kimberly-clark worldwide, inc., dallas, tex.), was chemically modified by coating with a synthetic polymer, i.e., poly(ethylenimine) (pei) (tiliket et al. ) or grafting of an antiviral agent, i.e., catechin (catel-ferreira et al. ) . the filtration efficiency of the modified filter media was first tested on aerosolized t d viruses (enterobacteria phage t , doermann's strain t d). then the treated filter was inserted inside a commercial medical mask in place of its cellulosic layer (kolmi m mask, kolmi-hopen, saint-barthélémy-d'anjou, france), and the reconstructed mask was challenged with td aerosols to evaluate its virus removal efficiency. both treatments significantly improved the virus capture factor (ratio of upstream to downstream pfu contents) of kw cellulose wipes and of reconstructed commercial masks compared to original masks and to masks reconstructed with untreated wipes. in these studies, the breathability of reconstructed masks was not quantified, nor was the virus survivability on the modified filter media. however, additional filtration experiments using aerosols of influenza a virus (low pathogenic h n strain) showed that challenged viral particles accumulated in pei functionalized kw layers with no loss in number and infectivity (tiliket et al. ) . thus these modified masks showed no virucidal effect, the peimodified layers behaving like an electret media owing to the polycationic character of pei. on the other hand, since catechin and catechin-grafted wipes showed antiviral activity against t d viruses in liquid media (catel-ferreira et al. ) , it may be assumed that part of viruses accumulated in excess in the treated wipes were inactivated by the polyphenol agent, i.e., the reconstructed masks were actually virucidal. despite these few examples, the use of cellulosic media in filtration devices aimed at the capture of airborne viral particles remains now very limited compared to filtration of virus-contaminated liquid samples. while patented, several cellulose-based, virucidal filter media designed for insertion in air filtering face masks have not been commercially developed yet. besides the wearing of personal respiratory protective equipment, another aspect of the fight against airborne infections lies in the decontamination of indoor air in healthcare facilities, and, more generally, of air processed through heating, ventilating and air conditioning (hvac) systems in the built environment. high efficiency particulate air (hepa) filters are the primary technology used for particulate removal in these collective protection devices-allied with uv irradiation, ozonation or air ionization to yield indoor air decontamination (bolashikov and melikov ; jacob et al. )-and may be complemented with a photytocatalyst such as tio to improve the inactivation of accumulated microorganisms pigeot-remy et al. ). since hepa filters are essentially made of glass fibers, they are beyond the scope of this review and their virus-retentive properties will not be detailed further. most frequently associated with other virus reduction (virus clearance of biopharmaceuticals) or concentration (viral analysis of water) steps, size-exclusion and/ or adsorptive (charge mediated) filtration is an essential tool to fight against viral contaminants in aqueous media. owing to inherent properties of cellulose, including mechanical strength and hydrophilicity (that opposes protein adsorption and biofouling), allied with widespread availability and biocompatibility, cellulose-based materials are still widely present in viral filters. developed commercially over the past thirty years, several cellulosic filter media have been and continue to be used for both routine analyses in laboratories and academic research studies aimed at improving the filtration performance, generating a wealth of data quantifying the efficiency of virus removal or recovery/concentration from biologic or water samples, respectively. as concerns biopharmaceutical compounds, these data arise from validation studies of the filtration step performed by spiking intermediate biologic solutions (mostly blood proteins) from multistage purification processes with known titers of viral particles. regenerated cellulose hf planova filters n -n , operated in the dead-end mode as a single filter or two units in series, were shown to provide lrv values ranging between and , on the average. increasingly tested since their launching in , these cellulosic filters compete with filter media made of synthetic polymers such as pvdf (e.g., viresolve Ò nfp-normal flow parvovirus-from emd millipore; ultipor Ò vf grade dv /dv from pall corporation, port washington, ny) or pes (e.g., millipore viresolve Ò pro; virosart Ò hs/hc/cpv from sartorius stedim biotech, aubagne, france). the hydrophilic nature of cellulose fibers is a definite advantage of planova filters over others for virus removal from protein solutions, minimizing flux decline due to proteindominated filter plugging (cake formation) at constant pressure (normal-flow) filtration (rathore et al. ). however, they are usually operated at lower flow rate than filters made of synthetic polymers whose operating pressure is higher. hydrophilic modified pvdf membrane filters designed for virus removal from high-concentration protein solutions at high flow rate are being developed by several manufacturers, including asahi-kasei (i.e., planova tm bioex filters, launched in ). many studies have also been reported to assess the virus concentration efficiency of the viradel method using spiked water samples, prior to the detection of waterborne viral contaminants in field water samples. in this method, cellulosic filters, whether positively or negatively charged, are used for adsorptive capture of viruses before virus recovery by elution with a lowvolume eluent. electropositive filters are more particularly suitable for virus capture in low-contaminated, high-volume water samples, e.g., samples of groundwater or source water for drinking water production ( table ) . though routinely used, these filters are expensive and face competition with nanoalumina fiber filters (e.g., the patented ahlstrom disruptor Ò electroadsorptive filter) (levi ) and glass wool filters, which are cheaper. on the other hand, efficient virus adsorption by electronegative filters-the standard, cost-effective mixed cellulose ester membrane microfilters-requires impractical sample pre-treatment: their use is limited to viral analysis of highlycontaminated waters, for which small-volume samples are sufficient (table ) . cellulose nanomaterials may represent a promising perspective for cellulosic materials in their application to viral filtration of liquid samples, yielding filtration membranes with higher mechanical strength, water permeability, surface hydrophilicicy and resistance to biofouling (carpenter et al. ) . ma et al. ( ) have presented a composite membrane consisting of an electrospun poly(acrylonitrile) nanofibrous scaffold deposited on a non-woven poly(ethylene terephthalate) support. this two-layered membrane was coated by a layer of cellulose nanofibers and tested for retention of ms- phages. at acidic ph, phage particles were adsorbed by the negatively charged cellulose nanofiber coating, yielding a lrv value [ . . later on, the same nanofibrous composite membrane was doped with cellulose nanofibers functionalized by grafting with poly(vinylamine) . ms- phages were adsorbed at neutral ph onto positively charged amino-modified nanofibers, with a lrv value of . metreveli et al. ( ) and asper et al. ( ) have presented a size-exclusionbased filter paper made of pure cellulose nanofibers for removal of swine influenza a and murine leukemia viruses, respectively. these non-woven materials could be tailored to ensure efficient virus retention (gustafsson and mihranyan a). self assembled into nanosheets to yield ''mille feuille'' structures, they showed efficient removal of parvoviruses (gustafsson et al. b) : maybe a new filtration media for viral clearance? a number of air filtration devices designed for individual protection against airborne viruses also include cellulosic materials. while published data on the filtration performance of cellulosic media for removal of viruses from air are scarce, industrial research is more apparent, in particular in asian countries (china, japan) where airborne pollution is a matter of great concern-the main objective being to add virucidal properties to the filtration media so as to inactivate accumulated viral particles. therefore, some 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many roles of membranotropic peptides date: - - journal: biochim biophys acta biomembr doi: . /j.bbamem. . . sha: doc_id: cord_uid: gf asni here, we review the current knowledge about viral derived membranotropic peptides, and we discuss how they may be used for many therapeutic applications. while they have been initially discovered in viral fusion proteins and have been involved in the mechanism of viral entry, it is now clear that their features and their mode of interaction with membrane bilayers can be exploited to design viral inhibitors as well as to favor delivery of cargos across the cell membrane and across the blood–brain barrier. the peptide gh has been extensively used for all these purposes and provides a significant contribution to the field. we describe the roles of this sequence in order to close the gap between the many functions that are now emerging for membranotropic peptides. over the past few decades peptides have progressively achieved increased value in drug design and pharmaceutical delivery. moreover, great interest has been dedicated to the identification of peptides as drug candidates. the number of peptides in the pharmaceutical industry is continuously growing and about % of the entire drug market is represented by peptide based drugs [ , ] . bioactive peptides can be derived from natural sources or can be discovered through rational engineering, high-throughput screening, or structure-based design starting from defined protein regions [ ] . among the many peptides playing a relevant role in biology, some show a high propensity for binding to lipid membranes due to their simultaneous hydrophobic and amphipathic nature. this class of hydrophobic peptides is characterized by the presence of unusual conspicuous amounts of alanine and glycine residues and sometimes also prolines. such a degree of ala/gly content is uncommon for hydrophobic domains such as signal sequences and transmembrane anchors; in fact, their presence may account for the intrinsic conformational flexibility which is a typical feature of membrane interacting peptides. also aromatic residues are generally present and dominate the interactions that take place at this unique physical-chemical environment of the water-membrane interface [ ] . the favorable interactions of aromatic side chains with phospholipid moieties located at the membrane interface contribute to the insertion of the peptide into the bilayer. amphipathicity is a key feature of these peptides. the term amphipathicity generally refers to molecules with both hydrophilic and hydrophobic faces [ ] . peptides can be amphipathic in their primary structure or secondary structure. primary amphipathic peptides correspond to the sequential assembly of a domain of hydrophobic residues with a domain of hydrophilic residues divided by a spacer domain; while secondary amphipathic peptides are generated by the conformational state which allows positioning of hydrophobic and hydrophilic residues on opposite sides of the same molecule. in particular, amphipathic, hydrophobic peptides present one face with large and aromatic residues and the other with small residues such as ala/gly. this distribution of amino acid residues facilitates the membrane interaction and peptide insertion into the bilayer [ ] . conformational polymorphism plays a key role; in fact, the ability to shift from random to α/β conformations as a consequence of membrane composition and peptide concentration has emerged as a common structural pattern for this class of peptides [ ] . there are several types of membrane active peptides which can be roughly divided in antimicrobial peptides [ ] , viral peptides [ ] and cell penetrating peptides [ ] . although very different in primary sequence one from the other, it may be hypothesized that their common physical features could result in a shared mechanism of action and essentially determines the many roles that they can play in nature. among the hydrophobic peptides with a propensity for membrane binding, characterized by a high interfacial hydrophobicity or amphipathicity, the ones derived from enveloped virus glycoproteins are attracting considerable attention. these peptides can interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on membranes and/or fusion proteins and are, thus, critical for both fusion and entry. viral glycoproteins undergo conformational changes as a consequence of either low endosomal ph or receptor binding which leads to the exposure of hydrophobic peptides, loops or patches, which then interact with and destabilize one or both the opposing membranes. crystallographic data on the post-fusion structures of viral fusion proteins have allowed the identification and characterization of three different classes [ , ] . class i fusion proteins are characterized by trimers of hairpins with a central α-helical coiled-coil structure and have been identified in orthomyxoviruses, paramyxoviruses, retroviruses, filoviruses and coronaviruses [ ] [ ] [ ] [ ] [ ] . class ii fusion proteins are present on viral envelopes as pre-fusion dimers which convert into post-fusion trimers of hairpins composed of β structures and have main representatives in the flaviviridae and togaviridae families [ , ] . class iii fusion proteins are characterized by a central α-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated β-sheet similar to class ii fusion proteins and members are present in herpesviridae and rhabdoviridae families [ ] . despite several differences in the mechanism of entry elicited by the three classes of fusion glycoproteins, they all induce membrane fusion in a similar manner through the formation of an analogous hairpin structure which allows fusion peptides to insert into cell membranes and to drive membrane destabilization. thus, during the viral entry process, the hydrophobic surfaces that become exposed are characterized by somewhat variable but at the same time detailed physical characteristics which include the size, shape and secondary structure of exposed hydrophobic patches, as well as the nature of the neighboring polar or charged residues. the wimley-white interfacial hydrophobicity scale (wwihs) is an experimentally-determined free energy scale that calculates the propensity of individual amino acids in peptide sequences to partition from water into a phosphatidylcholine interface [ , ] and has been effectively used to identify fusion peptides in viral glycoproteins. the hydropathy analysis allows calculating a hydrophobicity score along the sequence of a protein, identifying segments with a propensity to interact with membrane interfaces. wwihs values are calculated assuming random coil peptides partitioned into the bilayer interface; the values are minimum possible values and the Δg may get more favorable if peptide binding also promotes an increase in secondary structure [ , ] . the interfacial helical hydrophobic moment (ihhm) is a further physico-chemical factor that is important for membrane interactions and secondary structure formation of peptides bound to membrane interfaces. the ihhm describes the degree to which a peptide sequence would have segregated hydrophobic and hydrophilic faces if it folded into an α-helix [ ] . a peptide with a large ihhm can interact strongly with membranes as a helix due to partitioning-folding coupling [ , ] even with a wwihs score that is not positive overall. the presence of the fusion peptide within the ectodomain exposed to the aqueous phase is a feature shared by all viral fusion proteins and constitutes an absolute requirement for their fusogenic activity. fusion peptides are typically - residues long and potentially fold into amphipathic helices and are rich in glycines and alanines, providing them a high degree of conformational flexibility. thus, their structure is polymorphic and strongly dependent on the environment. the fusion peptide of influenza virus, for example, has been observed in random coil, α-helical and β-sheet conformations in different environments [ ] (fig. ) . it has been proposed that all three forms have some physiological relevance; the peptide may be unstructured in solution on the way to the target membrane; it may be helical at low concentrations but may self-associate in β-sheets at higher concentrations in the membrane interface. the fusion peptide of hiv also undergoes conformational transitions; it adopts α helical or β sheet structures depending on concentration, lipids and ionic conditions [ ] . the helical form of the influenza fusion peptide is probably a key determinant to promote fusion. the structure of the peptide in membrane shows a kink which separates the n terminal and the short c terminal helices which together form a boomerang shaped structure [ ] . both helical arms are amphipathic with bulky hydrophobic residues facing the membrane interior. the conserved n-terminal glycine residue is critical for fusion and for the correct structure of the peptide inside the membrane; in fact, when it is mutated to a valine the n-terminal helix is partially unwound and the fusion peptide is inactive [ ] . actually, there are numerous studies demonstrating that a delicate balance between α and β structures, is essential for membrane fusion and is influenced by environmental conditions such as ph, ionic strength, peptide sequence, presence or absence of divalent cations, cholesterol content and also by the lipid/peptide ratio. for instance, studies performed on the ebola fusion peptide, show that the conformational transition from an α-helix to a β-sheet is induced by a change in the peptide to lipid ratio in the membrane [ ] . at low peptide concentration in lipids, it is essentially an α-helix; while as the local peptide concentration increases in the membrane, the proportion of α-helix drops off in favor of a mainly antiparallel β-sheet structure. this concentration dependent effect on peptide conformation might be of biological relevance [ ] . the functional meaning of the conformational polymorphism is unclear, although it is believed to be fundamental to enable backbone reorientation of the fusion protein; therefore, the ability to insert at various levels might be required for the evolving of final stages of the fusion cascade [ ] . independently from the principal conformational organization, the degree of insertion plays a key role for inducing membrane fusion. it has also been hypothesized that [ ] fusion domains first assemble as β-sheets on the surface of the membrane and later convert into α-helices to complete fusion. aromatic residues are generally present in fusion peptides and may help in overcoming the energy cost of peptide bond partitioning into membranes. the interactions with phospholipid moieties located at the membrane interfaces may also help in stabilizing the insertion into just one leaflet of the bilayer. the initial interaction with the external leaflet is thought to generate elastic stresses which drive to bilayer fusion, helping to overcome the hydration repulsion forces between approaching bilayers by orienting the poorly solvated face toward the external medium [ ] . the asymmetric insertion into one membrane monolayer may promote expansion of the polar head region and determine a curvature stress onto the overall lipid bilayer; the created bulges that protrude from the membrane can facilitate the formation of lipid contacts between fusing bilayers [ ] . particular attention has also been devoted to the effect of additional membranotropic sequences on the overall fusogenicity. the presence of additional fusogenic sequences was evidenced in sendai f [ ] , measles f [ ] , sars-cov s [ ] , hepatitic c virus e and e [ ] , dengue e [ , ] and herpes virus gb and gh [ ] [ ] [ ] . the idea that a single fusion peptide is the solely responsible for the complete membrane fusion event has been substituted by the assumption that a concerted action of different membranotropic regions is necessary for membrane interacting/perturbing activity. as a matter of fact, also membrane proximal regions (pre-tm) play a key role in fusion [ , [ ] [ ] [ ] . the pre-tm domains are particularly rich in aromatic residues which enable them to insert into the membrane interface. effective therapeutics against enveloped viruses are still scarcely represented. a few drugs have been developed against hiv, influenza virus, hepatitis virus and a few other viruses but they are still not ideal and in some cases have proved to induce resistance [ ] . as a consequence, for most enveloped viruses, there are no effective therapies and entry inhibitors represent an interesting and underutilized target. peptides with a propensity for membrane binding can also interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on cell membranes and/or fusion proteins. as recently reviewed by badani et al. [ ] , there are many peptide inhibitors that are somewhat hydrophobic and/or amphipathic with a propensity to bind to bilayer membrane interfaces and other hydrophobic surfaces. it is not known whether membrane binding directly affects viral fusion, or whether interaction with the fusion protein itself is an absolute requirement for entry inhibition. it is widely accepted that membrane binding of an inhibitory peptide will greatly increase the effective concentration of the peptide close to the fusion protein, indicating that the interaction with membrane and the interaction with the fusion protein may be effectively coupled [ ] . as a matter of fact, the potential of numerous fusion peptides and/or membranotropic peptides derived from proteins of enveloped viruses as entry inhibitors has been widely described in literature [ ] [ ] [ ] [ ] . the accepted view is that the inhibition of infectivity may be due to the formation of inactive aggregates between the fusogenic stretches present in both the viral protein and the synthetic peptides. these aggregates are formed as a consequence of their ability to oligomerize or to mimic the modes of binding of their original domains in their partner protein. it has been hypothesized that they stabilize a pre-fusion intermediate and prevent merging of the bilayers. it is now evident that several domains are essential for membrane fusion and thus peptides involved in the fusion mechanism may all interfere with the intramolecular interactions between the several domains and may represent interesting targets for the design of entry inhibitors. thus, membrane physical properties could be critically important to the events that drive viral entry and it is conceivable that peptides interfere with the function of viral fusion proteins by changing the physical chemistry of the membrane itself by direct interaction. many laboratories are working to unravel the mechanism of action of viral membranotropic peptides and several hypotheses have been proposed. many studies suggest that multiple mechanisms may take place simultaneously. indirect ways in which interfacial binding peptides can affect viral entry have also been hypothesized. selfoligomerization of membrane embedded fusion peptides has been proposed to be responsible of inhibition [ , ] . the most studied case is that of hiv, where the inhibition has been attributed to the formation of structurally defined oligomeric complexes [ , ] ; while mutants with a lower helical content and tendency to self-associate into β-sheets [ ] are able to inhibit membrane fusion at various stages. it is interesting to note that there are clinical studies on a peptide called virip, which is designed as an inhibitor of the hiv fusion peptide [ , ] . this sequence was able to block hiv- infection by targeting gp fusion peptide [ ] and optimized versions of this sequence proved to be as potent as inhibitors targeting the coiled coil sequences and moreover were devoided of cellular toxicity. a -day monotherapy clinical trial enrolling hiv- infected patients [ ] showed that the drug can be well tolerated by patients and reduces their plasma viral load. its identification and clinical evaluation represent the first proof of concept that membranotropic sequences could suppress viral replication in infected individuals and have potential clinical effectiveness. the hypothesis that peptide entry inhibitors act by a physicalchemical interaction with hydrophobic surfaces exposed during the fusion process suggests that this novel approach may be a general rule; moreover, instead of focussing on the structure-based design, it would be possible to design novel hydrophobic/amphipathic inhibitors which could be easily made protease resistant by the introduction of nonnatural or d-amino acids. the membrane bilayer represents a semi-permeable barrier, defining the interior of an individual cell; its existence confers cells their potential to survive and function properly. nevertheless, crossing of the cellular membranes remains one of the major obstacles for the proper delivery of therapeutics [ , ] . the lipophilic nature of biological membranes restricts the direct intracellular delivery of most compounds; whereas small molecules and ions can diffuse across the bilayer, larger molecules are generally excluded from simple diffusion into the cell. the differing hydrophobicity/hydrophilicity of the lipid membrane renders the transfer across this barrier extremely difficult due to differences in solubility. notwithstanding the therapeutic potential of a number of novel molecules, their pharmaco-distribution properties hamper the possibility to reach the stage of pharmaceutical preparations and stimulate industrial interest; in fact, these molecules need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm or onto individual organelles. it is, thus, evident that the therapeutic potential of a drug is largely dependent on the development of delivery tools able to selectively and efficiently carry it to target cells with minimal toxicity. the translocation across the membrane is by far less well understood than the binding step. there is a significant similarity in the physico-chemical parameters between membrane partitioning peptides and membrane translocating peptides [ ] . a novel intriguing hypothesis is that hydrophobic peptides that partition into membranes may also be able to cross cell membranes and enter cells. therefore, these peptides may also cross endothelial layers in vivo, including the blood-brain barrier [ , ] . delivery across cellular membranes involves several membrane reorganization processes such as transient permeabilization of the cell membrane, which are similar to the ones involved in the entry of viruses. membrane fusion and its disruption are related processes, although leakage and fusion capacities of peptides do not always correlate and the features/activities of membranotropic peptides may depend on particular environmental and temporal conditions. since not all membranotropic peptides are able to cross the membrane bilayer, it is essential to identify structural characteristics of hydrophobic peptides know to enter the cell membrane to highlight any feature that is involved in the penetration which may help in the design of novel delivery tools. thus, an important feature to consider is the structural requirements for cellular uptake and the ability of membranotropic peptides to interact with the cell surface and lipid moieties of the cell membrane. a very complete review describing the binding and translocation of membrane active peptides has been recently published [ ] which highlights the fact that peptide translocation is not coupled with dye flux. graded dye flux would occur concomitant with peptide translocation, which would explain incomplete dye release; whereas all-or-none flux would occur with peptides unable to translocate, and therefore these peptides accumulate on the membrane until a rupture point is reached, resulting in complete dye release [ ] . cell-penetrating peptides (cpps) have been widely used due to their capability to transport several kinds of macromolecules across the membrane bilayer in vitro and in vivo [ ] [ ] [ ] . cpps are short and usually basic amino acid rich peptides originating from proteins that are able to cross biological barriers, such as the viral tat protein. although the uptake mechanism of cpps is still debated, it seems to involve mainly the endocytic pathway, trapping the conjugated cargo in endosomes eventually ending in lysosomes where common enzymatic degradation mechanisms take place, therefore leading to a limited delivery of therapeutic agents to the intracellular target. hydrophobic peptides that efficiently traverse biological membranes, promoting lipid-membrane reorganizing processes, such as fusion or pore formation and involving temporary membrane destabilization and subsequent reorganization [ , ] , may be able to circumvent the endosomal entrapment either favoring the escape from the endosome or by translocating a cargo through the plasma membrane directly into the cytosol. this idea has been exploited to design the drug delivery tool called mpg. mpg is an amphipathic peptide whose primary sequence is composed of the hydrophobic amino acids of the hiv- fusion peptide (galflgflgaagstmga) associated to a hydrophilic domain derived from the nuclear localization sequence (nls) of simian virus (sv ) large t antigen (pkkkrkv). these hydrophilic and hydrophobic segments are separated by a three amino-acid spacer (wsq) [ , ] . this peptide exploits the known properties of the glycine-rich hiv fusion peptide essential for membrane fusion activity and the nls of the sv large t antigen to improve the nuclear addressing of the peptide [ , ] . at the moment this is the only viral fusion peptide that has been widely exploited for applications in drug delivery. a milestone in understanding the role of hydrophobic viral peptides is represented by the sequence "gh " derived from glycoprotein h of herpes simplex virus type i. herpes simplex virus (hsv) is an important human pathogen, responsible for significant morbidity and mortality worldwide and is characterized by a complex multi-component entry machinery. hsv enters host cells by fusion of the viral envelope with either the plasma membrane or an endosomal membrane, and the entry pathway is likely determined by both virus and host cell factors and involves multiple viral glycoproteins and cellular receptors in a cascade of molecular interactions [ ] [ ] [ ] [ ] . the envelope glycoproteins gh/gl, gb and gd are all essential for the entry process and their expression is able to induce the fusion of cellular membranes in a virus-free system [ , ] . both gh/gl and gb constitute the core fusion machinery and cooperate to induce the initial lipid destabilization that ends in fusion [ ] and both gb and gh contain several membranotropic sequences [ ] [ ] [ ] , , [ ] [ ] [ ] . although it has recently become available the crystal structure of the gh-gl complex [ ] , it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process and many studies suggest that the gh-gl complex may undergo dynamic rearrangements [ , ] . in particular, some peptides derived from the gh ectodomain block virus entry, while others have the ability to bind and disrupt model membranes. gb is considered a canonical class iii fusion protein and has been demonstrated to be involved in virus attachment, penetration and cell-to-cell spread. the crystal structure of gb is a trimer in which multiple contacts between protomers throughout the molecule contribute to its stability [ ] . it has been hypothesized [ ] that gb refolds similarly to class i fusion proteins and that the packing of the c-terminal arm against the coiled-coil provides the driving force for gb refolding from the prefusion to the post-fusion conformation. the gb structure corresponds to a post fusion conformation and it is now widely accepted that gb undergoes conformational changes upon variations of ph in order to bring about fusion [ ] [ ] [ ] [ ] . several synthetic gb peptides induced the fusion of large unilamellar vesicles and inhibited herpes virus infection [ , ] . when the crystal structures of gb and of gh/gl were not yet available, we reported the identification of several sequences in gh and gb with the ability to interact with the membrane and among these sequences there was also the canonical fusion peptide of gb [ , , , ] . although it is not yet well understood the role played by the other membranotropic sequences and in particular by the glycoprotein gh in the whole fusion process, it is now widely accepted that several regions in the fusion glycoproteins are involved in the local destabilization of the membrane bilayer which ends in the fusion of the viral envelope with the host cell membrane. gh was first selected and characterized by our group in [ ] and still is the hsv- peptide with the highest fusion capability and the most widely studied. it was initially identified using the wwih scale and subsequent works allowed determining the many applications of this sequence from membrane fusion, to viral inhibition and drug delivery [ , , ] . the twenty residue peptide gh (from aa to aa ) is a membrane-perturbing domain, (fig. ) which interacts with biological membranes and is implicated in the merging of the viral envelope and the cellular membrane [ , ] . the peptide contains residues crucial for its capacity to interact and destabilize target lipid membranes. it is rich in hydrophobic residues including glycines, leucines, alanines, and aromatic residues such as tryptophan and tyrosines, which are known to be located preferentially at the membrane interface. the peptidelipid interactions are initiated by the arginine residue located at the c-terminus; in fact, when the arginine is mutated, the fusogenic activity of the peptide is strongly impaired. the hydrophobic domain is also crucial for its insertion into the membrane and further supports the view that hydrophobic interactions between fusion proteins and cellmembrane phospholipids initiate membrane perturbation in the early stages of viral fusion. the many biophysical experiments performed on gh have shown that the peptide interacts with model membranes, penetrates the bilayer from its n-terminal side, has a tryptophan residue buried inside the bilayer, and adopts a helical conformation with its hydrophobic residues on one face of the helix and polar or charged residues on the opposite face [ , , ] . the analysis of peptides with longer and shorter sequences derived from this region and of their interactions with membranes clearly demonstrated that the activity of this region depends on the amino acid sequence and on its length. the presence of a histidine residue at the n-terminus of the native sequence strongly increases the fusion activity [ ] . the importance of a single histidine residue as a switch for triggering viral fusion was also reported for other viruses [ ] such as paramyxoviruses, therefore supporting the importance and specificity of the histidine moiety in activating fusion. furthermore, a conserved histidine in one of the fusion loops of semliki forest virus e protein was found to be fundamental [ ] . the histidine in gh both helps the initial interactions with the membrane and the oligomerization process [ ] . this hypothesis is further supported by the fact that the histidine is located at the n-terminus and correct configuration of the n-terminal fusion peptide appears to be crucial for the fusogenic function of several fusion proteins as well as its location in the membrane core after peptide-bilayer interaction. the addition of one histidine at the n-terminus of gh is sufficient to make the peptide approximatively -fold more active. in particular, the addition of any other residue at the n-terminus impaired the fusion ability of the sequence (data not published). gh strongly interacts and spontaneously penetrates the lipid-phase and inserts into membranes with a α-helical structure [ , , ] . both the tryptophan and tyrosine are on the same side of the helix in the three-dimensional structure, forming an amphiphilic helix in which one side is constituted by aromatic and hydrophobic residues, whereas the other side is formed by hydrophilic or small residues. the interaction between the aromatic ring of tryptophan and the side chain of tyrosine is important for maintenance of structural stability during the interaction with the membrane. an amphipathic α-helix is believed to be an important feature of membranotropic peptides playing a crucial role for mediating lipid-protein interactions during the binding of proteins to membranes and once bound, the hydrophobic face of the amphipathic peptide would allow the peptide to enter the membrane interior, thereby triggering local fusion of the gh has the ability to penetrate deep into the bilayer as a helix without causing significant bilayer perturbations which may help explaining its ability to perform several different roles. gh showed a significant inhibitory effect and this effect appears conditioned by its ability to partition into membranes and aggregate within them. since the peptide self-associates in aqueous and lipid solutions, it is possible that it binds to its counterpart in the gh protein. gh may also interact with the host cell membrane, therefore its ability to moderately inhibit viral entry when cells are treated first, is dependent on the possibility that the virus will find a modified cell membrane still exhibiting on its surface the peptide [ ] . moreover, gh does not have any activity in virus preincubation experiments, indicating that an eventual binding partner site on the pre-fusion gh protein is probably hidden and not available to interactions with free peptides, as also demonstrated by the analysis of the gh crystallographic structure. while the n-terminal histidine residue was proven to be fundamental for the interaction with the membrane bilayer and for translocation across the membrane, the absence of this residue induced similar levels of viral inhibition when compared with the full length peptide. the substitution of leu with a valine residue does not alter the hydrophobicity of the peptide, and does not influence its infectivity inhibition properties while its substitution with a polar residue (serine) substantially reduces its inhibitory activity [ ] . recently, poly(amide)-based dendrimers functionalized at their termini with gh were shown to inhibit both hsv- and hsv- at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite for viral internalization [ ] . the % inhibitory concentration was and nm against hsv- and hsv- respectively, with no evidence of cell toxicity at these concentrations, indicating that the functionalization of a dendrimer with a membranotropic peptide represents a promising strategy for inhibition of viruses of the herpesviridae family. the multivalent display of gh on the dendrimer scaffold results in an almost six fold increase of antiviral activity for hsv- and two fold for hsv- in comparison to the activity of the dendrimer itself, and more than -fold increase in the activity of the unsupported peptide. the cytotoxicity profile measured by the mtt assay showed that the peptidodendrimer is not toxic to vero cells up to the highest concentration investigated in antiviral testing, while some toxicity was observed for the unfunctionalized dendrimer, especially at higher concentrations, demonstrating another advantage of the peptide functionalization [ ] . any inhibitory activity was excluded when the compounds were added at a post-entry step and also when cells were pre-treated with the dendrimer derivatives, indicating that both the peptidodendrimer and the dendrimer are not able to interfere with viral replication once the virus has gained access to the cellular milieu. the peptidodendrimer might sterically hinder the gh relative domain, either in a pre-fusogenic or in an intermediate conformation, preventing a complete and functional interaction between gh and the membrane to fuse. the mechanism of inhibition may involve binding to gh itself through oligomerization of the gh domain present on the glycoprotein or interaction with other glycoproteins present on the virion envelope, such as gb or gd. the modification of a dendrimer scaffold with membranotropic peptides represents an attractive strategy for the design of a new class of antiviral drugs that exert their effect, coupling the intrinsic anti-viral properties of the dendrimer with the activity of membranotropic peptides and have the potential of being developed as multifunctionalized scaffolds to provide a therapeutic molecule to directly deliver to its target [ ] . the inhibition of membrane fusion represents an attractive target for drug design and although further studies are needed to better define the exact mechanism of inhibition by hydrophobic peptides and the specific nature or location of their interactions with viral targets, the data obtained for gh suggest that hydrophobic domains play a significant role in membrane fusion and provide an alternative approach to the development of viral peptide inhibitors outside of the classical inhibitory heptad repeat regions. gh cellular uptake is associated with its hydrophobic and amphipathic characters which provide the necessary ability to interact with membrane lipids and to form a transient helical structure that temporarily affects membrane organization, thereby facilitating insertion into the membrane and translocation [ ] . compared to tat peptide (a positively charged cpp) which mainly exploits the endocytic pathway, gh crosses membrane bilayers mainly through a translocation mechanism. a one amino acid shorter version of this fusogenic peptide was also found to improve the endosomal release of dna/lipofectamine lipoplexes and transgene expression up to -fold in human cell lines [ ] . it has been recently demonstrated that gh is able to traverse the membrane bilayer and to transport into the cytosol several compounds, such as qds [ ] , liposomes [ ] , nps [ ] , dendrimers [ ] , and proteins [ ] . examples of using gh as an intracellular delivery enhancer are provided in the remaining part of the paragraph (fig. ) . qds are fluorescent probes under intense research and development for broad applications in molecular, cellular and in vivo imaging [ ] . although considerable success has been achieved in using qds for labeling fixed cells and for imaging cell membrane proteins, only limited progress has been made for molecular imaging inside living cells because of their insufficient ability to traverse cell membranes. several authors have recently reported on the functionalization of qds with positively charged cpps and established that the main route of entrance is via endosomal uptake, therefore, escape from the endosomal system is of paramount importance [ ] [ ] [ ] . gh -qd internalization was demonstrated to be highly successful and to involve the endocytic pathway only to a minor extent [ ] . liposomal aggregates have also attracted great attention due to their success as in vivo carriers of drugs [ ] . to enhance the antitumor efficacy of liposomal drugs, the efforts of many research groups are directed toward the improvement of cellular internalization of liposomes through the addition of surface ligands and cell penetrating peptides. liposomes decorated with gh and loaded with doxorubicin (dox) [ ] , were able to penetrate inside living hela cells. the results obtained suggest that the functionalization of liposomes with gh could affect the uptake mechanism of liposomes and their intracellular distribution and dox release. this evidence could be useful in the design of carriers for a controlled delivery and release of dox in order to avoid side effects associated to dox itself. dendrimers [ , ] also represent a very promising tool for drug delivery, combining the advantageous features of nanoparticles (ideal size as in vivo carriers, multivalency), of polymeric materials (low cost, tunable properties, biocompatibility) and of small molecules (monodispersity and detailed control of their properties) [ , ] . their surface modification by means of conjugation or adsorption of a biospecific ligand, may allow their delivery to specific sites and modulation of drug release minimizing toxic effects and increasing intracellular bioavailability [ ] . thus, the dendrimeric scaffolds may be a promising tool for an efficient drug delivery engine. little information is available on the mechanism of dendrimer uptake and intracellular trafficking [ ] . studies performed on pamam dendrimers [ ] and pamam dendrimers functionalized with the tat [ ] indicate that endocytosis mechanisms contribute to the internalization and intracellular trafficking and that adding the tat failed to enhance delivery efficiency. the attachment of gh to the termini of a poly(amide)-based dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. the peptide-functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism [ ] . the combination of the benefits of dendrimers and peptides chemistry could be useful for the development of a selective carrier which could cross the membrane and be efficiently internalized into the cellular targets. many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain-barrier (bbb) [ ] . the development of new strategies for enhancing drug delivery to the brain is fundamental in diagnostics and therapeutics of central nervous diseases (cns). most strategies to transport drugs inside the cns cause disruption of the anatomical texture of the bbb, therefore impairing its natural function; as a consequence, effective delivery approaches should be cautiously assessed considering their impact on the overall protective function of the bbb [ ] . targeted delivery of a therapeutic cargo to the intended site of action in the brain appears to be one of the most promising non-invasive approach to overcome the bbb, combining the advantages of brain targeting, high incorporation capacity, reduction of side effects and circumvention of the multidrug efflux system [ ] [ ] [ ] [ ] . polystyrene nanoparticles (nps) decorated on their surface by gh showed that the uptake of nps with gh by brain endothelial cells was greater than that of the nps without the peptide and functionalized nps were free to move intracellularly [ ] . most importantly, gh decreased np intracellular accumulation as large aggregates and enhanced the np bbb crossing. the surface functionalization with gh may change nps fate and provides a good strategy for the design of promising carriers to deliver drugs across the bbb for the treatment of brain diseases. whether multifunctional nanosystems, designed and tested in vitro, are able to properly work in vivo into mammalian hosts, is not fully granted. to address this issue, in vivo studies are necessary, thus validating design strategies and facilitating optimization and further functionalization. although numerous studies showed that gh is an efficient carrier for bioactive cargoes in vitro [ ] , these results did not guarantee that it can be developed into a useful pharmaceutical delivery platform. the ability of gh to cross the bbb in vivo was also recently evaluated [ ] . gh was administered in vivo to rats and its presence in the liver and in the brain was detected. within . h from its i.v. administration, gh can be found beyond the bbb in proximity fig. . the many applications of gh to drug delivery. confocal microscopy images showing the internalization of gh functionalized: a) proteins [ ] ; b) liposomes [ ] ; c) qdots [ ] ; d) dendrimers [ ] . e) scanning electron microscopy images of functionalized polystyrene nanoparticles [ ] . of cell neurites. gh has no toxic effect in vivo, since it does not affect brain maximal oxidative capacity and mitochondrial respiration rate. the data suggest that gh , for its ability to cross the bbb, represents a novel nano-carrier system for drug delivery to the central nervous system. these results open new possibilities for direct delivery of drugs into patients in the context of theranostics and might address the treatment of several human diseases. other peptides have been proposed as a drug delivery system; it was demonstrated that tat was able to enter tissues in vivo in mice [ ] ; antp was able to activate endogenous t cells in mice [ ] . these peptides are highly positively charged, and absorptive-mediated transcytosis has been proposed for their transport across the bbb. a bradykinin analogue has also been reported to increase the penetration of small molecules by transitory opening of the bbb [ ] . gh is the first viral membranotropic peptide which was shown to be a potential delivery system for macromolecules in vivo; these results coupled with previous in vitro data support the view that gh enters the bbb without involving endocytic processes. hence, the eventual cargo may be immediately and completely available [ ] . the presence of multiple metabolic barriers may restrict the application of such peptide-based ligand for targeted drug delivery in vivo. peptides alone or conjugated on the surface of nanocarriers are subject to proteolysis in the blood after systemic administration. in addition, the bbb is also a metabolic barrier due to the presence of various enzymes in brain capillary endothelial cells. gh starts to be degraded after h of incubation but the intact peptide is still present after . h of incubation and thus holds the potential for extending brain targeting efficiency due to its resistance to proteolysis for . h [ ] . it is still under-recognized that some amino acid sequences in virtue of their specific features can play many different roles in nature. membranotropic viral peptides derived from fusion glycoproteins are widely studied especially for their ability to fuse membranes but there are many literature data also describing other roles besides membrane fusion. gh is an example of how these sequences can be employed for completely different purposes: fusion of membranes, viral inhibition and drug delivery. till now, gh is the only membranotropic peptide that has been extensively used for many applications and among them as a drug delivery system for the brain. in the development of new therapies to treat brain pathologies, the bbb represents a major obstacle against the use of potential drugs for treating disorders of the cns due to the impermeable nature of the cell membranes of this compartment to several molecules [ , ] . the data reported on the in vivo application of gh for brain delivery, support the novel view that synthetic peptides derived from viral membranotropic sequences can be used successfully to deliver biologically active substances inside the bbb. the exact molecular mechanism of gh entry remains to be established but it appears to be a general feature of membranotropic peptides, which may be used for mediating delivery to virtually any tissue and in particular across the bbb, conveying a wide variety of cargoes with intact bioactivity into virtually any tissue or organ. other sequences have been found to be useful for drug delivery which happens to have the same features of the viral fusion peptide, indicating that it may be possible to design novel sequences with pre-determined characteristics which can be useful to treat many diseases. however, still much work has to be done on this type of peptides; we should not forget that their mechanism of perturbation of membrane bilayers may also allow the design of new membranotropic peptides with the ability to denature the membrane bilayer of bacteria and thus we may add to their many roles also the antibacterial activity which may represent an alternative to classical antibiotics in order to combat the antibiotic resistance problem. much of the vast literature on membranotropic peptides is devoted to single activities of these sequences; yet compelling structure-function relationship studies bridging the gap among all these activities are necessary. the future of peptide-based drugs synthetic therapeutic peptides: science and market therapeutic peptides: technological advances driving peptides into development the preference of tryptophan for 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translocation and applications to the delivery of quantum dots a fusogenic segment of glycoprotein h from herpes simplex virus enhances transfection efficiency of cationic liposomes clickable functionalization of liposomes with the gh peptide from herpes simplex virus type i for intracellular drug delivery dendrimer functionalization with a membrane-interacting domain of herpes simplex virus type : towards intracellular delivery pedone, gh is a viral derived peptide for effective delivery of intrinsically disordered proteins probing cellular events, one quantum dot at a time intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides intracellular trafficking and unpacking of sirna/quantum dot-pei complexes modified with and without cell penetrating peptide: confocal and flow cytometric fret analysis spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques the benefits and challenges associated with the use of drug delivery systems in cancer therapy dendrimers and dendrons: concepts, synthesis, applications designing dendrimers for biological applications dendrimers and dendritic polymers in drug delivery surface modifications of nanocarriers for effective intracellular delivery of anti-hiv drugs crossing cellular barriers using dendrimer nanotechnologies comparison of the endocytic properties of linear and branched peis, and cationic pamam dendrimers in b f melanoma cells tat-conjugated pamam dendrimers as delivery agents for antisense and sirna oligonucleotides current approaches to enhance cns delivery of drugs across the brain barriers astrocyte-endothelial interactions at the blood-brain barrier nanoparticle-mediated targeted delivery of antiretrovirals to the brain nanobiotechnology-based strategies for crossing the blood-brain barrier biomaterial-based technologies for brain anti-cancer therapeutics and imaging receptor-mediated delivery of magnetic nanoparticles across the blood-brain barrier review of a viral peptide nanosystem for intracellular delivery the peptide gh enters into neuron and astrocyte cell lines and crosses the blood brain barrier in rats tat-mediated delivery of heterologous proteins into cells introduction of exogenous antigens into the mhc class i processing and presentation pathway by drosophila antennapedia homeodomain primes cytotoxic t cells in vivo facilitation of drug entry into the cns via transient permeation of blood brain barrier: laboratory and preliminary clinical evidence from bradykinin receptor agonist, cereport getting into the brain: approaches to enhance brain drug delivery the authors thank luca de luca for excellent technical assistance. key: cord- -gf bglm authors: scutigliani, enzo maxim; kikkert, marjolein title: interaction of the innate immune system with positive-strand rna virus replication organelles date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: gf bglm the potential health risks associated with (re-)emerging positive-strand rna (+rna) viruses emphasizes the need for understanding host-pathogen interactions for these viruses. the innate immune system forms the first line of defense against pathogenic organisms like these and is responsible for detecting pathogen-associated molecular patterns (pamps). viral rna is a potent inducer of antiviral innate immune signaling, provoking an antiviral state by directing expression of interferons (ifns) and pro-inflammatory cytokines. however, +rna viruses developed various methods to avoid detection and downstream signaling, including isolation of viral rna replication in membranous viral replication organelles (ros). these structures therefore play a central role in infection, and consequently, loss of ro integrity might simultaneously result in impaired viral replication and enhanced antiviral signaling. this review summarizes the first indications that the innate immune system indeed has tools to disrupt viral ros and other non- or aberrant-self membrane structures, and may do this by marking these membranes with proteins such as microtubule-associated protein a/ b-light chain (lc ) and ubiquitin, resulting in the recruitment of ifn-inducible gtpases. further studies should evaluate whether this process forms a general effector mechanism in +rna virus infection, thereby creating the opportunity for development of novel antiviral therapies. the innate immune system forms the first line of defense against pathogens and its initial function is to recognize pathogenassociated molecular patterns (pamps) [ ] , which ultimately leads to induction of the antiviral state that effectively hampers spread of the infection. the adaptive immune system then kicks in to (in most cases) fully clear the virus and build up memory. viral rna is a very potent inducer of innate antiviral signaling [ , ] . therefore, detection of viral rna and the subsequent induction of antiviral effector mechanisms play an important part in the onset of an antiviral state in the context of rna virus infections. the study of pamp recognition and signaling by cytosolic and membrane bound sensors has intensified tremendously in the last decade. several comprehensive reviews on this subject have been published lately [ , ] . additionally, investigation of the involvement of intracellular organelle membranes of mitochondria, er, and peroxisomes and their mutual interactions indicated that these are important signaling platforms in innate immunity [ , ] . together, this has resulted in a better understanding of the details of innate immune responses that target rna viruses. in this review, we will focus on the interaction of the innate immune system with viruses that have a positive-strand rna (+rna) genome. in response to innate immune reactions, virtually all +rna viruses interfere to delay antiviral innate immune signaling in several ways [ , , ] . a key feature of immune evasion by +rna viruses, and simultaneously the hallmark of +rna virus infection, is rearrangement of host membranes into viral replication organelles (ros). as these structures are thought to shield viral rna from the host innate immune system and additionally seem to play a fundamental role in viral rna replication, ros in this sense seem to have a central and dual function in viral replication [ ] [ ] [ ] [ ] [ ] . therefore, disrupting integrity of ros might simultaneously result in impaired viral replication and enhanced antiviral immune signaling, which would be a beneficial effect from an antiviral immunity point-of-view. however, whether host cells possess effector mechanisms to disrupt +rna virus ros has hardly been investigated yet, and only quite recently some studies have shed more light on this, which will be the focus of this review together with the body of literature that surrounds it. positive-strand rna viruses have caused multiple outbreaks during the past decade: severe acute respiratory coronavirus (sars-cov) infected more than individuals in of which almost died [ ] . its close relative middle east respiratory syndrome coronavirus (mers-cov) emerged in saudi arabia in . while sars-cov infections have not been reported in humans after , mers-cov currently still regularly occurs in dromedary camels as well as in humans, and the virus displays a lethality rate of around % in the latter [ ] . zika virus, which received a lot of societal attention lately, is an example of a reemerged +rna virus with significant impact [ ] . these outbreaks, and the lack of tailored antiviral strategies against them, clearly illustrate the potential health risks associated with (re-)emerging +rna viruses, and emphasize the need for a precise understanding of host-pathogen interactions to facilitate development of novel antiviral therapies or vaccination methods. over the last decades, many +rna viruses have been extensively studied for their intriguing biology, including coronaviruses as those mentioned above, picornaviruses such as coxsackievirus and poliovirus, and flaviviruses such as west nile virus (wnv), hepatitis c virus (hcv), dengue virus (denv), zika virus, and japanese encephalitis virus (jev), as well as the family of togaviridae, including rubella virus and chikungunya virus. one of the intriguing features that is shared by all +rna viruses is the rearrangement of host membranes into viral ros, whereby particular cellular organelles (depending on the virus) are used as membrane donors. concerning their role in viral rna replication, it is thought that the structures form a scaffold that improves efficiency of enzymatic reactions and provides a spatiotemporal regulation of the different stages in the virus life cycle. besides this, it is thought that ros have an important role in shielding viral rna products from the rna sensors of the innate immune system. ros are therefore believed to have a dual role in +rna virus infection and innate immune evasion, which will be elaborated on further in this review. several comprehensive reviews have recently described the current knowledge of these structures [ , [ ] [ ] [ ] , therefore we will only briefly summarize this below. most studies until now focused on the architecture of structures induced by different viruses, and based on electron microscopy and electron tomography, ros were categorized into two different classes: the invaginated vesicle/spherule (inv) type, and the double membrane vesicle (dmv) type (fig. ) . inv ros are predominantly observed during infection with alphaviruses, such as semliki forest virus and sindbis virus, nodavirideae such as flock house virus, bromoviridae such as bromovirus, and flaviviridae such as rubella virus, denv, and wnv [ , ] . although the location and dynamics of the formation of inv ros varies per virus, the result is a single membrane invagination of which the content is connected to the cytosol. in addition, replicase proteins and newly synthesized viral rna were observed in these spherules, strongly suggesting that inv ros are sites of viral rna replication [ ] [ ] [ ] . furthermore, ribosomes are found in close proximity of some inv ros [ ] , suggesting that viral rna replication and translation are spatially separated. a similar organization of inv ros was found for flock house virus, rubella virus, denv, and wnv. moreover, a recent study visualized virus budding in close association with inv ros, suggesting a direct role for these structures in coordinating virus assembly [ ] . alternatively, dmv ros are known to be formed by enteroviruses such as coxsackievirus b and poliovirus, coronaviruses such as sars-cov and mers-cov, arteriviruses such as equine arteritis virus (eav), and the flavivirus hcv [ , ] . besides dmvs, for most of these viruses other kinds of structures are found during infection, mostly in close proximity to the dmvs themselves, such as single membrane vesicles, multi-membrane vesicles, vesicle packets, tubular structures or zippered er membrane. as for inv ros, replicases and viral rna of poliovirus, eav, and coxsackievirus b were demonstrated to localize to dmvs, suggesting that these structures might also support viral rna replication. likewise, for hcv replicase proteins and viral rna were predominantly found in dmvs, and the number of dmvs positively correlated to the amount of viral rna produced, suggesting that dmvs indeed serve as sites of rna replication. however, for coronaviruses, the number of dmvs is not necessarily proportional to replication capacity [ , ] . also, whereas sars-cov dsrna (a replication intermediate) was found inside dmvs, replicase proteins were more abundant in surrounding convoluted membranes, raising questions regarding the spatial organization of sars-cov rna replication. the lack of clear connections between the inside of corona-and arterivirus-induced dmvs and the cytosol (where the rna products have to go for translation and particle formation) further complicates our current understanding of coronavirus dmv functionality [ , ] . together, these data continue to cause debate on the exact function of dmvs and other features of dmv ros. the notion that expression of combinations of non-structural viral proteins (nsps) for some of these viruses mimics the formation of membrane alterations as observed during infection confirms the viral induction of these structures and opens possibilities for detailed study of this particular feature of the infection [ ] [ ] [ ] . . innate immune recognition and responses targeted towards viral rna replication, transcription, and translation rapid production of interferons (ifns) and pro-inflammatory cytokines is an important consequence of virus detection, as it contributes to an antiviral state in both the infected host cell and the (un)infected surrounding cells. in addition, ifns play an essential role in coordinating the antiviral adaptive immune response, which has been reviewed elsewhere [ ] . three types of ifns have been described. type i ifns consist of subtypes of ifna and a single subtype of ifn-b, ifn-d, ifn-e, ifn-k, ifn-t and ifnv. type ii ifn only contains one subtype of ifn-g, and type iii ifns comprise of ifn-l through Àl . whereas it is known that most cell types produce type i ifns in response to viral infection, type ii ifns are only produced after antigenic stimulation of an expanding group of certain immune cells, including t-cells, natural killer cells, dendritic cells, and macrophages [ , ] . in contrast, little is known about type iii ifn production in vitro and in vivo, although it is believed that most cell types that produce type i ifns are capable of producing type iii ifns as well [ ] . most cells are able to respond to ifn-i and Àii, whereas ifn-iii receptors are mainly found on epithelial cells [ ] . the protective role of ifns during viral infection is for example illustrated by inhibitory effects of ifna, ifn-b, and ifn-g on sars-cov replication in vitro and in vivo [ ] [ ] [ ] [ ] . importantly, correct timing and amount of ifn and subsequent pro-inflammatory cytokine expression is essential for an effective antiviral immune response, as delayed and/or elevated induction may stimulate immunopathological outcomes [ ] . below we will focus on the interactions of the innate immune system with the rna replication stages of +rna virus infection, including exposure of viral rna to the cytosol after the unpacking of virus particles, and formation of ros by the virus. we will also discuss examples of viral evasion surrounding these processes. ifn-i and -iii production is triggered after host cells detect viral rna, primarily using cytosolic rig-i like receptors (rlrs) and membrane-bound toll-like receptors (tlrs). rlrs retinoic acidinducible gene i (rig-i) and melanoma differentiation associated factor (mda ) have been studied extensively (reviewed in [ , ] ). besides their role in rna metabolism, rig-i and mda recognize viral rna by binding phosphorylated termini ( ppp-rna) in combination with dsrna or ssrna motifs. detection of viral rna activates rlrs and triggers downstream signaling through the mitochondrial antiviral signaling (mavs) adaptor, which is localized on the outer mitochondrial membrane [ ] [ ] [ ] [ ] . the importance of mavs is underscored by the observation that silencing of this protein by rna interference abolishes expression of type i ifns [ ] . subsequently, mavs recruits various adaptor molecules such as stimulator of interferon genes (sting) and tnf receptor-associated factors, resulting in the formation of large signaling complexes [ ] . ultimately, this leads to activation of kinase complexes ikke/tbk and ikka/ikkb/ikkg, resulting in activation of interferon regulating factor (irf ), (irf ) and nf-k b. these transcription factors then translocate to the nucleus and initiate the expression of ifns and pro-inflammatory cytokines. besides the role of rlrs, other rna sensing molecules have been shown to participate in antiviral responses. for example, of the types of tlrs described in humans, endosomal tlrs , , and are known to have a role in viral rna detection. a detailed overview on the role of tlrs in antiviral signaling can be found elsewhere [ ] . briefly, tlrs interact with various adaptor molecules after stimulation, and the combination of recruited adaptors influences downstream signaling events. tlr eventually recruits common adaptor molecule tir-domain-containing adapter-inducing interferon-b (trif), whereas tlrs and engage myeloid differentiation primary response gene (myd ). as in rlr signaling, this leads to activation of kinase complexes ikke/ tbk and ikka/ikkb/ikkg. furthermore, protein kinase r (pkr) and , -oligoadenylate synthetase (oas) have been demonstrated to induce antiviral activities upon binding of dsrna, as is described in detail elsewhere [ ] . well-established examples of antiviral activity provoked by pkr include inhibition of translation and inflammasome activation, and oas activates rnase l to initiate degradation of host and viral rna. in addition, pkr and oas have been suggested to amplify rlr-mediated antiviral immune signaling [ ] . the production and release of ifns and pro-inflammatory cytokines contributes to an antiviral state in both infected host cells and in (un)infected surrounding cells. despite the presence of multiple ifn and receptor types [ ] , the janus kinase-signal transducer and activator of transcription (jak/stat) pathway is utilized by all ifns to establish expression of interferon-stimulated genes (isgs). the observation that a deficiency in separate ifn receptors did not affect disease progression during murine sars-cov infection, in contrast to a deficiency in common signaling molecule stat , illustrates this high degree of redundancy [ ] . at the moment, hundreds of isgs have been identified. however, an exact function has only been clarified for a relatively low number of the corresponding proteins, a description of which can be found elsewhere [ ] [ ] [ ] . nonetheless, these studies suggest that isgs exhibit overlapping inhibitory activity towards most components of virus replication, including virus entry, uncoating, translation of viral proteins, rna replication, and egress [ ] . in summary, host cells detect viral rna by several detection mechanisms to establish an antiviral state via the induction of ifns and pro-inflammatory cytokines, leading to expression of isgs. as mentioned earlier, mavs localized on mitochondria is a key player in antiviral signaling. therefore, studies demonstrating that the mavs adaptor molecule sting is located on the er implied that earlier identified contacts between mitochondrial membranes and er membranes (mitochondrion-associated membranes: mams) were possibly involved in antiviral signaling [ ] . in support of this theory, expression of a mam-enriched marker protein followed by cell fractionation revealed that mavs localizes to mams, which was supported by immunofluorescence assays of mavs and mamenriched proteins [ ] . additionally, immunoprecipitation analysis of the mam fraction of sendai virus-infected hepatocytes confirmed that mam-localized mavs interacts with rig-i and signaling cofactor traf [ ] , indicating that mams are involved in the rlr-mediated antiviral immune signaling pathway. therefore, mams function as important signaling platforms that govern expression of type i and iii ifns. interestingly, mavs is also expressed on peroxisomal membranes, and interacts with stimulated rlrs during viral infection [ ] . however, in contrast to its mitochondrial counterpart, peroxisomal mavs was found to induce expression of isgs by an ifn-independent mechanism, and this relatively rapid process seems to provide short-term protection until the ifn-dependent expression of isgs mediated by other viral rna-sensing mechanisms is established [ ] . interestingly, recent studies found that direct contacts between peroxisomes and er membranes were involved in maintaining peroxisomal function [ ] [ ] [ ] , raising the question whether these contact sites might also regulate antiviral immunity-related processes. in summary, peroxisomal and mitochondrial membranes and their contactsites with the er are important regions for converting viral rna detection by rlrs to downstream events required for establishing an antiviral state. besides mams, increasing evidence suggest a role for stress granules (sgs) in antiviral immune signaling. sgs are nonmembranous compartments in the cell that contain aggregates of ribonucleoprotein (rnp) and mrna, and are formed as a physiological response to various stress stimuli that cause temporal stalling of mrna translation, suggestively preventing accumulation [ ] . consistent with the function of this structure, inhibition of translation through the inactivation of eukaryotic translation initiation factor (eif) a by phosphorylation is an important cue for sg formation [ ] . considering that various rna virus infections result in shutdown of host translation by inactivation of eif a by for example pkr, it is now known that formation of sgs occurs to different extends during infection with various rna viruses, including hcv, denv, and sfv [ , ] . interestingly, multiple lines of evidence have demonstrated that sgs in encephalomyocarditis virus-or influenza virus-infected cells contain multiple rna sensing molecules that were found to contribute to ifn production in vitro, including rig-i, mda , pkr, oas, rnasel, and dsrna [ , ] . however, it should be noted that the contribution of sgs to antiviral signaling differs per virus, as sg-localized mda , an rlr that greatly contributes to ifn induction upon encephalomyocarditis virus infection, was not found to support ifn expression upon infection with this virus [ ] . nonetheless, these findings suggest an important role for sgs as detection platforms for viral rna. taken together, increasing evidence suggests that the antiviral innate immune signaling cascade is spatially organized, and since different structures in the cell seem to be specialized in a particular aspect of this process, it can be hypothesized that interaction between these structures plays a fundamental role in orchestrating the antiviral innate immune response. positive-strand rna viruses developed multiple mechanisms to impair rna recognition and antiviral signaling (also reviewed in [ ] ). as mentioned above, the presence of viral rna in ros suggests a role for ros in shielding viral rna from the innate immune detection machinery. while there is no unequivocal evidence supporting this theory, the finding that isolated viral rnacontaining membrane alterations only become sensitive to nuclease treatment after membrane disruption by nonionic detergents supports this hypothesis [ ] [ ] [ ] [ ] . in addition, a positive correlation between cytosolic exposure of viral rna and ifn induction was recently found by an in vitro comparison of jev and denv infections [ ] . another important strategy by which rna viruses avoid recognition is their modification of viral rna molecules to mimic eukaryotic mrna. for example, the formation of a cap is catalyzed by viral phosphatases and methyltransferases in some viral families such as the coronaviruses. from work on mouse hepatitis virus (mhv) it became clear that coronaviruses need to take care of n-linked as well as o-linked methylation on the cap structure, in order to avoid recognition by innate immune sensors [ ] . mimicry of eukaryotic rna in this way was shown to be an important immune evasion strategy by +rna viruses such as sars-cov and jev [ , ] . a recent report indicated that coronaviruses also evade dsrna mediated recognition by pkr and oas by using their endonuclease (endou), which cleaves free viral (and cellular) rna that is somehow exposed to the cytosol, in order to prevent antiviral signaling [ ] . in addition to avoidance of recognition, +rna viruses actively interfere with antiviral signaling components to impair expression of ifns and pro-inflammatory cytokines. very often, the viral proteases that are expressed by +rna viruses, and which mostly have a primary function in cleavage of viral replicase polyproteins, seem to have a prominent role in this. for example, hcv ns / a and denv ns a cleave mavs [ , [ ] [ ] [ ] [ ] [ ] [ ] , and the denv protease complex and hcv ns b inhibit adaptor molecule sting by cleaving it [ ] [ ] [ ] [ ] [ ] . additionally, human cov nl and sars-cov nsp possess a papain-like protease (plp) domain that prevents dimerization of sting and its complex formation with mavs and ikke, and almost all nidovirus-encoded plps have been shown to display deubiquitinating activity [ ] . several have been suggested to facilitate deubiquitination of innate immune factors such as sting, rig-i, tbk and irf in order to halt innate immune signaling [ ] . moreover, in collaboration with others, our lab demonstrated that eav and mers-cov virus mutants that lack the dub activity of their plps while retaining polyprotein cleavage functions suppress ifn production less efficiently during infection ( [ , ] and our unpublished data). furthermore, viral proteins such as denv ns b/ and the sars-cov membrane glycoprotein interfere with complex formation of ikke/tbk and ikka/ikkb/ ikkg or downstream transcription factors, thereby effectively inhibiting both rlr and tlr signaling [ , ] . interestingly, accessory proteins encoded by open reading frames of covs have been shown to be involved in inhibition of irf , irf , and nf-kb by undefined mechanisms [ , ] . in addition, several methods are employed by +rna viruses to interfere with jak/stat signaling. for instance, jev ns and wnv ns b inhibit phosphorylation and activation of jak , and suppressors of jak are induced by viruses such as wnv, jev, and chikungunya virus [ ] [ ] [ ] [ ] [ ] . however, stat and stat are most heavily targeted. jev ns , wnv and denv ns b, and jev ns a have been shown to inhibit stat activation. in addition, denv ns promotes proteasomal degradation of stat . interestingly, sars-cov inhibits stat signaling in three different ways. firstly, sars-cov nsp binds stat to inhibit its phosphorylation [ ] . secondly, the accessory protein encoded by sars-cov open reading frame prevents nuclear transport of stat [ , ] . thirdly, sars-cov plp induces expression of e ubiquitin ligase e - k, which promotes proteasomal degradation of extracellular signal-regulated kinase , a protein responsible for activation of stat [ ] . to our knowledge, no studies have been published (yet) about specific interference of +rna viruses with ifn-ii production. interference of +rna viruses with antiviral signaling also has consequences for the spatial organization of the antiviral innate immune system. for instance, since sgs suggestively have an antiviral role, interfering with sg formation might constitute an opportunity for viruses to hamper the innate immune response. indeed, various proteins of poliovirus, coxsackievirus, encephalomyocarditis virus, denv, wnv, mers-cov, chikungunya virus, and jev have been shown to prevent the formation of sgs and thereby suppress ifn production [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . thus, dysregulation of sgs seems to be a common strategy for +rna viruses in order to dampen the innate antiviral immune response. in addition, the finding that mams are involved in the rlr-mediated antiviral immune signaling pathway shedded new light on the immunosuppressive role of viral proteases. by performing colocalization analysis with a mam-enriched marker protein and cell fractionation experiments in both uninfected and hcv-infected cells, it was found that ns / a also localizes to mams [ ] . intriguingly, despite the clear function for peroxisomal mavs in antiviral signaling, cleaved mavs was found solely in mam-enriched cell fractions. in addition, recent findings suggest that mams are also physically disrupted during denv infection [ ] . thus, these data indicate that mams are critical locations for antiviral signaling and have an important role in expression of type i and iii ifns. moreover, increasing evidence suggests that at least some +rna viruses in fact occupy or hijack mam-membranes during infection, as mams of hcv-infected cells were found to contain proteins involved in virus assembly and fully assembled virions [ ] . it remains to be investigated whether mams also serve as platforms for viral assembly of other +rna viruses. in addition, mam disruption as a consequence of denv infection was followed by the formation of convoluted membranes at the same location, which supported denv replication [ ] . moreover, promoting the formation of convoluted membranes further repressed the ifn response, underscoring the importance of mam disruption in both the replication and immune evasion of +rna viruses [ ] . taken together, these studies underscore the importance of mams as key antiviral signaling platforms, and disruption of mams constitutes an effective immune evasion strategy for +rna viruses. in conclusion, +rna viruses developed divergent methods to delay antiviral innate immune signaling at multiple levels. in addition, interference of +rna viruses with antiviral signaling leads to spatial disorganization of the antiviral innate immune system, and future studies will hopefully elucidate the consequences of this phenomenon in the context of other cellular compartments and viruses. assuming that ros impair antiviral signaling by shielding viral rna from the host, it is tempting to hypothesize the existence of host cell mechanisms aimed to disrupt ros, thereby exposing viral rna and promoting antiviral signaling. the fact that +rna viruses display such elaborate activities to inhibit viral rna recognition and subsequent signaling, as detailed in the former paragraphs, supports the view that disruption of ro integrity is a situation these viruses anticipate dealing with. however, evidence suggesting targeting of ros by the innate immune system is still scarce, although it was reported that hcv ros are attacked by the isg viperin [ ] . additionally, -hydroxycholesterol, a product produced by the isg cholesterol -hydroxylase also modifies hcv replication organelles [ , ] . recently, our lab published a study suggesting that ifn-b signaling also negatively influences arterivirus ros, since significantly less were formed and remaining structures showed drastically different morphology after ifn-b treatment. the results suggested that the treatment interrupted biogenesis of these membrane structures rather than breaking down structures that were already made [ ] . interestingly, neither viperin nor -hydroxycholesterol was involved in the observed effects, and the mechanistic details therefore have to be further investigated. other recent data suggest that there may indeed be specific mechanisms by which the innate immune system tags and disrupts so-called non-or aberrant-self membrane structures in the cell [ ] , which could include viral ros. most data originate from studies focusing on bacteria, fungi, or parasites that reside in rearranged membranes, known as pathogen-containing vacuoles (pvs), which prevent detection of cytosolic innate immune sensors in a similar way as viral ros are thought to do. multiple studies found that enzymes capable of disrupting pvs are gtpases, such as effector immunity-related p gtpases (irgs) and guanylatebinding proteins (gbps), which are part of a family which we will refer to as ifn-inducible gtpases [ ] . expression of irgs and gbps is induced upon ifn-g stimulation, and leads to their accumulation on pvs. subsequently, activation of these gtpases due to the exchange of gdp for gtp results in membrane disruption as a consequence of their dynamin-like activity [ ] . to prevent aspecific disruption, host cell membranes contain a set of proteins that inhibit gtpase activity, known as ''guard proteins'' [ ] . wellstudied pathogens in the context of this process are the protozoan parasite toxoplasma gondii (t. gondii), the bacterium chlamydia trachomatis and the microsporidian encephalitozoon cuniculi [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although it remains unclear for most of these pathogens how ifn-inducible gtpases are recruited towards pvs, recent studies on t. gondii infection demonstrated that host cells label these membrane structures, for example with various forms of microtubule-associated protein a/ b-light chain (lc ) to initiate this process [ ] [ ] [ ] [ ] (fig. ) . lc is a well-known factor in the autophagy pathway, and several forms of lc exist in the human proteome, which seem to have overlapping as well as distinct functions. the human genome encodes homologs lc a, lc b, and lc c and lc -like homologs gabarap, gabarapl , gabarapl , and gabarapl . we will refer to all of these as lc unless stated otherwise. lc was initially discovered as an essential protein for autophagosome formation, which requires a covalent interaction between cytosolic lc (lc -i) and the phospholipid phosphatidylethanolamine (pe), after which lc is referred to as lc -ii or lipidated lc [ ] . however, recent studies have now revealed that labeling of (foreign) membrane compartments by lc conjugation can also have various autophagyunrelated consequences, as has been reviewed elsewhere [ , ] . interestingly, multiple studies demonstrated that labeling of lc on pvs recruits ifn-inducible gtpases upon ifn-g expression during t. gondii infection, resulting in exposure of t. gondii to the host cell cytosol, thereby triggering anti-bacterial immune responses [ , , ] . considering the function of lc in autophagosome formation, jayoung choi and co-workers [ ] termed this process targeting by autophagy proteins (tag). in addition, a similar role was recently found for ubiquitin, the versatile regulator of numerous important cellular processes, and also a well-known autophagy-related protein [ ] . a study on t. gondii and chlamydia trachomatis infection demonstrated that pvs are also recognized and labeled by the ubiquitination pathway upon ifn-g expression, resulting in recruitment and activation of ifn-inducible gtpases [ ] . importantly, it was shown that virulent strains of t. gondii and chlamydia trachomatis interfere with the deposition of ifn-inducible gtpases on pvs, underscoring the importance of this mechanism in clearance of associated infections [ , , ] . collectively, these studies demonstrate the existence of various innate immune responses that result in tagging and subsequent disruption of non-or aberrant-self membrane structures in the cell, thereby exposing pamps and promoting immune signaling. importantly, the findings suggest that ifn-mediated membrane disruption might be a common principle in clearance of pathogens that use rearranged membranes to support replication and avoid innate immune detection. like pvs, ros induced by +rna viruses are membranous compartments that constitute an environment for efficient replication, simultaneously avoiding immune detection by the host. therefore, similar mechanisms might be aimed towards viral ros. the observation that ros induced by various +rna viruses consist of a double membrane initially suggested a role for the autophagy machinery in the formation of these structures. however, only nonlipidated lc , and not an intact autophagy pathway, was found to be essential for viral ro formation and viral replication upon infection with eav and mhv [ , ] , thereby contradicting this hypothesis. interestingly, lipidated lc was found to support ifn-g mediated protection against murine norovirus (mnv) infection by autophagy-unrelated means [ ] . furthermore, the induction of viral membranous structures by expression of mnv nsps led to a high colocalization between atg l , a protein involved in lc conjugation, and the mnv polymerase. since atg l determines the site for lc conjugation [ ] , and the mnv polymerase is associated with ros, this further supports the possibility that lc conjugation occurs on ros. other supporting in vitro experiments show that conditional ko of atg b inhibits replication of mnv and t. gondii more effectively compared to wildtype cells [ , ] . in contrast to the multiple autophagins involved in pre-processing of lc for conjugation, atg b is the only human homolog of yeast atg that efficiently deconjugates lc from membranes [ ] , and therefore plays a major role in negatively regulating the fraction of conjugated lc . several groups have reported enhanced conjugation of lc to membranes during knockdown of atg b in various cell types [ ] [ ] [ ] [ ] , including those used by aforementioned studies [ , ] . thus, it is likely that increased lipidated lc was present on ros and pvms during experiments in atg b-deficient recent studies demonstrate that lc is conjugated to the pvm by the lc conjugation system, leading to recruitment of ifn-g inducible gtpases to the pvm upon ifn-g stimulation. as a consequence, activation of ifn-g inducible gtpases leads to membrane disruption of the pvm, and exposure of t. gondii to the innate immune system. cells, thereby explaining the enhanced inhibition of replication. interestingly, findings in other studies are contradictory in the sense that only lc -i was observed on ros of mnv, eav, and mhvinfected cells [ ] [ ] [ ] . on this note, it is worth mentioning that lc -i does not support ifn-g mediated inhibition of viral replication, as ifn-g mediated inhibition of mnv-infected cells does not occur in the case of atg deficiency, one of the proteins responsible for lc conjugation [ ] . in conclusion, although there is no unequivocal evidence regarding the presence of conjugated lc on +rna virus induced ros, the studies described above provoke the hypothesis that membrane disruption of viral ros mediated by ifn-g inducible gtpases may be part of the innate immune response against +rna viruses (figure ). interestingly, current knowledge of ifn-inducible gtpases suggests that membrane disruption of ros by ifn-g inducible gtpases might be part of a common antiviral mechanism that utilizes ifn-inducible gtpases to combat viral infection. aside from irgs and gbps, other families of dynamin-like ifn-inducible gtpases exist, such as myxovirus resistance proteins (mx) and the very large ifn-inducible gtpases (vligs) [ ] . these enzymes are also known to have antiviral properties against a variety of viruses (reviewed in [ ] ). for example, the protective role of mx proteins has been demonstrated for orthomyxoviruses such as influenza, lentiviruses such as human immunodeficiency virus (hiv) , and, interestingly, several +rna viruses belonging to the picornaviridae and togaviridae [ ] . moreover, based on evolutionary studies, it was recently suggested that mx proteins mediate protection against even a wider variety of viruses [ ] . in contrast to ifn-g inducible irgs and gbps, expression of mx proteins is induced by ifns type i and iii, implying that all ifn types are capable of inducing ifn-inducible gtpases [ ] . therefore, all cell types known to respond to ifns could theoretically induce a variety of ifn-inducible gtpases in response to viral infection. in the case of +rna viruses, it is intriguing that multiple studies demonstrated how mxb suppresses replication of hiv- by targeting the viral capsid protein [ ] [ ] [ ] , given the parallels between retroviral capsids and +rna virus ros [ ] . in conclusion, these findings again suggest a general function for ifn-inducible gtpase effectors in the targeting of viral intracellular membrane structures that function to shield away viral components away from the cytosol. rearrangement of host membranes into ros is considered a hallmark of +rna virus infection. ros are believed to have at least a dual role in +rna virus infection and innate immune evasion, as they facilitate efficient rna replication while shielding viral rna from the host antiviral response machinery. as viral rna is a very potent inducer of antiviral signaling, we focused on how both the biochemical and spatial organization allows the innate immune system to convert detection of viral rna to an antiviral state. in addition, we described part of the divergent methods by which +rna viruses impair antiviral signaling surrounding their rna replication, transcription, and translation in order to establish infection. at last, based on recent studies that demonstrated how ifn-g inducible gtpases are capable of disrupting pvs, we discussed the possibility of a general function of ifn-inducible gtpases in the targeting of viral ros. in summary, upon infection, +rna viruses hamper ifn and isg induction at multiple levels to decelerate antiviral innate immune signaling. in this process, the formation of ros enables rapid viral rna replication while masking it from the host. however, in case of sufficient activation of ifn-inducible gtpases by ifn-i and -iii originating from various cell types, or ifn-ii produced by specialized immune cells, disruption of ros by ifn-inducible gtpases may result in enhanced exposure of viral rna, thereby amplifying the innate immune response and ensuring efficient clearance of the virus. however, considering the differences in morphology and origin of ros, the variety of infection dynamics within the category of +rna viruses, and a lack of knowledge regarding the exact mechanism(s) of disruption of non/aberrant-self membrane structures by ifninducible gtpases and possibly other factors, extensive further research is required to elucidate whether this process plays a general role in +rna virus infection, and might open possibilities for development of novel antiviral therapies. none funding this research did not receive any specific grant from funding 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endoplasmic reticulum/golgi membrane severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferon-induced responses through downregulation of extracellular signal-regulated kinase -mediated signalling pathways inhibition of cytoplasmic mrna stress granule formation by a viral proteinase hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress japanese encephalitis virus core protein inhibits stress granule formation through an interaction with caprin- and facilitates viral propagation interaction of tia- /tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection stress granule components g bp and g bp play a proviral role early in chikungunya virus replication middle east respiratory coronavirus accessory protein a inhibits pkr-mediated antiviral stress responses dengue virus perturbs mitochondrial morphodynamics to dampen innate immune responses the hepatitis c virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites interferon-inducible cholesterol- -hydroxylase restricts hepatitis c virus replication through blockage of membranous web formation the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a +rna virus self and non-self discrimination of intracellular membranes by the innate immune system interferon-inducible gtpases in host resistance, inflammation and disease ifn-inducible gtpases in host cell defense irg and gbp host resistance factors target aberrant, non-self vacuoles characterized by the missing of self irgm proteins chlamydia muridarum evades growth restriction by the ifn-gamma-inducible host resistance factor irgb identification of the microsporidian encephalitozoon cuniculi as a new target of the ifn-g inducible irg resistance system the irg protein-based resistance mechanism in mice and its relation to virulence in toxoplasma gondii autophagosome-independent essential function for the autophagy protein atg in cellular immunity to intracellular pathogens the parasitophorous vacuole membrane of toxoplasma gondii is targeted for disruption by ubiquitin-like conjugation systems of autophagy interferon-inducible gtpases go to their target membranes via the lc -conjugation system of autophagy targeting by autophagy proteins (tag): targeting of ifng-inducible gtpases to membranes by the lc conjugation system of autophagy dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescenttagged lc selective autophagy against membranous compartments: canonical and unconventional purposes and mechanisms hidden behind autophagy: the unconventional roles of atg proteins ubiquitin and ubiquitin-like proteins as multifunctional signals ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins virulent toxoplasma gondii evade immunity-related gtpase (irg)-mediated parasite vacuole disruption within primed macrophages coordinated loading of irg resistance gtpases on to the toxoplasma gondii parasitophorous vacuole an autophagy-independent role for lc in equine arteritis virus replication coronaviruses hijack the lc -ipositive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication nondegradative role of atg -atg /atg l autophagy protein complex in antiviral activity of interferon gamma the atg l complex specifies the site of lc lipidation for membrane biogenesis in autophagy gate localize to autophagosomal membrane depending on form-ii formation human light chain /map lc b is cleaved at its carboxyl-terminal met to expose gly for lipidation and targeting to autophagosomal membranes hsatg b/hsapg b/autophagin- cleaves the carboxyl termini of three human atg homologues and delipidates microtubule-associated protein light chain -and gabaa receptor-associated protein-phospholipid conjugates autophagy proteins regulate erk phosphorylation autophagin- ) phosphorylation modulates autophagy evolutionary analyses suggest a function of mxb immunity proteins beyond lentivirus restriction mx is an interferon-induced inhibitor of hiv- infection human mx is an interferon-induced postentry inhibitor of hiv- infection the interferon-inducible mxb protein inhibits hiv- infection parallels among positive-strand rna viruses, reversetranscribing viruses and double-stranded rna viruses enzo scutigliani ( ) obtained a bsc in biomedical sciences with honor at the university of amsterdam (the netherlands) in , and is currently in the final process of becoming a msc with a specialization in cell biology and advanced microscopy. over the last years, he developed a particular interest for microbiology, and his ambition is to develop novel treatment or vaccination methods by understanding pathogens at the molecular level. to achieve this goal, he is currently specializing in combining microbiological research and advanced microscopy.a molecular sciences study at wageningen university and research center (the netherlands) motivated marjolein kikkert ( ) to pursue a ph.d. degree, which she achieved in at the department of virology of this university. her thesis was entitled "role of the envelope glycoproteins in the infection of tomato spotted wilt virus ". she then switched to mammalian virology, became a post-doc at the national institute of health and the environment in bilthoven, the netherlands with prof. emmanuel wiertz, and subsequently at leiden university medical center (lumc), working on immune evasion strategies of human cytomegalovirus. after this she started working with prof. eric snijder at the lumc as a post-doc, and developed into an assistant professor in the department of medical microbiology. her research currently focuses on the replication organelles and related virus-host interactions of nidoviruses and other +rna viruses, and the innate immune evasion mechanisms which these viruses employ. the knowledge gained from her research is being used for development of novel antiviral vaccines and Àdrugs. key: cord- -kzdp edn authors: domingo, esteban title: quasispecies dynamics in disease prevention and control date: - - journal: virus as populations doi: . /b - - - - . - sha: doc_id: cord_uid: kzdp edn medical interventions to prevent and treat viral disease constitute evolutionary forces that may modify the genetic composition of viral populations that replicate in an infected host and influence the genomic composition of those viruses that are transmitted and progress at the epidemiological level. given the adaptive potential of viruses in general and the rna viruses in particular, the selection of viral mutants that display some degree of resistance to inhibitors or vaccines is a tangible challenge. mutant selection may jeopardize control of the viral disease. strategies intended to minimize vaccination and treatment failures are proposed and justified based on fundamental features of viral dynamics explained in the preceding chapters. the recommended use of complex, multiepitopic vaccines, and combination therapies as early as possible after initiation of infection falls under the general concept that complexity cannot be combated with simplicity. it also follows that sociopolitical action to interrupt virus replication and spread as soon as possible is as important as scientifically sound treatment designs to control viral disease on a global scale. medical interventions have dramatically increased over the last century, and in the case of infectious diseases, the discovery and development of antibiotics and antiviral agents have represented a very powerful external selective constraint imposed upon replicating microbes. hundreds of antiviral agents have been developed since the second half of the th century, and viruses can generally find evolutionary pathways to continue replication in their presence. the same is true of antibiotics and replication of bacteria. the belief that bacterial diseases were on their way toward extinction was quite widespread in the middle of the th century. sir m. burnet wrote in his textbook: "and since bacterial infections are, with unimportant exceptions, amenable to treatment with one or other of the new drugs, our real problems are likely to be concerned with virus diseases" (burnet, ) . in , a prominent spanish medical doctor, g. marañ on, declared: "in the year cancer will be a historical disease. infections will be almost entirely absent as a cause of mortality." (on a personal note, when i joined the university of california, irvine in , to work as postdoctoral student with r.c. warner, i attended some biology courses in which the teachers expressed to students that infectious diseases would disappear in a few decades as a consequence of the use of antibiotics and antiviral agents). predictions in science tend to fail. the optimistic view was not unanimous. a. fleming, the discoverer of penicillin, recognized the adaptive capacity of bacteria and suggested virus as populations https://doi.org/ . /b - - - - . - that bacteria would inevitably find ways of resisting the damage to them caused by antimicrobial drugs [quoted from the document "new antimicrobial drugs" from the european academies science advisory council, november (www.easac.eu); see also chapter ]. furthermore, there was early evidence of selection of mycobacterium tuberculosis mutants resistant to streptomycin (mitchison, ) . antibiotic resistance in bacteria has similarities and differences with antiviral resistance in viruses, and they are compared in chapter . we are now very aware that one of the major problems in antiviral therapy is the nearly systematic selection of drug-resistant virus mutants, which is often associated with treatment failure. other external influences, such as vaccination or immunotherapy, particularly using monoclonal antibodies, can also evoke the selection of viral subpopulations capable of replicating in the presence of those components inherent to an immune response. thus, selective constraints intended to limit rna virus replication meet with the broad and dynamic repertoire of variants ingrained in quasispecies dynamics. two space-time levels of the effects of drugs or vaccines are distinguished in coming sections: (i) short-term consequences for the individual in the form of treatment or vaccination failure and (ii) long-term consequences at the population level in the field, or vaccine-driven evolution of the antigenic properties of viruses. there are other medical interventions that may alter virus survival. individuals who are immunocompromised as a consequence of treatment after organ transplantation or those subjected to anticancer chemotherapy become particularly vulnerable to viral infections. enhanced viral replication can favor pathological manifestations in the affected individual as well as the spread of a large number of viruses into the environment, with consequences for the emergence and reemergence of viral disease (section . in chapter ). viral diseases are an important burden for human health and agriculture (bloom and lambert, ) . virus evolution, through the basic mechanisms exposed in previous chapters, can influence the two major strategies to combat viral infections: prevention by vaccination and treatment by antiviral inhibitors. for the design of new antiviral vaccines, a critical issue is the diversity displayed in the field by the virus to be controlled. the natural evolution of the virus may result in the circulation of one major antigenic type or the cocirculation of multiple antigenic forms. the vaccine composition (independently of the type of vaccine; see section . . ) must match the antigenic composition of the virus to be controlled. hepatitis a virus (hav) circulates as a single serotype, while foot-and-mouth disease virus (fmdv) circulates as seven serotypes and diverse subtypes, and the antigenic types are unevenly distributed in different geographical locations. a monovalent vaccine made of the prevailing antigenic type of hav should be sufficient to confer protection, while a multivalent vaccine composed of several types or subtypes is required to confer protection against fmdv, and the antigenic composition of the vaccine should match the circulating viruses in each geographical region. this is why antifmd vaccines of different compositions are used in different world areas at a given time, and vaccine composition must be periodically updated to maintain its efficacy. thus, one effect of virus evolution relevant to vaccine design derives from the necessity to prepare a vaccine that mirrors the antigenic composition of the virus to be controlled. in the case of live-attenuated antiviral vaccines, the evolution of the vaccine virus while it replicates in the vaccinee is a risk factor to produce virulent derivatives. the invasion of a susceptible host by a virus and the ensuing viral replication can be regarded as a step-wise process during which the virus must adapt to a series of selective pressures presented by the host, notably the immune response. the outcome can be either viral clearance (elimination of the infection) or virus survival and progression toward an acute or a persistent infection. administration of antiviral agents is an additional selective constraint that limits viral replication. evolutionary mechanisms may either succeed in the selection of mutants resistant to the antiviral agent that will permit the infection to continue or fail in sustaining the infection, resulting in the clearing of the virus from the organism. treatment planning, one of the aims of the new antiviral pharmacological interventions, based on information of viral genomic sequences present in each infected patient, has parallels with vaccine composition design. for vaccines, the information comes from the analyses of antigenic composition of circulating viruses, and for antiviral agents, the information comes from the mutant spectrum composition of the virus to be controlled in the infected patient. world-wide vaccination campaigns made possible the eradication of human smallpox [with the official declaration by the world health organization (who) in ] and animal rinderpest [with the official declaration by the world organization for animal health, office international des epizooties (oie) in ]. the number of new cases has decreased as a result of vaccination programs against several viral diseases, including measles or hepatitis b (bloom and lambert, ) , and substantial progress has been made toward the eradication of poliomyelitis (chumakov and kew, ; himman, ) . only the deliberate decision not to vaccinate (for religious reasons or misinformation campaigns) or lack of vaccine accessibility (for socioeconomic circumstances) jeopardizes vaccine efficacy. these facts demonstrate that at least some viral diseases can be controlled on a global basis by vaccination, an unprecedented achievement of human and animal health. despite the huge economic investment, however, there are important viral diseases such as acquired immunodeficiency syndrome (aids), hepatitis c, or viral hemorrhagic fevers for which no effective vaccines are available. for some diseases such as human influenza or animal fmd, vaccines are accessible, but they require periodic updating to approximate the antigenic composition of the vaccine to that of the circulating virus (section . ). in the case of influenza virus (iv), a major change in antigenic composition can occur through antigenic shift, in which the virus acquires new hemagglutinin and neuraminidase genes by genome segment reassortment (section . in chapter ), with the first evidence obtained by g. laver as early as (for the early history of influenza, its causative virus, and vaccine designs, see beveridge, ; kilbourne, ) . antigenic variation of viruses, whatever the mechanism might be, can affect vaccine efficacy and in some cases, the extreme rapid intra and interhost evolution of a virus may render an effective vaccine unfeasible at least with the current tools of vaccinology. cd þ t cell responses may act soon after infection and promote the selection of escape mutants (bull et al., ) . the difficulties for the control of virus disease derived from the adaptive potential of viruses (domingo, ; domingo and holland, ; bailey et al., ; hamelaar et al., ) require the judicious application of existing tools and innovative approaches that are still in their infancy. a first basic requisite for the preparation of a vaccine against a viral agent is the understanding . antiviral vaccines and the adaptive potential of viruses of the immune response evoked by the virus when it infects the organism to be protected (activation of b and t lymphocytes for antibody production, cellular responses, and generation of memory cells) and correlates of protection (bloom and lambert, ; van regenmortel, ; hagan et al., ; cunningham et al., ; rolland, ) . despite social pressure to rapidly obtain a vaccine, for each virus-host system, well-designed experiments are necessary to try to establish the determinants of protection, which is not a simple issue. the discussions in coming paragraphs are focused on the relevance of virus evolution in vaccine efficacy, irrespective of the type of protection afforded by the vaccine. what we term "protection" may mean the total absence of replication of the infecting virus (termed "sterilizing" immunity) or absence of disease manifestations despite infection and virus replication. as a general initial statement, which is widely accepted by vaccinologists, a vaccine is likely to be effective when it evokes an immune response that is similar to the response elicited by the authentic viral pathogen when it produces disease successfully overcome by the infected organism (evans and kaslow, ; bloom and lambert, ) . we refer to this as the basic principle of vaccinology. when infection by an antigenically constant virus produces lifelong immunity (i.e., measles virus infection), a vaccine is likely to evoke longlasting protection. in contrast, if a patient cured of a virus can be reinfected by the same (or a closely related) virus (i.e., hepatitis c virus infection) a vaccinedat least one prepared by standard methodologydis unlikely to evoke protection. some points to be considered in the design of antiviral vaccines are listed in box . . they are intended to minimize the selection of vaccineescape mutants and favor the success of vaccination campaigns. some of the recommendations deserve further comment. first, a basic knowledge of virus evolutionary dynamics and how it affects virus antigenic stability (or lack of) is essential. the fact that a methodology is available (i.e., vectors that can express large amounts of based on domingo and holland ( ) . . quasispecies dynamics in disease prevention and control antigens displaying good immunogenicity) does not guarantee vaccine efficacy, and even less if correlates of protection are not understood. the order of efficacy of different vaccine designs proposed in box . is justified both by the basic principle of vaccinology and by the mechanisms of selection of antibody (ab)-and cytotoxic tcell (ctl)-escape mutants by viruses. single amino acid substitutions at b-and t-cell epitopes in viral proteins are often sufficient to elude neutralization by the corresponding cognatespecific antibody or to escape recognition by a clonal ctl population. for many viruses, the frequency of monoclonal antibody-escape mutants has been measured in À to À , even in clonal populations obtained under controlled laboratory conditions and that have undergone a limited number of replication rounds (section . . in chapter ). generation of immune-escape variants can result in lack of vaccine efficacy, contribute to viral persistence (pircher et al., ; weidt et al., ; ciurea et al., ciurea et al., , richman et al., ; pawlotsky, ) , and provoke vaccination-induced virus evolution (section . . ). in human immunodeficiency virus type (hiv- ), antibody-escape variants are incessantly being produced in vivo to the point that virus replication continues despite the antibody response (richman et al., ; bailey et al., ) . the frequency of selection of mutants that can escape a number (n) of components in which we could hypothetically separate a global immune response is far lower than the frequency of escape to a single (a, b, c, etc.) of the i components of the response. making a simple mathematical abstraction that is applicable also to antiviral-escape mutants (section . ), the frequency of mutants that escape n components of an immune response is the product of frequencies of escape to each individual component [ Àa  Àb  Àc  . . this is an oversimplification because it is not realistic to dissect the selective impact of a complex immune response into discrete components. a virus generally includes multiple antigenic sites and each of them is often composed of several overlapping or nonoverlapping epitopes; in addition, a virus has several t-cell epitopes in different structural and nonstructural proteins, and each epitope displays a different degree of relative dominance. however, the above abstraction reflects the advantage of stimulating the host immune system with a sufficiently broad array of b-and t-cell epitopes to prevent selection of vaccineescape mutants due to a high genetic and phenotypic barrier (compare with the barrier to drug resistance described in section . . ). therefore, selection of vaccine-escape viral mutants is more likely with synthetic peptidic vaccines, than with whole virus-attenuated or inactivated vaccines because the latter present a broad epitope repertoire to the immune system. selection of escape-mutants by peptidic vaccines that evoked partial protection of cattle was documented with fmdv (taboga et al., ; tami et al., ) . the arguments in favor of multiepitopic presentation are also endorsed by a notorious scarcity of licensed peptidic vaccines for viral diseases despite horrendous economic investments (orders of magnitude greater than investments in quasispecies research!). use of a complex, multiepitopic vaccine, however, need not prevent long-term selection of antigenic virus variants as a result of vaccine usage, an important still largely underexplored topic discussed in section . . . experimental evolution with fmdv has opened the way to a new generation of antiviral vaccines that share features of attenuated and inactivated vaccines. this new design is based on the conversion of the monopartite fmdv genome into a segmented genome version that dominated the population after extensive high moi passages (section . in chapter and section . in chapter ). for the segmented virus version to be able to produce progeny, the two genome classes (which are encapsidated into separate particles) must reach the same cell. therefore, administration of the segmented virus preparation should lead to an . antiviral vaccines and the adaptive potential of viruses immune response akin to that evoked by inactivated viral particles, followed by a self-limiting infection. the vaccine was tested successfully in mice and swine, the authentic host of the parental virus (rodriguez-calvo et al., ) . potential concerns with this type of vaccine are that the standard (monopartite) genome can be reconstructed by recombination in the early stages of replication in the animal. a safety level is included in that particular fmd vaccine because of multiple mutations that accumulated during the transition toward genome segmentation, which deviated the genome sequence from the one present in the original swine isolate . exploration of evolutionary mechanisms that result in altered forms of viruses may open new possibilities for vaccine design. new prospects for attenuated vaccines have been opened with the engineering of viruses with suboptimal replication fidelity or deoptimized codon or codon pair usage (coleman et al., ; vignuzzi et al., ; cheng et al., ) . altered polymerase copying fidelity often leads to virus attenuation (gnadig et al., ; graham et al., ; korbouk et al., ; rozen-gagnon et al., ; van slyke et al., ) . a critical issue with live-attenuated vaccines based on deviation from the standard mutation rate is the stability of the attenuation trait. not only true revertants or other site revertants of the polymerase may arise and displace the vaccine virus, but other viral proteins may affect nucleotide incorporation (smith et al., stapleford et al., ; agudo et al., ). when a virus circulates in a population where vaccinated and unvaccinated host individuals coexist, and the vaccine does not induce sterilizing immunity, viruses with an altered antigenic profile might be selected. the larger the overall effective population size of the circulating virus, and the longer the virus is allowed to replicate in such a scenario, the higher the probability of incorporation of compensatory mutations that yield high-fitness antigenic variants. these events in the case of vaccines used in veterinary medicine are particularly significant because they may alter the cell tropism and host range of viruses, thus increasing the possibilities of their zoonotic transmission into humans (schat and baranowski, ) . evidence of vaccination-induced dna and rna virus evolution is increasing, and it has been documented with bovine respiratory syncytial virus, bovine herpesvirus- , marek's disease virus, porcine circovirus , and classical swine fever virus, among others [ (valarcher et al., ; muylkens et al., ; ji et al., ; kekarainen et al., ; constans et al., ; yoo et al., ) ; reviews in gandon et al., ; schat and baranowski, ) ]. the timing of dominance of ctl-escape mutants of the simian immunodeficiency virus (siv) was influenced by vaccination, and the process could be analyzed by penetration into the mutant spectra of the relevant viral populations (loh et al., ) . for human viruses, evidence of vaccineescape mutants has been obtained for hepatitis a and b viruses. vaccination-associated escape mutants of hav with substitutions around the immunodominant site of the virus were identified in a cohort of hiv- , hav doubly infected individuals (perez-sautu et al., ) . the study suggested that an incomplete vaccination schedule, combined with the hiv- -produced immunosuppression might have contributed to high-hav loads, thus facilitating the generation and dominance of antigenic variants. in taiwan, the prevalence of mutants at a major antigenic determinant of the surface antigen of hepatitis b virus (hbv) tripled in decade, and it has been suggested that this increase of prevalence might be due to the ample vaccination coverage in the region (hsu et al., ) . other studies also suggest the circulation of hbv mutants . quasispecies dynamics in disease prevention and control associated with vaccine escape and diagnosis failures (di lello et al., ) . vaccines can rarely afford protection to all vaccinated individuals due to many factors that include variations in vaccine receptivity factors due to polymorphisms in genes involved in the adaptive immune response, immunosuppression of the vaccine recipient, the insufficient time between vaccination and exposure to the viral pathogen, and antigenic differences between the vaccine strain and circulating viruses. in addition, for massive vaccinations in veterinary medicine, damage to the vaccine (during transport, storage, etc.) and improper administration are additional problems. vaccination may occasionally promote the selection not only of antigenic variants but also host cell tropism, host range, or virulent variants (swayne and kapczynski, ; kirkwood, ; read et al., ; rolland, ) . it is not known to what extent the widespread use of vaccination can contribute to antigenic variation relative to other factors (persistence of antibodies from previous infections, genetic drift due to genetic bottlenecks, etc.). however, our current understanding of virus dynamics should encourage investigations on the genetic and antigenic modifications of breakthrough viruses that arise from vaccinated individuals as compared with changes in viruses from unvaccinated host populations. reversion of live-attenuated vaccine viruses into virulent forms is a cause of disease derived from the evolutionary potential of viruses. in the case of attenuated sabin poliovirus vaccine, the rate of vaccine-associated poliomyelitis among those vaccinated for the first time was one per , to , vaccinees, and the rate of those receiving the second vaccine dose was about one in million (reviewed in rowlands and minor, ) . attenuated antifmd vaccines were used in some countries during the second half of the th century, but a reversion to virulence forced the halting of the vaccination programs. vaccine-escape mutants may arise due to ineffective vaccines, and concomitant factors, such as immunosuppression. the escape mutants may remain confined to the unsuccessfully vaccinated host or may spread to other susceptible individuals, and attain different degrees of epidemiological relevance. escape mutants may be direct mutants of the infecting virus or may originate by recombination between the infecting virus and other coinfecting related viruses, as observed with poliovirus and bovine herpesvirus- [kew et al., ; thiry et al., ; among other studies]. reiteration of vaccine selection and fitness increase processes over many generations of vaccinees (be it humans or animals) may result in accelerated virus evolution. since systematic use of vaccines for humans and animals in intensive production units is relatively recent in terms of evolutionary time (less than years, and in some cases even only a few decades), it is still premature to evaluate whether vaccination is a significant factor in promoting long-term virus evolution. the first description of virus resistant to an antiviral inhibitor was by j. barrera-oro, h.j. eggers, i. tamm, and colleagues working with enteroviruses and guanidine hydrochloride and -(alpha-hydroxybenzyl)-benzimidazole as inhibitors (eggers and tamm, ; melnick et al., ) . these early results that suggested that antiviral-resistant mutants could be readily selected have been amply confirmed with many viruses and inhibitors in cell culture and in vivo. indeed, the selection of viral mutants resistant to antiviral agents is an extremely frequent occurrence that has been known for decades, although it became widely recognized in the course of development and clinical use of antiretroviral agents to treat hiv- infections and aids. the description of drug-escape mutants has been based on three main groups of observations: • detection of antiviral-resistant mutants in patients during treatment. when a reverse genetics system is available, the suspected mutation should be introduced in an infectious clone and resistance ascertained and quantified in cell culture or in vitro enzyme assays. • selection of resistant mutants in cell culture, by subjecting the viruses to passages in the presence of inhibitors. the viral population size is an important variable in this type of experiment (section . . ). • calculation of the frequency of resistant mutants by plating a virus in the absence and presence of the antiviral agent similar to the assays to calculate the frequency of monoclonal antibody (mab)-resistant mutants (described in chapter , section . . ). in the three groups of observations, the frequency at which a specific escape mutant is found depends on a number of barriers to resistance (section . . ). traditionally, the fact that a drug can select virus-resistant mutants is regarded as a proof of the selectivity of the drug, as opposed to unspecific or toxic effects on the host cell that indirectly impair virus replication (herrmann and herrmann, ; golan and tashjian, ) . selection of viral mutants resistant to antiviral inhibitors is a major problem for the control of viral disease for two main reasons: (i) because it often results in virus breakthrough (increase of viral load) resulting in treatment failure and (ii) because resistant virus variants may become epidemiologically relevant, with the consequent decrease of inhibitor efficacy at the population level (domingo and holland, ; huang et al., ) . increasing numbers of antiviral agents have been developed based on the three-dimensional structure of viral proteins and their complexes with natural and synthetic ligands, in efforts that have engaged academic institutions and pharmaceutical companies. others have been incorporated as a result of drug repositioning, that is, the discovery of antiviral activity of compounds licensed for other medical purposes, often with the help of computational tools (ab ghani et al., ) . antiviral agents may target viral or cellular proteins involved in any step of the virus life cycle. they may interact with virions and inhibit an early step of infection, such as the attachment to the host cell, penetration into the cell, or uncoating to liberate the genetic material of the virus inside the cell. other agents interfere with the synthesis of viral nucleic acids or viral protein processing, particle assembly, or virus release from cells. selection of resistant mutants has been described for virtually any chemical type of antiviral agent directed to any step of the infectious cycle of dna or rna viruses, including important pathogens, such as herpesviruses, picornaviruses, iv, hbv, and hepatitis c virus (hcv). several reviews and articles have covered the theoretical basis of drug resistance, and consequences for treatment management [as examples see (domingo et al., b) and previous versions in progress in drug research (richman, (richman, , ribeiro and bonhoeffer, ; domingo et al., a domingo et al., , menendez-arias, ; perales, ; mokaya et al., ; nitta et al., ; pawlotsky, ) , and the articles in the current opinion of virology volume edited by l. menendez-arias and d. richman (menendez-arias and richman, ) ]. therefore, the general mechanisms that confer adaptability to viruses are very effective in finding drug-escape pathways through molecular mechanisms that are summarized in section . . considering the implications of quasispecies dynamics explained in previous chapters, the . quasispecies dynamics in disease prevention and control following statement will be obvious to the reader: "if a single mutation is able to confer resistance to an antiviral agent, and the mutation does not cause a significant selective disadvantage to the virus (fitness decrease) in the considered environment, a drug-resistant virus mutant will be present in most, if not all, virus populations" (domingo, ) . if a virus replicates in such a way that a population size of can never be achieved in a single population, it is extremely unlikely that any drug-resistance mutation (or any mutation associated with a phenotypic change) that is generated at a frequency of À or lower will be propagated from that viral population (perales et al., ) . selection of escape mutants depends on the replicative load, and the concentration of inhibitor attained at the sites of virus replication. consider different cell or tissue compartments in which an antiviral inhibitor reaches different concentrations (exerts different intensity of selection) ( fig. . ). in each compartment, there are multiple replication complexes. a mutation conferring resistance to the inhibitor will occur at the same rate in each of them, assuming that the mutation rate is independent of the presence of the inhibitor. however, after its occurrence, the proportion of viral rnas harboring the mutation will decrease depending on the inhibitor concentration. the time at which the effect of the inhibitor will be manifested depends on the inhibitor target. in the example of fig. . we assume that the concentration of inhibitorresistant mutants will decrease in the replication complexes, reaching resistant mutant frequencies of À , À , and À in compartments , , and , respectively. the frequency of inhibitor-resistant mutants in the entire cell, tissue, or organism at that time will be given by the weighted average of mutation frequencies at the individual compartments. in the case of a virus-producing viremia, assuming no bottleneck effects or differential selection for other figure . frequency of a drug-resistant mutant in different compartments (subcellular site, cell, tissue, or organ) of the same organism. three compartments labeled , , and three are drawn. replication complexes are depicted as ellipses and replicating genomes as lines. a replicative unit is defined here as a set of replication complexes. the inhibitor-resistant mutant is represented by a red circle in a genome. compartments , , and reach increasing concentration of the inhibitor, rendering inhibitor-resistant mutant frequencies of À , À , and À , respectively. see text for the difference between occurrence and presence of the resistant mutant, and implications of compartmentalization. . resistance to antiviral inhibitors traits, the frequency of resistant mutants calculated for the virus in blood should reflect the average frequency in all compartments that supply virus to blood. low inhibitor concentration in a compartment will favor the selection of the resistant mutant that can either be archived as an adaptive reservoir or penetrate other compartments, depending on the sequence of events of virus spread. if two or more independent mutations can confer resistance to an inhibitor, the probability of occurrence of an inhibitor-resistant mutant is equal to the sum of probabilities of occurrence of each mutation. for multiple mutations, the probability will be the sum of probabilities of the different mutations, a frequent case in viruses since they often display several evolutionary pathways to drug resistance. the probability of finding a viral genome resistant to two or more inhibitors directed to different targets is given by the product of probabilities of resistance to each of the individual inhibitors. the basic probability considerations regarding the frequency of occurrence of inhibitor-resistant viral mutants are summarized in box . . when two or more mutations occur in the same genome, they may be subjected to epistatic effects, meaning either increase (positive epistasis) or decrease (negative epistasis) of viral fitness (see section . of chapter for the concept of epistasis). the diversity of chemical structures of the antiviral compounds that can select for escape mutants is illustrated in figs. . and . with the formulae of some antiviral agents in current or historical use. they include relatively simple organic molecules, nucleoside analogs, and complex heterocyclic compounds with a variety of residues (ch -, c═ o, nh, nh , f, and cl) that may contribute to interactions with viral proteins or alter the electronic structure of neighbor bonds thus modifying the interaction behavior of some atoms. for all of them, resistant viral mutants have been identified, despite barriers imposed upon the virus to reach a drug-resistance phenotype. • if there are two or more different mutations that produce the same inhibitor-resistance phenotype, and once one of the mutations is present additional mutations are no longer necessary to produce the phenotype, the probability of achieving the phenotypic change is equal to the sum of probabilities of finding each mutation individually. • if two or more independent mutations must happen to produce resistance to an inhibitor, the probability of occurrence of the necessary mutations is equal to the product of probabilities of occurrence of each mutation individually. • if a virus is inhibited by an inhibitor combination, and the mutations that confer resistance to each inhibitor are independent (no cross-resistance is involved), the probability of a combination-resistant mutant to arise is equal to the product of probabilities of resistance to the individual mutations. these probability calculations are applicable to other mutation-dependent virus variations. the impediments for a virus to attain resistance to an inhibitor are divided into genetic, phenotypic, and mutant swarm (population) barriers to resistance (box . ). the genetic barrier to resistance to a specific inhibitor is not a universal value for a virus group, since it may be affected by genetic differences among natural viral isolates. the diversification of hcv into genotypes a and b influenced the genetic barrier to resistance to the ns / a protease inhibitor telaprevir [formula ( ) in fig. . ]. one of the amino acid substitutions that confer resistance to telaprevir is r k in ns . in genotype a, the triplet encoding r is aga; therefore, a single nucleotide transition g / a can yield the triplet aaa, which encodes k. in genotype b, the triplet encoding r- is cga; therefore, two nucleotide changes (transversion c / a and transition g / a) are required to reach aaa, the triplet encoding k. reaching the alternative aag codon for k would require the same or a larger number of mutations (see section . . in chapter for another example of how the synonymous codon usage can influence an ) rimantadine, (a-methyl- -adamantane methylamine). ( ) oseltamivir (trade name tamiflu), ethyl ( r, r, s)- -amino- -acetamido- -(pentan- -yloxy)-cyclohex- -ene- -carboxylate. ( ) zanamivir (trade name relenza), ( r, r, s)- -guanidino- -(prop- -en- -ylamino)- -(( r, r)- , , -trihydroxypropyl)- , -dihydro- h-pyran- -carboxylic acid. . resistance to antiviral inhibitors figure . some inhibitors of human immunodeficiency virus type (antiretroviral agents) and hepatitis c virus. the inhibitors are ( ) zidovudine (azt), -[( r, s, s)- -azido- (hydroxymethyl)oxolan- -yl]- -methylpyrimidine- , -dione. ( ) zalcitabine (ddc), -amino- -(( r, s)- -(hydroxymethyl)tetrahydrofuran- -yl)pyrimidin- ( h)-one. ( ) r, r , r, r)- -( , -dioxopyrimidin- -yl)- -fluoro- -hydroxy- methyl-tetrahydrofuran- -yl]methoxy-phenoxy-phosphoryl]amino]propanoate. additional drugs currently used in antiviral therapy can be found in the references quoted in the text and in chapter . . quasispecies dynamics in disease prevention and control evolutionary outcome). the requirement of transitions versus transversions to reach the resistant phenotype will affect the genetic barrier to resistance. most viral polymerases tend to produce transition mutations more readily than transversions presumably because in the course of rna elongation it is easier to misincorporate a purine by another purine than by a pyrimidine, and the same for pyrimidine misincorporations (chapter ). mutation preference is one of several factors that determine the frequency of drug-escape mutants. thus, evolution may diversify viruses to display different genetic barriers to the same drugs. since in many cases, several independent mutations may confer resistance to the same drug to complicate matters even more, it also has to be considered that the genetic barrier to one inhibitor may be affected by the presence of other inhibitors (beerenwinkel et al., ) . the phenotypic barrier to drug resistance is equivalent to the fitness cost inflicted upon the virus by the mutations and corresponding amino acid substitution(s) required for resistance [fitness cost is treated in chapter (section . ) and in chapter (section . . ) in connection with the frequency of monoclonal antibody-or cytotoxic t-cell-escape mutants in viral populations]. when a drug-resistance mutation inflicts a high fitness cost, a likely result is a reversion of the mutation when the virus replicates in the absence of the drug. an alternative outcome is that compensatory mutations are introduced in the genome so that viral fitness increases while maintaining the inhibitor-resistance mutation. the two outcomes are not mutually exclusive and may contribute to the multiple, transient selection pathways observed by the application of deep sequencing to monitor the response of a viral population to specific selective force (tsibris et al., ; fischer et al., ; cale et al., ; kortenhoeven et al., ) (see section . in chapter ). a high fitness cost may prevent or delay the selection of escape mutants. sofosbuvir [formula ( ) in fig. . ] is a very effective ns b (viral polymerase) inhibitor of hcv. amino acid substitution s t in ns b has been associated with sofosbuvir resistance, and the substitution has been detected in patients and in some natural isolates of hcv. in one of several clinical studies on sofosbuvir efficacy, the mutant spectrum composition of hcv genotype b in an infected patient treated with the drug was followed by deep sequencing of the virus at baseline (prior to initiation of treatment), in the course of treatment, and posttreatment. the frequency of s t was . % at baseline, indicating preexistence of resistance mutations despite no exposure of the virus to the drug (section . ). two days after initiation of sofosbuvir treatment, the level of s t decreased to . %, and viral breakthrough was detected weeks later when . % of the viral population included s t. during the posttreatment period, genomes with the wild-type s amino acid regained dominance that was attributed to the true reversion of mutant genomes rather than the outgrowth of baseline wild-type genomes (hedskog et al., ) . this result suggests a high phenotypic barrier for sofosbuvir, and that hcv has mechanisms to overcome the barrier. the complexities of virus-host interactions render the elucidation of the pathways exploited by a virus to overcome the phenotypic barrier to a drug a highly empirical endeavor. the hope is that a combination of inhibitors that display a high barrier to resistance may impede escape and drive the virus to extinction (chapter ). the mutant swarm barrier to resistance is a consequence of the interfering interactions that operate within quasispecies, and that are described in chapter (section . ). it is a particular case of interference that can delay or impede the increase of frequency of a resistance mutation (crowder and kirkegaard, ; kirkegaard et al., ) . the possible contribution of mutant swarms to facilitate or prevent the dominance of drug-resistant mutants in infected patients is still largely unexplored. it is difficult to anticipate how the three types of barrier listed in box . may result in a level of drug resistance for a particular virus, in a particular host individual, in a specific target organ, at a given time. additional influences are drug pharmacokinetics, drug penetration into different cells, tissues, and organs where the virus replicates (see fig. . ), and prior history of virus replication in the infected host. it is not surprising that the study of drug resistance in viruses remains fundamentally descriptive. when independent amino acid substitutions can lead to resistance to the same drug, alternative evolutionary pathways may be followed depending on trna abundances, mutational preferences, and relative nucleotide substrate concentrations at the virus replication sites. if resistance requires two or more amino acid substitutions, the genetic barrier will be correspondingly increased (section . . and box . ). quantification of barriers to resistance in experiments in cell culture requires a prior characterization of the drug to be tested when acting on the cell culture-adapted virus as it infects a specific cell line. the two basic parameters to be determined are the toxicity of the drug for the host cell, and its capacity to inhibit the production of infectious virus. toxicity is quantified by the concentration of drug that kills a given percentage (generally %, but sometimes another value) of cells under the conditions used in the infection. it is expressed as the cytotoxic concentration (cc ), as depicted in fig. . . toxicity may depend on the cell concentration, the extent of confluence in a cell monolayer, and the metabolic state of the cell (resting vs. actively dividing). the capacity of inhibition is quantified by the concentration of inhibitor that reduces the infectious progeny production by a given percentage (generally %, but sometimes another value) under the defined conditions of the infection, including a multiplicity of infection (moi). it is expressed as the inhibitory concentration (ic ), as depicted in fig. . . the therapeutic index (ti) is given by the quotient cc /ic , and although generally used for in vivo experiments of drug efficacy testing, it can also be applied to cell culture measurements. the three parameters, cc , ic , and ti, are not universal for a virus and a drug since they may be influenced by the composition of the viral population and environmental factors, as repeatedly expressed for other features of viruses in the present book. as a guide, ti values of or more suggest the excellent performance of an antiviral agent; values higher than ten are acceptable, but values lower than ten predict limited efficacy. the quantitative effects of a drug may vary when analyzing a single round of infection versus multiple rounds in serial passages, or when comparing in vivo versus cell culture experiments. cc and ic values serve as a guide to decide the range of the drug concentration to be used in serial passage experiments to evaluate the possible selection of inhibitor-resistant mutants and to estimate the genetic barrier. the possibility to overcome a genetic barrier depends on the virus population size. for viruses that replicate in cell culture, it is possible to estimate the minimal viral population size needed to select a drug-resistant mutant which is generally positively correlated with the genetic barrier ( fig. . ). in the hypothetical example of the figure, a viral population is composed of inhibitor-sensitive viruses (blue spheres), and a low level of inhibitor-resistant viruses (red spheres). the proportion of inhibitorresistant viruses is given by the mutational pressure (e.g., at a frequency of À , which is increased in the picture for clarity). passage of a small amount of virus (e.g., infectious virus in the small circle at the upper part of the figure) will exclude the mutant virus (red spheres) that will be maintained at the basal level dictated by mutational pressure in the course of passages (limited to two in the figure for simplicity). selection of escape mutants is precluded by the limited population size at each transfer. in contrast, if the population size used for the successive infections is sufficiently large (> , larger circles at the bottom that surround both sensitive and resistant viruses), the resistant mutant can become figure . schematic representation of two experiments to determine the concentration of an inhibitor needed to kill % of cells in culture (cc value, left) and the concentration of inhibitor that reduces the viral production to % (ic value, right). the therapeutic index is the quotient between cc and ic (box at the bottom). similar tests can be performed with tissue explants or animals, under controlled environmental conditions. see text for pharmacological implications. . resistance to antiviral inhibitors gradually dominant and can be isolated for further studies. to give another example, a single amino acid replacement that requires two mutations (the change from cag to aug to attain substitution q m in hiv- reverse transcriptase, associated with resistance to multiple antiretroviral nucleosides) will occur at a lower frequency than replacements that require a single mutation. if each of the two mutations reaches a frequency of  À , the expected frequency of the drug-resistant genomes (ignoring fitness effects) will be (  À )  (  À ) ¼  À . thus, at least  viral genomes must undergo one round of copying (or a lower number of genomes a proportionally higher number of rounds of copying) to approach a good probability to obtain a drug-resistant genome in that viral population. population size limitation of a drug selection event is a specific example of how random events may intervene in the process of positive selection (compare with section . and fig. . in chapter ; in that figure, the random event that excludes the positively selected population is conceptually equivalent to the insufficient population size depicted in the upper infection series of fig. . ). when two or more mutations are needed to confer the resistance phenotype, drug resistance will be less likely not only due to the lower probability of generating the two required mutations but also because of the increased chances of two mutations in the same genome entailing a fitness cost. a virus that requires three or more mutations to overcome a selective constraint may occur at a frequency in the range of À or lower which will often be insufficient for the mutant to be present in the mutant spectrum of the infected host ( fig. . ). failure to select for a drug-resistant mutant in cell culture does not necessarily mean that the resistant mutant is not present in the population. it may mean that due to a high genetic barrier, the selection experiment was designed to infect with an insufficient amount of virus. similar and even more accentuated problems are encountered in selection experiments in vivo, since not only the phenotypic barrier or fitness cost inflicted by a drug-resistance mutation (box . ) is often estimated empirically from the frequency of the relevant substitution in patients treated with the drug or in cell culture assays. an adequate procedure to quantify the phenotypic barrier to resistance is to determine the fitness of the virus expressing the protein with the wild-type amino acid (the one that confers drug sensitivity) relative to the virus expressing the protein with the substituted amino acid that confers resistance; fitness is measured in the absence and presence of the drug (double assay). this is an extension of the determination of fitness vectors described in section . . of chapter , as depicted in fig. . ; the assays are best performed in cell culture, although the use of explants or in vivo assays is also feasible. two parameters can be calculated: the fitness cost inflicted by the amino acid substitution associated with resistance, in the absence of the drug (reflected in a fitness value f À drug relative to the wild type), and the selective advantage conferred by the substitution in the presence of the drug (reflected in a fitness value f þ drug > relative to the wild type). the lower the value of f À drug , the higher the fitness cost. we define the selective strength of the resistance mutation as f þ drug /f À drug . for example, if we put arbitrary numbers (unrelated to values shown in ordinate) to the fitness values in the first graph of fig. . , figure . decreased frequency and fitness of mutant genomes resistant to one, two, or three inhibitors. the genome frequency level decreases by several orders of magnitude when resistance to one inhibitor (red asterisk in the upper mutant spectrum), or two inhibitors (red and green asterisks in the middle-mutant spectrum), or three inhibitors (red, green, and blue asterisk in the bottom mutant spectrum) must occur in the same genome (left part of the figure and numerical values at the center). increased number of mutations generally implies fitness decrease (right part of the figure) . see text for implications. . resistance to antiviral inhibitors f þ drug ¼ . and f À drug ¼ . , we obtain a selective strength of . . for the vectors in the second graph, if f þ drug ¼ . and f À drug ¼ . , the selective strength is . high selective strength means an important selective advantage conferred by the amino acid substitution for virus replication in the presence of the drug despite a high fitness cost inflicted by the substitution (compare with section . in chapter for the trade-off and "no free lunch" concepts). if the substitution does not entail any fitness cost (f À drug ¼ ), the fitness value in the presence of the drug equals the selective strength. selective strength can be calculated for a mutation or group of mutations that confer resistance to a drug used at a given concentration in a defined environment [example in (de la higuera et al., ) ]. the limitations of fitness measurements (environment dependence, etc.) described in section . . of chapter apply here. since viral genomic sequences may vary in the course of fitness assays, a limited number of passages and triplicate parallel assays are recommended. if a substitution entails a high fitness cost, direct reversion of the substitution or incorporation of compensatory mutations may occur. nucleotide sequence monitoring in the course of the assay should reinforce the conclusions. most drug-resistance mutations inflict a fitness cost upon the virus and yet very rarely drug resistance represents an unsurmountable barrier to maintain viral infectivity. several possibilities can account for the pertinacious occurrence and selection of drug-resistant, viable viral mutants. one possibility, supported by some experimental and clinical observations, is that a drug-resistant phenotype may be achieved through a number of alternative genetic modifications. even if a specific amino acid substitutiondthat would serve as the most direct and effective determinant of drug resistancedwere highly detrimental or lethal for the virus, alternative mutations can often be found that lead to a similar resistance phenotype, or at least a sufficient resistance to permit virus replication and exploration of sequence space to find compensatory mutations. the connectivity among points of sequence space and the fact that several space positions map into the same (or similar) drug-resistance phenotype contribute to the extended occurrence of drug resistance. this phenotypic redundancy applies to both standard nonmutagenic inhibitors and to mutagenic inhibitors. the cascade of mutations that confer resistance of picornaviruses to the mutagenic purine analog ribavirin illustrates how alternative amino acid substitutions in the viral polymerase (some being genuine resistance mutations and others acting as compensatory substitutions to maintain polymerase function) can lead to the ribavirin-resistance phenotype (discussed in chapter ). a speculative interpretation of the systematic occurrence of drug-resistant viral mutants is that the majority of the chemicals used in antiviral therapy (figs. . and . ) have a structure which may be related to natural compounds that viruses and their ancestral replicative machineries encountered in their continuous struggle to survive. in this view, drug resistance would have been gradually built as a consequence of coevolution (section . of chapter ) between virus replicative and gene expression machineries and the "space" of chemical compounds that interacted with them. mechanisms of drug resistance might have had their roots in molecular events repeatedly experienced as viruses evolved in an interactive manner with protocellular and cellular metabolites in our biosphere. discrimination in favor of small molecule substrates compatible with a flow of genome replication and gene expression and avoidance of perturbing intruders that could alter catalytic activities should have been positively selected. unfortunately, this is a possibility we will never be able to test. whatever the reasons behind, the unfortunate reality is that drug resistance is an extremely frequent event that complicates enormously the control of the viral disease. multiple drug evasion mechanisms have been identified or proposed for rna and dna viruses, and they can operate depending on basic replicative parameters in connection with drug levels and their variation with time. delayed lysis of bacteriophage vx that reduced the replication rounds in the presence of -fluorouracil (fu) produced resistance to this analog (pereira-g omez and sanju an, ). in this line, synchronization of a virus life cycle so that replication occurs when drug levels are minimal has been proposed as a potential resistance mechanism (neagu et al., ) . virus plasticity favors modification of life cycle parameters for virus survival in the presence of drugs, provided time for the relevant selection events is allowed once the treatment has been implemented. the great majority of inhibitor-resistance mechanisms involve amino acid substitutions in viral proteins that directly or indirectly diminish the binding of the drug to the viral target. the following major mechanisms have been documented: • substitutions in the protein targeted by the drug that decreases the affinity of the protein for the drug. mutations that modify nucleotide selectivity in viral polymerases belong to this group. substitutions may affect the neighborhood of the polymerase catalytic site, other polymerase domains, or even nonstructural proteins that interact with the polymerase. • if the inhibitor acts on a viral protein that itself has some other viral protein or genomic structure as a target, amino acid substitutions, or mutations that affect that target may also contribute to resistance. correlated mutations . molecular mechanisms of antiviral resistance in the first and second target may also yield the resistance phenotype. this is the case of some protease inhibitor-resistance mutations in hiv- . • in the case of viral polymerases, some mutations permit the excision of a chainterminating nucleotide at the -end of the primer. it is achieved through phosphorolysis mediated by a pyrophosphate donor, probably atp. resistance to nucleoside/nucleotide reverse transcriptase (rt) inhibitors (nrtis) is achieved by one of at least two mechanisms: (i) discrimination against the incorporation of the triphosphate form of the nrti and (ii) excision of the chain-terminating nucleotide once incorporated at the -end of the growing dna chain. this occurs with thymidine analog resistance mutation (or tams) that are typically selected under treatment with azt or d t [formulae ( ) and ( ) in fig. . ]. groups of amino acid substitutions may yield a multidrugresistance phenotype. a well-studied example in hiv- is the q m complex in the reverse transcriptase, which includes substitutions a v, v i, f l, f y, and q m. the phenotype consists of the limitation of incorporation of several nucleotide analogs. these and other mutants are characterized by a decrease in the catalytic rate constant (k pol ) of incorporation of the analog or analogs relative to standard nucleotides (see section . in chapter for the basic kinetic parameters for polymerase activity). the combination of enzymological and structural studies has provided a molecular interpretation of the mechanism of inhibition of hiv- by nrtis (reviews in men endez-arias, ; men endez-arias and alvarez, ; clutter et al., ; g€ unthard et al., ) ; for a predictive model that includes nucleotide levels, see von kleist et al. ( ) . nonnucleoside rt inhibitors (nnrtis) bind to an rt pocket Å away (a considerable distance) from the catalytic site composed of residues of the p and p subunits of the enzyme. the hydrophobic nature of nnrtis is illustrated in fig. . with the structures of nevirapine, efavirenz, and delavirdine [formulae ( ), ( ), and ( ), respectively]. several mechanisms have been proposed to explain their inhibitory activity, including alteration of the catalytic amino acids ymdd at the rt active site, distortion of the nucleotidebinding site, or modification of the position of the primer that receives the incoming nucleotides (men endez-arias, ). amino acid substitutions that confer resistance to nnrtis block the access of the inhibitors to their binding sites or alter the conformation and volume of the binding pocket. the essential viral proteases are a target for the development of specific antiviral inhibitors. examples are saquinavir and ritonavir for the hiv- [formulae ( ) and ( ), respectively, in fig. . ]. multiple resistance mutations have been described for protease inhibitors. they can affect the substrate-binding site or neighbor positions, often accompanied of compensatory substitutions at distant positions including sites of the target viral protein [e.g., the gag cleavage sites in hiv- (fun et al., ; flynn et al., ) ]. some hiv- protease inhibitor combinations display a high genetic barrier to resistance but, significantly for the capacity of viruses to explore sequence space, resistant mutants with e amino acid substitutions in the proteasecoding region have been isolated (rhee et al., ) . the reviews by men endez-arias ( ) and men endez-arias and alvarez ( ) provide excellent and detailed accounts of mechanisms of resistance of hiv- and hiv- to inhibitors that include virus entry and integrase inhibitors, in addition to the rt and protease inhibitor briefly described here. . quasispecies dynamics in disease prevention and control because of the distance effects that can be exerted among amino acids on the structure of proteins, substitutions that confer resistance to inhibitors of viral enzymes may lie far from the catalytic site. the effect of drug-resistance mutations on the general catalytic efficiency of a viral enzyme is one of the determinants of the functional barrier to resistance, since it may diminish replicative fitness. when an enzyme activity assay in vitro is available, the effects of specific drug-resistance amino acid substitutions on enzyme activity can be tested, although the observed alteration may not be the only influence on the fitness modification of the corresponding mutant virus. the reason is that most viral proteins (including viral enzymes) are multifunctional, and an enzyme activity assay may not capture the range of influences exerted by the enzyme. regarding direct-acting antiviral (daa) agents for hcv, the inhibitors that target the hcv polymerase (ns b) generally display a higher functional barrier to resistance than the protease inhibitors and manifest a broader genotype coverage. fitness decreases entail reductions in viral load and, consequently, lower probability of viral breakthrough (treatment failure). nucleotide analogs that bind to conserved residues at or near the active site of the viral polymerase tend to show subtypeindependent antiviral activity. because amino acid substitutions at or near the active site of viral enzymes are likely to inflict a fitness cost, such substitutions may not preexist in treatment-naive patients (margeridon-thermet and shafer, ; sarrazin and zeuzem, ) . interestingly, in the case of hiv- , mutations conferring resistance to rt inhibitors inflict a lower fitness cost than mutations that confer resistance to protease inhibitors (reviewed in martinez-picado and martinez, ) . thus, enzymes that perform similar functions for different viruses may have evolved to display different tolerance to amino acid substitutions. it is not possible to generalize which types of resistance mutations will display high or low functional barriers. there is a broad range of frequencies of antibody-and drug-escape mutants in viral populations, although values of À to À mutants per infectious unit are frequent for many rna and dna viruses (see table . in chapter for antibody-escape mutants). a few estimates for drug-escape mutants are listed in table . ; several observations and characterization of escape mutants have not been accompanied by frequency measurements. a point worth emphasizing is that quantification of viruses harboring biologically relevant mutations has been possible because an adequate biological assay is available. an antiviral agent or a neutralizing antibody measures the proportion of infectious viral particles that differ from the majority of the population in the relevant resistance trait. there is no reason to suspect that the viral amino acid residues (that are the target of an inhibitor or an antibody) that are substituted to confer the resistance phenotype are more prone to accept variations than many other amino acids in viral proteins. if we had additional selective agents to probe other viral sites, we expect a similar range of variant amino acids than using inhibitors or antibodies. this quite straightforward prediction is another way to state that there is general agreement in the mutation rates and frequencies for viruses being in the range of À to À substitutions per nucleotide (s/nt), calculated using a variety of biochemical and genetic methods (chapter ). a quite general observation is that in antibody neutralization experiments, a fraction of the virus population remains infectious despite the . molecular mechanisms of antiviral resistance addition of high antibody concentrations. although the resistance mechanism is unclear, a possibility is that the heterogeneous viral population includes a small proportion of antigenic variants with decreased affinity for antibodies. incomplete neutralization with the nonsigmoidal slope in the neutralization curves has been characterized for broadly neutralizing antibodies directed to hiv- (mccoy et al., ) . this general observation is an added complication to preventive designs that consider administration of neutralizing antibodies either as vaccine additives or in combination with antiviral inhibitors (section . in chapter ). the calculated frequencies of antibody-or drug-resistant mutants in viral populations may be lower than the rate at which they originate by mutation due to the fitness cost of the mutation (section . . ) . the argument is parallel to that used to justify why mutation rates and frequencies differ due to the fitness effects of mutations (chapter ). another prediction derived from the above considerations is that mutations conferring resistance to antiviral agents are expected to be detected in viral populations never exposed to the relevant drugs. all forms of genetic variation of viruses can contribute to dominance and spread of drugresistant mutants, including the combined effect of mutation, recombination, and genome segment reassortment (richman, ; neher and leitner, ; rogers et al., ) . the basal level of mutational pressure may be sufficient to provide a detectable proportion of escape mutants without the need for selection by the selective agent. this is an important aspect of antiviral therapy that is addressed next. the first demonstration that the baseline mutation level in viral quasispecies can include a detectable level of mutations that confer resistance to inhibitors in the absence of selection by the inhibitors, was obtained by d.d. ho, i. n ajera, c. l opez-galíndez, and their colleagues working with hiv- (mohri et al., ; n ajera et al., , . one of the studies examined the pol gene of hiv- genomes obtained directly from lymphocytes of infected patients. mutation frequencies for independent viral isolates were in eggers and tamm ( ) amantadine and rimantadine iv  À to  À measurements in cell culture appleyard ( ) , lubeck et al. ( ) rimantadine a iv % percentage of children treated with rimantadine that shed resistant iv belshe et al. ( ) disoxaril a hrv  À to  À low-level resistance in cell culture heinz et al. ( )  À high-level resistance in cell culture guanidine d pv .  À to  À measurements in cell culture pincus and wimmer ( ) a the formula of these drugs is included in figure . with the following number in parenthesis: amantadine ( ); rimantadine ( ); disoxaril ( ) the range of . . À to . . À s/nt, while for mutant spectrum components of individual isolates the values were . . À to . . À s/nt. in the virus from these patients, mutation frequencies at the codons for amino acids involved in antiretroviral resistance were very similar to the average mutation frequency for the entire pol gene. consistently with the mutation frequency values, several mutations that led to amino acid substitutions that conferred resistance to reverse transcriptase inhibitors were identified in patients not subjected to therapy. at the time of the study, the number of antiretroviral agents was still limited, and a considerable number of patients were not treated. the authors gave convincing epidemiological arguments that the background of mutations related to antiretroviral resistance was a consequence of high mutation rates and quasispecies dynamics, and not due to the transmission of resistant virus from individuals that had been subjected to therapy (primary resistance) (n ajera et al., ) . the presence of inhibitor-resistance mutations in viral populations never exposed to the corresponding inhibitor has been confirmed for hiv- and for several other viruses, including hcv, and it is supported by the calculated mutant frequencies in viral quasispecies (havlir et al., ; lech et al., ; ribeiro et al., ; ribeiro and bonhoeffer, ; cubero et al., ; johnson et al., ; toni et al., ; tsibris et al., ; peres-da-silva et al., ) . ample support has also come from deep sequencing analyses of mutant spectra, opening a point of debate on the basal frequency of inhibitor-resistance mutations that constitutes an indication to avoid the use of the corresponding inhibitors in therapy. at least in the case of hcv (and probably applicable to other viruses), there is no basis to suggest that the presence of a drug resistance mutation below a certain level in a patient will not have relevance for treatment failure when the inhibitor is administered. the understanding of quasispecies dynamics cautions against such reductionist arguments as evidenced by clinical cases (perales et al., ) . the data underline the relevance of mutant spectra as phenotypic reservoirs to confront selective constraints before constraints are in operation. for treatments, including a drug that has already been administered to a patient in the past, the influence of quasispecies memory should also be considered (section . . in chapter ). mutant spectra can be viewed as an anticipatory reservoir of phenotypes. in addition to the presence of drug resistance mutations in viral populations due to mutant frequency levels, the transmission of drug-resistant mutants from treated to na € ive patients may contribute to epidemiological relevance of resistance mutations. such primary resistance has been amply documented with hiv- , and it appears to increase in the case of hcv (franco et al., ; echeverría et al., ; huang et al., ) . higher levels of resistance mutations as a function of time in untreated patients is an indication that the mutations are not due to basal mutant frequencies but to the epidemiological expansion of virus mutants that originated in treated patients. from the clinical data available, the rate of expansion of virus harboring resistance mutations may vary depending on transmission and epidemiological features of each pathogen, but it seems unavoidable in the face of extended treatments for genetically variable pathogenic viruses. we confront a situation with parallels with antibiotic resistance in bacteria (chapter ). the major mechanism of drug resistance in viruses is based on amino acid substitutions that render the drug ineffective through the several molecular mechanisms summarized in section . . application of the cell culture system of hcv replication in human hepatoma cells (lindenbach et al., ; wakita et al., ; zhong et al., ) to examine the effects of . fitness or a fitness-associated trait as a multidrug-resistance mechanism long-term evolution has indicated that viral fitness can be an additional mechanism of drug resistance. the evidence was obtained when addressing the important issue of hcv resistance to interferon-alpha (ifn-a). ifn-a and ribavirin were the two components of the standard of care treatment against hcv infections until the advent of new therapies based on daa agents in . natural hcv isolates differ in ifn-a sensitivity, and the molecular basis of the difference is largely unknown. the study in cell culture consisted in subjecting a clonal population of hcv (termed hcvp , prepared by electroporation of hepatoma cells with rna encoding the viral genome, transcribed from a plasmid) to serial passages (of the type described in section . of chapter ) in the absence or presence of increasing concentrations of ifn-a added to the culture medium. several mutations scattered throughout the hcv genome were associated with ifn-a resistance (perales et al., ) . the selection of multiple alternative mutations is most likely influenced by the fact that ifn-a evokes a multicomponent antiviral response, which is not focused toward a single viral protein (perales et al., ) . unexpectedly, even the control hcv populations (those passaged times in the absence of ifn-a) displayed a partial (but statistically significant) resistance to ifn-a that could not be attributed to endogenous ifn production by the hepatoma cells (perales et al., ) . in view of this intriguing result, the initial hcvp population and the hcv population passaged and times in the absence of ifn-a (termed hcvp and hcvp , respectively) were tested for their resistance to other inhibitors of hcv replication: the protease inhibitor telaprevir [formula ( ) in fig. . ], the ns a inhibitor daclatasvir, the cellular protein cyclophilin a inhibitor cyclosporin a, the mutagenic purine nucleoside ribavirin, and the high barrier inhibitor sofosbuvir [formula ( ) in fig. . ]. hcvp and hcvp displayed significantly increased resistance to all inhibitors tested, as compared with the parental population hcvp gallego et al., ) (fig. . ) . passage of hcv entailed a -to -fold increase of viral fitness and a broadening of the mutant spectrum that might have increased the frequency of mutations associated with drug resistance, thus explaining the behavior of the multiply passaged hcv populations. the search for the resistant mutations was easier for telaprevir, daclatasvir, and cyclosporin a than for the other drugs because amino acid substitutions in the target protein had been previously identified as responsible for drug resistance. (in the case of cyclosporin a resistance, substitutions map in ns a and ns b, because the drug binds to cyclophilin a, which in turn interacts with ns a). analysis of the mutant spectra of hcvp and hcvp by molecular cloning and sanger sequencing and by deep sequencing failed to identify specific drugresistance mutations. since it could not be excluded that the broadening of the mutant spectrum might have increased the frequency of resistance mutations still to be characterized, two additional tests were performed. one was to determine the kinetics of viral production over a -fold range of moi in the absence and presence of telaprevir. both the unpassaged and multiply passaged hcv displayed parallel kinetics at the different mois, which excludes that drug resistance was due to the presence of resistance mutations in minority components of the mutant spectrum ( fig. . ). to further substantiate the findings, biological clones obtained by end-point dilution of the corresponding hcvp and hcvp populations were tested regarding drug resistance. a biological clone should have eliminated the minority genomes that harbored drug-resistance mutations since biological cloning is the most severe form of bottleneck event (sections . and . in chapter ). the biological clones did not display any decrease in drug resistance as compared . quasispecies dynamics in disease prevention and control moreno, e., gallego, i., pineiro, d., et al., . increased replicative fitness can lead to decreased drug sensitivity of hepatitis c virus. j. virol. , e , with permission from the american society for microbiology, washington dc, usa. . fitness or a fitness-associated trait as a multidrug-resistance mechanism with their corresponding parental, uncloned populations . the above observations have established viral fitness as a multidrug-resistance determinant in hcv that may also apply to other viruses. one possible molecular mechanism may consist of competition between replicative complexes and inhibitory molecules inside the infected cells. this model implies that fitness increase is reflected either in more replicating molecules per each replicative unit or in an increase in the number of replicative units per cell, without any influence on the number of inhibitor molecules that reach the replication sites. exploration of this competition model and alternative models and the extension to other viral-host systems are important challenges in the field of antiviral research. to sum up, mutant spectra and quasispecies dynamics can mediate antiviral resistance by at least two mechanisms: (i) by increases in the proportion of resistance mutations in the mutant spectra and (ii) by a fitness increase promoted by continued viral replication in the same environment. both mechanisms may act conjointly during viral infections in vivo. some studies with hcv have documented drugresistance phenotypes in infected patients, in the absence of specific drug-resistance mutations (sullivan et al., ; svarovskaia et al., ; sato et al., ; stross et al., ; di maio et al., ; dietz et al., ) . in fact, prolonged chronic hcv infections represent an adequate scenario for fitness increase due to extended rounds of infections in the same host liver. as a consequence, chronic infections may be prone to display fitness-associated multidrug-resistance phenotypes in the absence of drug-resistance mutations. the multiple mechanisms of drug resistance related to quasispecies dynamics justify even further the need for new antiviral strategies, as presented in chapter . high viral loads are predictors of disease progression. for hiv- and other lentiviruses, efficient early control of virus replication by the host immune response is generally associated with limited disease severity. the viral load that follows after the initial immune response to hiv- is referred to as the "viral set point." in the absence of early therapy, low set points in hiv- are generally attributed to a strong cellular immune response, likely influenced by additional host and viral factors. [this and other aspects of hiv- replication and pathogenesis have been reviewed in excellent monographs by levy ( levy ( , ]. a low set point predicts an asymptomatic outcome, and this is generally the case for viruses that establish persistent rather than acute infections. high viral fitness during the early stages of viral replication can promote disease manifestations. this was suggested by the progression toward the disease of a cohort of individuals that were infected during blood transfusion with an hiv- containing a large deletion in nef, an adaptor protein that mediates replication and pathogenesis (reviewed in arien and verhasselt, ) . after more than years, some of the infected individuals showed clinical signs, probably as a result of the accumulation of mutations in the hiv- genome that compensated for the lack of nef. more generally, fitness-decreasing (but not lethal) genetic lesions in a viral genome may be compensated by additional genomic mutations that become increasingly dominant in the course of further viral replication. the kinetics of fitness gain will depend on the nature of the lesion and the functional implications of the altered protein or genomic regulatory region (chapter ). fitness, replicative capacity, and viral load are directly interconnected parameters, and they . quasispecies dynamics in disease prevention and control affect disease progression (domingo et al., ) ( fig. . ). fitness gain will be more effective with a high load of actively replicating virus in the infected organism. elevated replicative capacity and fitness sustain high viral loads. the reason for this basic feature of viral population dynamics is that given a basal mutation rate, a large number of replicating genomes entails a correspondingly higher probability that a required mutation for fitness gain can be produced. the events involved are a specific case of search for adaptive mutations in terms of exploration of sequence space, as discussed in section . of chapter . while active viral replication, high load, and high fitness favor progression of the infection and disease manifestations, the fourth parameter included in the large arrow of fig. . , mutant spectrum diversity, has an optimal range. too low or too high intrapopulation diversity is detrimental to virus adaptability. insufficient diversity limits adaptability to complex environments (pfeiffer and kirkegaard, ; vignuzzi et al., ) , while excess diversity may lead the virus to cross an extinction threshold, and this is the basis of lethal mutagenesis as an antiviral therapy (chapter ). an additional implication of the parameters shown in fig. . for antiviral interventions is that fitness decrease is recognized as an alternative to inhibition of viral replication to control viral infections[ (clementi, ; clementi and lazzarin, ) ; reviewed in (domingo et al. ) ]. thus, key features of quasispecies dynamics have a direct implication on the management of viral infections. external interventions that have been applied or have been envisaged to limit or suppress virus infection include not only vaccination and administration of antiviral agents as described in previous sections, but also passive immunotherapy, antisense rnas, or oligonucleotides with various chemical modifications, interfering rnas, ribozymes, or their combinations. biotechnological developments have favored the design of chemically defined vaccines (consisting of expressed immunogenic proteins, synthetic peptides, or peptide arrays), without the need to handle or administer live virus. one of the most na € ive manifestations of trust in biotechnology in the middle of the th century was the belief that a catalog of plasmids encoding the antigenic proteins of the circulating types of pathogenic viruses would suffice to prepare the required vaccine as needed. concerning influenza vaccines, w.i. beveridge wrote the following: "the first objective would be to capture the full range of influenza a subtypes. their antigens would be studied by specialists at central laboratories and made available for the preparation of particular vaccines if and when required. it might be feasible to stockpile some vaccine against all the principal hemagglutinin antigens to be used in a figure . a schematic representation of interconnected parameters of viral replication that often relate to disease progression. an understanding of quasispecies dynamics has made it evident that the aim of antiviral therapy need be not only to directly diminish the viral load but also to affect other parameters that can then reduce the viral load. see text for justification and references and chapter for new antiviral strategies that follow the concept expressed in this figure. . limitations of simplified reagents and small molecules as antiviral agents fire brigade type of action as soon as an incipient pandemic is spotted" (beveridge, ) . naivety is also perceived in current designs of universal vaccines based on conserved antigens, considering the different escape mechanisms that operate in viruses to elude neutralization by antibodies (chai et al., ) . it is remarkable how our present understanding of viral populations renders obsolete the views expressed in the w.i. beveridge book, and in other writings at the boom of implementation of dna recombinant techniques. yet, attempts to produce vaccines against highly variable viruses, based on antigenic structures or some viral isolates, are ongoing (christiansen et al., ) . a critical issue is if the host immune response against a vaccine engineered with "universal" (conserved) antigenic motifs will be sufficient to prevent disease upon infection by other forms of the same pathogen (freeman and cox, ) . from our current knowledge of viral population dynamics, it seems unlikely but time will tell, since efforts toward the manufacturing of universal antiviral vaccines are under way. with a conceptual similarity to vaccines, in medical practice, monotherapy with an antiviral agent was traditionally preferred over drug mixtures. (the change of paradigm was largely a consequence of the aids epidemic, and it was publicly expressed by the pioneer hepatologist s. sherlock in a summary address of an international symposium on viral hepatitis held in madrid in ). the change of perspective is clear. some antiviral strategies, such as antisense nucleic acids or virus-directed ribozymes, were intensely investigated decades ago. it is unlikely that when used in isolation, they can be converted into useful antiviral therapies because resistant mutants are likely to be selected. yet, they could be part of combinations with other antiviral inhibitors to provide a larger antiviral barrier (chapter ). a similar fate is likely for interfering rnas (boden et al., ; gitlin et al., ; herrera-carrillo and berkhout, ; mcdonagh et al., ) . to a large extent, the failures of defined chemical entities (oligonucleotides, ribozymes, small molecule inhibitors, etc.) to control virus replication and spread are a consequence of their targeting a very defined viral genomic sequence combined with the adaptability of viral populations. combination of such multiple elements have been envisaged and tested, but off-target effects and the adaptive potential of viruses are likely to limit their efficacy. unfortunately, biotechnological developments that have been so positive for many research areas and practical applications tend to simplify the types of agents to prevent disease or inhibit virus replication, ignoring the inherent complexity of the object to be controlled. success is unlikely when "complexity" is combated with "simplicity." an increased understanding of viral population dynamics over the last decades has changed the picture dramatically by providing an interpretation of "virus escape" as a general and largely unavoidable phenomenon. such awareness has pushed the development of new antiviral designs, which are fundamentally centered in two strategies: a combination of multiple, independently acting elements or fitness decrease through excess mutations (chapter ). a pronouncement by p. ehrlich at the international congress of medicine held in london in , reflected old traditions on how to treat microbial infections ["here, therefore, the old therapeutic motto is applicable: frapper fort et frapper vitte" and "therefore, it is in my opinion necessary to allow the therapeutic treatment to come into action as early as possible, ..."; sentences taken from (ehrlich, ) , with emphases as in the original text]. this pronouncement fits our current understanding of viral populations. following ehrlich, the full implications of quasispecies-mediated adaptation of viruses for . quasispecies dynamics in disease prevention and control antiviral therapy were expressed by d.d. ho in an influential article entitled "time to hit hiv, early and hard" (ho, ) . the article title captures what is needed to prevent adaptation of a virus in the infected host. any opportunity to replicate is exploited by the virus to increase its fitness and to become less vulnerable to internal (intrahost) or external interventions such as antiviral therapy. treatment interruptions during chronic infections, such as "drug holidays" that in the case of hiv- -infected patients were justified to alleviate side effects associated with administration of antiretroviral agents, provided an opportunity for the virus to gain fitness. in principle, given our current understanding of viral quasispecies dynamics, the proposal of p. ehrlich and d.d. ho is applicable to other viral pathogens. one argument that tones down the strength of the "hit early and hit hard" proposal is that some infected patients may not progress to disease, but maintain an asymptomatic lifelong persistent infection. this is the case with elite controllers in the case of hiv- infection, and individuals infected with hcv who will not progress toward liver disease. in cases in which such nonprogression can be anticipated by viral and host parameters, it may be justified to exclude some patients from aggressive interventions (suthar and harries, ; casado et al., ) . as a general rule, however, the potential benefits of early treatment are obvious not only to avoid disease on an individual basis, but also to diminish the chances of virus transmission (reviewed in suthar and harries, ; see also sections . and . in chapter regarding the relevance of viral population numbers in transmission). restricting the number of treated patients for economic reasons will result in more expensive public health interventions when infected individuals develop the disease. box . includes recommendations for the use of antiviral agents and recapitulates concepts explained in this and preceding sections. despite the emphasis on evolutionary aspects, prevention and treatment of viral disease have many other angles some of which were box . • avoid monotherapy. ideally, use two or more antiviral agents which do not share a mechanism of action ( fig. . ). • treat as soon as possible after virus diagnosis, to avoid virus adaptability associated with high virus population size and to minimize transmission of inhibitor-resistant mutants. • individual patients should be treated only during the time at which the drug proves effective. when viral load rebounds, treatment should be discontinued. • use deep sequencing methodology to determine mutant spectrum composition for an adequate choice of inhibitors. the aim is to design personalized treatments that consider the probability of drug combination efficacy with minimal side effects. • consider temporary shelving of effective drugs when resistant mutants acquire epidemiological relevance. considered in chapter in connection with factors of disease emergence (smolinski et al., ) . three of them should be mentioned here because they are as important as the adequate treatment designs described in this chapter: (i) adequacy of public health measures, (ii) public information about virus sources and means of contagion and (iii) need of global political action. information to the public should aim at limiting the spread of disease that is, undertaking personal and collective actions to reduce the r value for a given virus (chapter , section . ). as an illustration of this key point, there was a quite extensive information campaign on hiv- and aids in developed countries during the early decades of hiv- spread, while the information about other potentially threatening viruses such as ebola or the severe acute or the middle east respiratory syndrome (sars and mers, respectively) coronaviruses was more limited. the need of a global response to limit the extension of disease episodes at the sites where they are initiated has been recognized for a long time, but it became obvious with the e west african ebola epidemic (see siedner et al., ) . there is a need for international organizations and governments of developed countries to provide the health-care workforce to assist low-and middle-income countries to control viral episodes at an early stage. "help early, help effectively" is the recognized need at a global scale, which is the parallel to "hit early, hit hard" for the treatment of infected patients. global early action and adequate information can be as important as an adequate treatment design to control viral disease. it can restrict viral replication rounds and consequent adaptability. information is thought to have been critical for the control of ebola epidemic in nigeria (siedner et al., ) . however, information must also be planned to reach the target population in a convincing manner, as learned from the poliovirus vaccination and eradication campaign (renne, ) . the uncertainties regarding whether an initial, limited episode of viral disease will expand or die out do not help in decision making. however, the best choice in the case of emerging and reemerging infections is to act assuming the worst scenario. medical interventions represent a totally new set of selective constraints that viruses are facing only since decades ago, an infinitesimal time of their existence as biological entities. however, the evolutionary mechanisms available to viruses have successfully coped with many selective pressures, notably the effect of vaccination when a broad immune response is not evoked, or treatment with antiviral agents. a common way to proceed is to test a new vaccine with an animal host, be it the authentic host or an animal model, and obtain full protection when the animal is challenged with a virus that matches the antigenic composition of the vaccine. following the initial excitement, very often the vaccine displays only partial protection when tested in the natural environment. somewhat parallel arguments can be made about clinical trials for antiviral agents, usually performed initially with selected groups of patients. clinicians have coined the term "reallife" treatment studies when patients are not selected for optimal results (often pushed by commercial and political interests). this chapter has emphasized how the complexity of viral populations is a serious (often underestimated) difficulty to prevent and treat viral disease. examination of the molecular mechanisms exploited by viruses to survive despite antiviral interventions suggests two major lines of action: first, more judicious use of existing tools that should consider the complexity of viral populations and their dynamics; complexity . quasispecies dynamics in disease prevention and control cannot be combated with simplicity. second, the need to design new antiviral strategies, a topic addressed in chapter . several interconnected parameters determine the probability of success of an antiviral intervention. most of them follow from the general concepts of darwinian evolution explained in preceding chapters. it is important, however, to quantify as much as possible the evolutionary events that determine therapy success or failure. for this reason, the importance of viral population size, basic probability calculations of developing resistance, and the selective strength of mutations, have been explained with numerical examples. hopefully, these simple quantifications will permit a higher awareness of when and why treatment may succeed or fail. we live in a very unequal society. the chapter closes with the recognition that there are many social economic issues that are as important as scientific planning to combat the pathogenic viruses around us (see summary box). • medical interventions represent a new class of selective constraints acting on viral populations. • viral evolution affects antiviral preventive and treatment strategies in two different ways: (i) through the molecular mechanisms of short-term response in treated individuals that select escape mutants, and (ii) through the epidemiological impact of viruses that have acquired escape mutations. • ineffective vaccines can contribute to the selection of antigenic viral variants. • selection of viral mutants resistant to 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resistance selection production of infectious hepatitis c virus in tissue culture from a cloned viral genome emergence of virus escape mutants after immunization with epitope vaccine genetic evolution of classical swine fever virus under immune environments conditioned by genotype -based modified live virus vaccine robust hepatitis c virus infection in vitro key: cord- -zwvr a authors: mukherjee, moumita; goswami, srikanta title: global cataloguing of variations in untranslated regions of viral genome and prediction of key host rna binding protein-microrna interactions modulating genome stability in sars-cov- date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: zwvr a background: the world is going through the critical phase of covid- pandemic, caused by human coronavirus, sars-cov- . worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. however, there have not been many studies focusing on the variations in the ’ and ’ untranslated regions and their consequences. considering the possible importance of these regions in host mediated regulation of viral rna genome, we wanted to explore the phenomenon. methods: to have an idea of the global changes in ’ and ’-utr sequences, we downloaded complete and high-coverage sars-cov- genome sequence information from human host in fasta format from global initiative on sharing all influenza data (gisaid) from different geographical regions. next, we aligned them using clustal omega software and investigated the utr variants. we also looked at the putative host rna binding protein (rbp) and microrna binding sites in these regions by ‘rbpmap’ and ‘rna v ’ respectively. expression status of selected rbps and micrornas were checked in lungs tissue. results: we identified unique variants in sars-cov- utr region based on a minimum variant percentage cut-off of . . along with c>t change the important ’-utr change identified was a>g, while g>c, g>a/t and c>t were the most familiar variants of ’utr among most of the continents. furthermore, we found that despite the variations in the utr regions, binding of host rbp to them remains mostly unaltered, which further influenced the functioning of specific mirnas. conclusion: our results, shows for the first time in sars-cov- infection, a possible cross-talk between host rbps-mirnas and viral utr variants, which ultimately could explain the mechanism of escaping host rna decay machinery by the virus. the knowledge might be helpful in developing anti-viral compounds in future. outbreak of novel coronavirus sars-cov- has been proclaimed as a pandemic by the world health organization (who). from the first report of infection in wuhan, china on december , , the virus has infected . m people worldwide till th june, . sars-cov- is an enveloped, positive-sense, single stranded rna virus of genus beta-coronavirus (β-covs) with the entire genome size of approximately kb. this viral genome is composed of about open reading frames (orf) which encodes both structural and non-structural proteins having a role in their transmission, survival and pathogenesis [ ] . the main structural proteins translated from sub-genomic mrnas include spike glycoprotein (s), envelope protein (e), membrane glycoprotein (m) and nucleocapsid protein (n) along with non-structural proteins (nsp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the genomic rna element of sars-cov- also includes '-untranslated region ( -utr) of bp length with a methylated cap and a ' polyadenylated utr of length bp, according to the reference sequence from wuhan. low fidelity of the rna polymerase makes the rna viruses prone to high frequency mutation and the mutation determines the virus evolution [ ] . systemic mutation analysis of the viral genome revealed that the virus had mutated several times in spatio-temporal variation and has evolved into numerous strains [ ] . this diversity of rna strains might be correlated to severity and mortality seen in covid- (corona virus disease- ). a recent phylogenetic network analysis on complete sars-cov- genome reported that the virus appeared to evolve in three distinct clusters a, b and c, with a being the ancestral type. type a and c seemed to be more prevalent in americas and europe, whereas type b was dominant in eastern asia [ ] . during viral replication, sub-genomic mrnas are synthesized by a process of discontinuous transcription with a common -untranslated leader sequence [ '-utr] and a '-noncoding co-termini [ 'utr] , identical to the viral genome [ ] . hence, highly structured ' utr of positive-strand rna viruses is indispensable control element in replication, transcription, and translation of rna viruses along with the ' utr [ ] . extent of structural and functional conservation in the '-terminal of genomic rna of different species of genus coronavirus has been found to a distinctive magnitude [ ] . these terminal untranslated regions are thus substantial site for rna-rna interaction and binding of viral and host cellular proteins for rna replication and translation [ ] . molecular evolution in the untranslated region i.e. the variation in the utr region leads the virus to evolve to a great extent. there are many studies which have considered the mutation in the coding region with respect to geographical location. here, we have enumerated the variants in the '-and '-utr regions of sars-cov- genome. we have studied a total of viral sequence samples worldwide for this purpose and found that there are some rare variants of utr that are distributed over a global spectrum, while some variants are specifically present in a population at a comparatively higher frequency. this drove us to make a systematic catalogue of the utr variants across six continents of the world that could have a role in emergence of different strains of sars-cov- . we have also looked at the possible regulation of viral genomic rna through binding of host rna binding proteins (rbps) and mir-nas in specific sequences of the viral utrs. there are experimentally validated evidences of human rbps binding to the regulated signals within the untranslated region of sars-cov rna in order to control the viral rna synthesis and turnover. polypyrimidine tract-binding protein (ptb) is found to bind to the leader sequence and hnrnpa , hnrnpq is bound to the 'utr of beta-coronavirus mhv. similarly, a host protein, madp (zinc finger cchctype and rna binding motif ) interacts with the -utr of sars-cov, influencing the rna synthesis machinery [ ] . likewise, there are reports of mirna binding to the viral genome, both in the coding and non-coding regions [ ] . as the mirna mediated decay of mrna is observed mainly through the 'utr of the gene in mammalian system, we are also interested to explore the host mirna-mediated regulation of the viral genome through the '-utr of sars-cov- via mrna decay and translational repression. interaction and/ or competition between rbp and mirna-risc is also well studied in mammals [ ] and investigation of this interplay of host rbp and mirna in the untranslated region of virus may delineate the role of utrs in sars-cov- virulence and survival and how variation in the utr can have an impact in the overall regulation. we have downloaded complete and high-coverage sars-cov- genome sequence data in fasta format from global initiative on sharing all influenza data (gisaid), which is a public repository of all influenza virus sequence. our data involved only the human-host specific viral sequence covering six continents of the world: asia, europe, oceania, north america, south america and africa. from asia, we have taken the viral sequence data from china (n = ), india (n = ), japan (n = ), thailand (n = ) and taiwan (n = ). from europe, the data is taken from italy (n = ), spain (n = ), england (n = ), russia (n = ) and germany (n = ). viral sequence data is taken only from australia (n = ), on behalf of oceania. representative countries from north america include usa (n = ) and canada (n = ). there are no further divisions for south america (n = ) and africa (n = ). initially the plan was to retrieve viral sequences collected in a span of one month, just after an interval of two month from the date of first report in the corresponding place, in order to catch the diversity in the viral sequence in that region. but, unfortunately, due to less number of sequence deposition in some countries, we had to deviate from the plan and retrieve all the available sequences till may , for them ( table ) . we have downloaded the sequences on th may, . the reference sequence from wuhan was taken as our reference from ncbi (nc_ . ). all the country specific sequences were aligned with the reference sequence separately using multiple sequence alignment tool clustal omega (embl-ebi). as we were interested only in the '-and '-untranslated region (utr) and these positions are located at the two extreme ends of the sequences, there is a chance of lower coverage and higher error-rate. to resolve this ambiguity, we have put a high filtering cut-off of at least % coverage in a particular location of that area. all the alignment files have been shared in 'figshare' and can be accessed using the link: https://figshare.com/articles/supplementary_data_alignment_files/ . for prediction of human rbps bound to the utr of a viral genome, we have used the webtool 'rbpmap' that enabled prediction of rbp binding on genome sequences from a huge list of experimentally validated motifs of rbpmap database. weighted-rank algorithm was used for mapping the motifs [ ] . we have put the reference and mutated '-and '-utr sequences separately as query sequence with the in-built human rbp motifs of the database. from the output, we have selected only the predicted rbps with high z-score and p-value. putative human mirna binding sites on viral utr was assessed using the 'rna v micro-rna target detection' tool [ ] . as a target sequence input, we provided the reference and mutated '-utr sequences of virus genome. for input mirna sequences, we have obtained a list of all annotated human mirnas with their sequences in fasta format from mirbase database [ ] . for all other criteria to be used for the prediction was given as per the default settings and additionally homology with seed sequence and its complementary target sequence were manually examined in the output file of interaction. expression of specific genes was obtained from 'the protein atlas' respective to normal lung tissue. rna-seq data from lung tissue generated by the genotype-tissue expression (gtex) project was reported as mean ptpm (protein-coding transcripts per million), corresponding to mean values of the different individual samples from each tissue. hpa rna-seq data from lung tissue was reported as mean ptpm (protein-coding transcripts per million), corresponding to mean values of the different individual samples from each tissue. tissue data for rna expression also obtained through cap analysis of gene expression (cage) generated by the fantom project, reported as scaled transcripts per million. gepia [ ] web-tool was used to obtain comparative expression of specific genes in lung adenocarcinoma (luad) and normal lungs. sequence alignment of over virus samples collected from over geographical locations with the sars-cov- reference genome (nc_ . ) has yielded a total of variants in the ' utr and variants in the 'utr. among them, unique variants have been summarized in the s table based on a minimum variant percentage of . . but for the variants that are present in at least four populations, no such variant percentage cut off was taken. the most common high frequency variant is the c>t, which lies in the leader sequence of the sars-cov- viral genome. china, being the ancestral domicile of the virus has a very low percentage of this variant (fig ) . europe has the maximum frequency of this variant (fig ) . other than china, other countries in asia also have a lower percentage of this variant (fig ) . thus, we can a see a clear effect of regional variation on the frequency of this variant. another study reported that this variant has co-evolved with three coding region mutations ( c > t, c > t, and a > g) of nsp , rna primase and spike glycoprotein. they have also related this mutation with the increased transmissibility of the virus [ ] . another common variation in the 'utr is a>g, that is present in seven of our populations being most prevalent in italy and canada. g>a/t and c>t are the most familiar variants of 'utr among all the continents. variant has two alternative alleles with g>a being more prevalent in india, usa and africa, whereas g>t variant is more dominant in thailand, taiwan, germany, australia and canada. china and england has both the variants of almost similar percentage. hence, this variant seems not be associated with any specific region. another important variant of 'utr is g>c, which is mostly seen in europe and australia, but italy has a quite high percentage of this variant than others (fig ) . most of the countries in this study seem to have a moderate to significant variation in the 'utr of the viral genome. furthermore, we have explored the relationship of these variants with the secondary structure of sars-cov- viral rna. 'utr variant a>g fall within the sl a stem-loop and c>t variant was within sl b stem-loop. the above mentioned three 'utr variants were also within the bulge portion of the stem-loop of viral rna secondary structure. wild type nucleotides of all these variants had high shape-reactivity value which suggested that these nucleotides were less likely to form base-pair and hence had an important role in affecting the secondary structure [ , ] . all the populations and sub-populations used in this study have a definite pattern of utr variants of their own (figs ; ) . even countries in a single continent differ in their utr variation. some variations are merely specific to a particular location with a quite considerable percentage. the overall sequence of 'utr of sars-cov- in italy is exceptionally variable, whereas the 'utr is substantially stable with a single variant in the position (g>t). but england, also being a part of europe, has a notable number of variations in the 'utr having a relatively stable '-end. from our analysis, it is evident that russia had no such frequent variation in the utr, except the c>t variant which was present in % of the population. india had two striking variations in the 'utr, a>t, g>t, which was found nowhere in this global mutational landscape (s table) . all the sequences under this study with these two variants are from indian state of maharashtra, which has the highest number of sars-cov- infected people within india. most interestingly, both of these variants occur simultaneously in majority of the cases. similarly, south america also had five unique mutations in the positions g>t, g>c, t>c, c>t and a>g of 'utr. rna binding proteins belong to a class of proteins which bind to target rna molecules through characteristic binding motifs and perform a variety of functions. in fact, irrespective of the type of rnas, whether is coding or non-coding, specific rbps get associated with rnas right after their birth and subsequently control the processing, stability, transport, translation, regulatory function and turnover. as mentioned earlier, exogeneous viral rna will also encounter host rna decay machinery when released into the cell and there are ample evidences that they attempt to escape the process in order to maintain their successful propagation within the host. one way to take care of the situation is to recruit host rna stabilization factors into the regulatory region, i.e. ' and '-utrs [ ] . table lists the predicted host rbps interacting with the '-utr of virus sars-cov- as obtained using 'rbpmap'. in the next step, the logical exploration was to find out whether the most prevalent variations identified in the '-utr of the viral rna genome could alter binding of any of these rbps. the 'a' to 'g' change at position coincided with the binding site of cug-bp (also known as celf ) (fig a) , implicating that the variation might have some effect on cug-bp binding. in order to test that, we used the mutated sequence as 'input' and found that the single nucleotide change didn't have any effect on binding of cug-bp to its target sequence. in other words, no matter whether the virus has 'a' or 'g' at position of its '-utr, cug-bp binds and probably performs its function. then, we focused on the most important change in the '-utr, the position at . looking at the distribution of this variation and the reports related to its association with virulence [ ] , we had a belief that it might be giving some selective advantage to the virus. however, we didn't find any rbp binding site overlapping with this region. we changed the nucleotide at position from 'c' to 't' (for corresponding 'u' in rna) and used the mutated sequence in rbpmap. to our surprise, we found a tardbp binding site created upon this change ( fig b) . tardbp (also called tdp- ) is a well characterized rbp which binds to specific 'ug' (and 'tg') rich sequences of rna (and dna) and reported to facilitate translation when bound to '-utr [ ] . from protein data bank (pdb) we retrieved the co-crystal structure of tardbp with single strand dna (fig c & d) which indicated strong binding of the protein to that particular nucleic acid stretch. hence, our finding showed strong binding of tardbp to viral '-utr having 'u' at position . this could be implicated in facilitating translation of viral proteins resulting in its effective propagation within human host. following the same principle and considering the fact that '-utr is the most important site for regulation of rna stability and turnover, we also wanted to find out what are the rbps interacting with this region. the '-utr reference sequence of sars-cov- was fed into rbpmap and we obtained a list of '-utr binding rbps selected on the basis of p-value and z-score ( table ) . as with '-utr, here also we focused on the major variations in '-utr to find out if specific nucleotide change could interfere with the binding of specific rbps. we found that '-utr variations at positions and could affect interaction of srsf and hnrnpa respectively. however, the regulation at '-utr is not that simple. another important factor which needs be taken into account is whether there are host mirna binding sites present within the viral '-utr region and how the combinatorial interaction of host rbps and mirnas could dictate the viral rna stability. in order to identify whether there are any host mirna binding site present in viral '-utr and if so, what they are, we used the viral '-utr sequence as 'input' in the mirna prediction software 'rna v '. the algorithm predicts several of such mirnas and we selected them based on folding energy for heteroduplex formation and p-value ( table ) . as the most important factor for the functional mirna-target rna interaction is to have the perfect homology between the seed sequence of mirna ( nd to th nucleotide from ' end) and the corresponding target rna sequence, we also explored the fact in the identified pairs. furthermore, whether the changes caused by the common variations interfere with the seed sequence https://doi.org/ . /journal.pone. .g binding was also investigated. the interesting part in this scenario is that the reference sequence at a particular position causes a mismatch in the seed resulting in inefficient binding of a mirna, but the variant base at that position creates a perfect binding site. however, considering the complexity of the region, all these probabilities could not be assessed only in terms of mirnas and hence we set out to probe into the interaction between the host rbp-mirnas and viral sequence variants in '-utr region in a comprehensive manner. we investigated the interaction in a stepwise manner. the first situation explored was the case where host mirna mir- b- p targets viral rna and no mutation has been identified in the 'seed'-corresponding region. this means that all the viral sub-types will be vulnerable to degradation by this mirna, provided it is expressed in host lungs tissue. careful scanning of the binding site and also looking at the rbp binding information we identified a rbms target sequence actually overlapping with the mir- b- p seed corresponding region (fig a) . this clearly indicated a prevalent competition between rbms and mir- b- p-risc complex for their respective binding site, with one of them simply presenting a steric hindrance to the other one by blocking the access. this is quite common scenario in regulation of mammalian mrna stability where the final verdict whether the mrna is degraded or stabilized largely depends on the spatio-temporal expression or the availability of both the molecules (rbp and mirna). second scenario was the variation in position . in this case, we identified that change from 'c' to 'a' disrupted the binding of mir- - p to its corresponding target sequence at the 'seed' region (fig b) . this is clearly an advantage to the virus as the variation would help it to get rid of the mirna mediated degradation. as this overlapping region also had a binding site for rbp hnrnpa , we wanted to find out what happens to hnrnpa binding when there is a change from 'c' to 'a'. interestingly enough, we discovered that hnrnpa binding is also not affected, implicating an ensured protection of viral rna by rbp hnrnpa , no matter whether mir- - p is functional or not. again, this situation is also quite common in mammalian mrna regulation where we see some amount of degeneracy among the rbp target sequences, when change of a single nucleotide is often tolerated. the virus uses this phenomenon to its benefit where in spite of utr sequence variation, protection from same rbp is ensured. in case of the third incidence, we looked at position , where the 'g' to 'a' change creates a binding site for mir- - p (fig c) , which means that the virus having the variant nucleotide will be prone to degradation by this mirna. we had already commented about the possible binding of srsf in this region and found that the variation under investigation had no impact on srsf binding. again, it indicated that despite creation of mir- - p binding site, the viral rna is safe until srsf is available to bind and compete with mir- - p-risc complex. however, as evident from our '-utr analysis, a significant proportion of viral sequence had 'g' to 't' change at position . while srsf binding site was still retained, the mir- - p site creation did not take place due to this change. the fourth scenario was found to be very different from what have been mentioned so far. we identified that change of 'g' to 'c' at position creates a binding site for mir- - p, but no rbp with a overlapping target sequence could be identified (fig d) . this means that there is probably no host factor to protect viral rna from mir- - p mediated the phenomenon of viral rna utilizing host stabilizing factors and escaping rna decay machinery is well-established. but, nothing is known so far for sars-cov- . as mentioned before, the actual incident what is taking place after viral infection largely depends on the expression status of the interacting rbps and mirnas in particular host tissue. since, lungs is the primary site of infection for sars-cov- , we wanted to find out what is the nature of expression of these molecules in normal lungs tissue. in the first approach we tested their expression in 'the protein atlas' portal (fig a and b ) and in the second approach we used the tcga dataset for lung adenocarcinoma (luad) from gepia and compared the expression of the selected rbps in adjacent normal lung tissue (fig c and d ). to our excitement, we found a decent to huge expression of all the rbps in normal lung tissues, clearly indicating their availability to protect viral rna from host decay machinery. as the viral infection will follow a huge inflammatory condition in the lungs and by the time we know that in severe cases this prolonged inflammation leads to pulmonary fibrosis, we also wanted to check whether there are supporting evidences relating the expression of these rbps and mirnas to inflammation or more specific to pulmonary inflammation. as shown in table , almost all the rbps under investigation were either reported to have induced by inflammation or responsible for fibrosis in lungs or other organ. expression of some of them was also reported to be modulated by viral infection. this finding supports our hypothesis and indicates that from the stages of early viral infection to severe advanced conditions sars-cov- could utilize the host stabilization factors for its own benefit. following the same tune, we looked at the mirnas also in greater details and several interesting observations were noted. apart from mir- - p, other three mirnas were detectable in lungs tissue and even were closely associated with pulmonary inflammation. in case of mir- - p also, its involvement in breast cancer tumorigenesis and associated inflammation was quite supportive. most of these supporting studies mentioned here for both rbps and mirnas are actually functional interrogations involving rigorous experimental procedures. hence, we are quite confident that that our predictions got validated by experimental reports asking the similar type of questions in a similar set up. it has been more than six months we are experiencing one of the biggest pandemics of the world. investigation of the viral subtypes across the globe has identified several variants in the coding region of the viral rna possibly resulting in key structural or functional changes in spike protein, nucleocapsid proteins or the rna dependent rna polymerase of the virus. as compared to the coding region variants, there have not been so many changes identified in the ' or ' untranslated regions of the viral genome. this made us interested about these regions and first we wanted to carry out a systematic exploration of the prevalent changes present in the region using more than viral sequences isolated from all the continents and explore their significance with respect to host rna decay machinery as well. in spite of some specific changes concentrated in some specific regions of the world, overall pattern of the ' and '-utr variants pinpoint on few predominant ones. the most important of it was at position , where we see a clear change of distribution of reference to variant nucleotide from china to south-east asia to europe and americas. this change has also been implicated to higher mortality and/ or infectivity of the virus [ ] . for the first time, we report the likelihood of tardbp binding to this region. tardbp has been known to promote translation and rna stability and it has also been shown to play important role in viral infection [ , ] . therefore, promotion of translation of sars-cov- viral proteins by tardbp fits well with possible selection of 't' base over time and its correlation to infectivity. the other rna binding proteins identified in the study to be interacting with viral ' and '-utr also has reported evidences of functioning as rna stabilizing factor or facilitator of translation. cug-bp has been shown to interact with eif a [ ] and promote translation in selected targets [ ] . rbms is a very well-characterized rna stabilizing factor and known to increase half-life of target mrnas as well as increase protein expression in many instances [ ] . similarly, hnrnpa is also a rbp known for its role in promoting mrna stability after binding to '-utr specific target sites and as mentioned before, this rbp has been experimentally identified to bind to sars-cov '-utr [ , ] . srsf , although primarily involved in splicing, has been attributed to functions like maintaining mrna stability and translation [ , ] . the most significant aspect of this study was to identify mirna binding site in the viral '-utr and deciphering the cross-talk between rbps and mirnas along with the nucleotide variations at specific sites. our results elaborated five distinct types of such interactions, as summarized in fig . we have seen cug-bp binding site is retained and tardbp binding site is created upon change in viral nucleotide sequences at target positions of '-utr, providing a continued stability and translational advantage to the virus (shown in arm:a). on the other hand, there exists multiple possibilities in case of '-utr, where we first see direct competition over access to target sites between rbms and mir- b- p (arm:b). lung inflammation causes induction of mir- b- p [ ] which could be a step to attenuate viral infection. however, the sustained expression of rbms in lungs tissue probably acts to prevent mir- b- p action after binding to the viral rna at overlapping site. the effect of variation in viral sequence could either disrupt or create mirna binding site, keeping the rbp binding site intact. this result in non-functional mirna site in one case (arm:c) and competition between rbp and mirna in the other (arm: d). expression of both hnrnpa and srsf is high in lungs, further supporting their possible protective effect on viral genome. the incidence described in arm: e is different as it creates a mirna binding site without an rbp protection. we have seen that expression of mir- - p is less in lungs, provoking the thought that despite having its site created, there might not be enough mirna to act on the viral rna at this site. thereby, we have characterized sars-cov- ' and '-utr sequences for existing variations in these regions prevailing at major countries of infection spread over all the continents. we further identified interactions between host rbps like cug-bp, tardbp, rbms , hnrnpa and srsf with host mirnas mir- b- p, mir- - p, mir- - p and mir- - p and showed how the interactions changed along with sequence variations at specific positions of untranslated regions of viral genome. our findings elaborate complex relationship between host rna stabilization/ decay factors and sars-cov- rna genome highlighting how the virus could manipulate host machinery. the knowledge could also be used to develop antiviral compounds following further experimental studies. supporting information s table. list of variations in ' and '-utr of sars-cov- across six continents of the world. (xlsx) severe acute respiratory syndrome coronavirus (sars-cov- ): an overview of viral structure and host response. diabetes & metabolic syndrome specific mutations in sars-cov rna dependent rna polymerase and helicase alter protein structure, dynamics and thus function: effect on viral rna replication phylogenetic clustering of the indian sars-cov- genomes reveals the presence of distinct clades of viral haplotypes among states phylogenetic network analysis of sars-cov- genomes coronavirus subgenomic minus-strand rnas and the potential for mrna replicons a phylogenetically conserved hairpin-type ' untranslated region pseudoknot functions in coronavirus rna replication structural and functional conservation of cis-acting rna elements in coronavirus '-terminal genome regions binding of the '-untranslated region of coronavirus rna to zinc finger cchc-type and rna-binding motif enhances viral replication and transcription mutations in sars-cov- viral rna identified in eastern india: possible implications for the ongoing outbreak in india and impact on viral structure and host susceptibility competing interactions of rna-binding proteins, micro-rnas, and their targets control neuronal development and function rbpmap: a web server for mapping binding sites of rna-binding proteins a pattern-based method for the identification of microrna binding sites and their corresponding heteroduplexes from microrna sequences to function gepia: a web server for cancer and normal gene expression profiling and interactive analyses genotyping coronavirus sars-cov- : methods and implications genome-wide mapping of therapeutically-relevant sars-cov- rna structures de novo d models of sars-cov- rna elements and small-molecule-binding rnas to guide drug discovery viruses: overturning rna turnover the crystal structure of tdp- rrm -dna complex reveals the specific recognition for ug-and tg-rich nucleic acids cugbp promotes cell proliferation and suppresses apoptosis via down-regulating c/ebpalpha in human non-small cell lung cancers mir- - p as rna decoy for cugbp stimulates human lung tumor growth by mpges- induction overexpression of cugbp is associated with the progression of nonsmall cell lung cancer cug-binding protein regulates hsc activation and liver fibrogenesis exercise, skeletal muscle and inflammation: are-binding proteins as key regulators in inflammatory and adaptive networks inflammation induces tdp- mislocalization and aggregation cytoplasmic translocation, aggregation, and cleavage of tdp- by enteroviral proteases modulate viral pathogenesis. cell death and differentiation rbms is a tumor suppressor gene that acts as a favorable prognostic marker in lung squamous cell carcinoma rna-binding protein rbms is expressed in activated hepatic stellate cells and liver fibrosis and increases expression of transcription factor prx knockdown of hnrnpa inhibits lung adenocarcinoma cell proliferation through cell cycle arrest at g /g phase prognostic value of heterogeneous ribonucleoprotein a expression and inflammatory indicators for patients with surgically resected hepatocellular carcinoma: perspectives from a high occurrence area of hepatocellular carcinoma in china viral modulation of cellular rna alternative splicing: a new key player in virus-host interactions? wiley interdisciplinary reviews rna global profiling of the cellular alternative rna splicing landscape during virus-host interactions trpv -dependent induction of a novel mammalian cold-inducible protein srsf as well as cirp and rbm . scientific reports srsf : a novel marker for small-cell lung cancer and pleural metastatic cancer mutually exclusive acetylation and ubiquitylation of the splicing factor srsf control tumor growth mir- b- p inhibition attenuates lung inflammation and apoptosis in an lps-induced acute lung injury mouse model by targeting progranulin mir- b- p knockdown attenuates bleomycin-induced pulmonary fibrosis by targeting tissue inhibitor of metalloproteinase (timp ) mir- - p promotes cell growth and metastasis in non-small cell lung cancer through the repression of tgfbr mir p suppresses the proliferation and metastasis of gastric cancer by attenuating the nfkappab signaling pathway through targeting mtdh. international journal of oncology analysis of differential expression profile of mirna in peripheral blood of patients with lung cancer tdp- enhances translation of specific mrnas linked to neurodegenerative disease unconventional rna-binding proteins step into the virus-host battlefront rna cug-binding protein increases translation of -kda isoform of ccaat/enhancer-binding protein beta by interacting with the alpha and beta subunits of eukaryotic initiation translation factor . the journal of biological chemistry the importance of celf control: molecular and biological roles of the cug-bp, elav-like family of rna-binding proteins viral and cellular proteins involved in coronavirus replication. current topics in microbiology and immunology heterogeneous nuclear ribonucleoprotein a regulates rna synthesis of a cytoplasmic virus proteasome-mediated proteolysis of srsf splicing factor intriguingly co-occurs with srsf mrna upregulation during late erythroid differentiation regulation of mcl- by srsf and srsf in cancer cells we acknowledge the support and advice of prof. saumitra das, director, nibmg and time to time suggestions from dr. bornali bhattacharjee and dr. bhaswati pandit, nibmg. conceptualization: srikanta goswami. cug-bp upregulated in lung cancer/ inflammation [ ] cugbp stimulates human lung tumor growth [ ] overexpression of cugbp is associated with the progression of non-small cell lung cancer [ ] cug-binding protein regulates hsc activation and liver fibrogenesis [ ] exercise, skeletal muscle and inflammation: are-binding proteins as key regulators in inflammatory and adaptive networks [ ] . tardbp upregulated in inflammation [ ] facilitation of viral pathogenesis [ ] . rbms rbms is a novel tumour suppressor in lungs squamous cell carcinoma, and its downregulation facilitates development and progression of lscc.[ ]rbms is upregulated by chronic inflammation and fibrosis [ ] . hnrnpa knockdown of hnrnpa inhibits lung adenocarcinoma cell proliferation through cell cycle arrest at g /g phase [ ] upregulated in inflammation [ ] sars-cov rna synthesis and turnover regulation [ ] . srsf expression upregulated by viral infection [ ] upregulated by host-viral interaction [ ] upregulated by intracellular stress [ ] upregulated in lung inflammation/ cancer [ , ] mirnas . mir- b- p upregulated in mouse model of lungs inflammation and fibrosis. [ ] upregulated in inflammation [ ] . mie- - p high expression level in normal lungs tissue. further overexpressed in nsclc/ lung inflammation [ ] . mir- - pno report of detectable expression in normal lungs tissue or in pathogenic condition involved in inflammation/ tumorigenesis associated with breast cancer [ ] . mir- - pupregulated in luad/ lung inflammation and very less amount of expression in normal lungs tissue [ ] https://doi.org/ . /journal.pone. .t key: cord- - vy i m authors: lou, zhiyong; sun, yuna; rao, zihe title: current progress in antiviral strategies date: - - journal: trends pharmacol sci doi: . /j.tips. . . sha: doc_id: cord_uid: vy i m the prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and influenza virus; the emergence and re-emergence of new viral infections, such as picornaviruses and coronaviruses; and, particularly, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. virus-targeting antivirals can function directly or indirectly to inhibit the biological functions of viral proteins, mostly enzymatic activities, or to block viral replication machinery. host-targeting antivirals target the host proteins that are involved in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells. here we review key targets and considerations for the development of both antiviral strategies. the prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and influenza virus; the emergence and re-emergence of new viral infections, such as picornaviruses and coronaviruses; and, particularly, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. virus-targeting antivirals can function directly or indirectly to inhibit the biological functions of viral proteins, mostly enzymatic activities, or to block viral replication machinery. host-targeting antivirals target the host proteins that are involved in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells. here we review key targets and considerations for the development of both antiviral strategies. viruses comprise a large group of pathogens that are responsible for causing severe infectious diseases. over the past years, antiviral agents that target viral proteins or host factors have been successfully developed. based on their inhibitory mechanisms, antiviral reagents can be divided into two groups: (i) inhibitors that target the viruses themselves or (ii) inhibitors that target host cell factors. virus-targeting antivirals (vtas) can function directly (dvtas) or indirectly (indvtas) to inhibit biological functions of viral proteins, mostly enzymatic activities, or they block the correct formation of the viral replication machinery (table ) . host-targeting antivirals (htas) include reagents that target the host proteins that are involved in the viral life cycle (figure ), regulating the function of the immune system or other cellular processes in host cells. with increased knowledge of viral protein and host factors, the scientific community has achieved great progress in mechanism-based antiviral discovery against chronic viral infectious diseases, and in understanding of the emergence of new viral diseases and of the resistance to traditional antivirals. this review will highlight recent achievements in antiviral development and discuss various strategies for preventing virus attachment and entry into the host cell, as well as strategies for preventing virus replication and transcription within the host cell. the first step in viral invasion is the attachment to host cells via an interaction with functional receptor(s). for enveloped viruses, the viral proteins located on the outer envelope of the virion are responsible for the recognition of receptors and the attachment to host cells. hiv (a member of the retroviridae family) is a typical enveloped virus, and its invasion is mediated by the envelope proteins gp and gp , which are arranged on the viral membrane as a trimer of three trans-membrane gp and three noncovalently attached gp surface subunits [ ] (figure ). gp recognizes the cd receptor and launches the conformational changes that expose the binding sites for the binding of a co-receptor, (i.e., ccr and cxcr [ ] ). antagonists that block the interactions between hiv and its receptor and co-receptors have therefore been developed as anti-hiv therapeutics. attempts to find specific inhibitors that block the interaction between hiv- and cd were initiated using a soluble extracellular domain of cd protein that retained the ability to bind gp . although the preliminary results revealed that either soluble cd protein or a cd -immunoglobulin fusion protein showed good in vitro anti-hiv activity, all failed in clinical trials due to poor pharmacokinetic features (e.g., the half-life of cd -immunoglobulin fusion protein in mice is only . h) [ ] [ ] [ ] . small molecule inhibitors that occupy a specific region within the cd -binding pocket of gp were subsequently developed to block the gp -cd interaction (figure a ). for example, bms [ ] and bms [ ] were found to significantly reduce hiv- proliferation and have good pharmaceutical characteristics. another success is influenza neuraminidase (na) inhibitor (nai). influenza na is a surface glycoprotein and functions at two steps of the viral life cycle: (i) cleaves the cell receptor sialic acid residues, which bind to influenza hemagglutinin (ha), and allows the release of the progeny virus; and (ii) cleaves the sialic acid moieties on the mucin that bathes the airway epithelial cells or cobinds the receptor with ha [ ] . in line with the structure of na [ , ] , several nais have been successfully developed to competitively occupy the sialic acid-binding pocket of na. among these nais, oseltamivir and zanamivir were first used clinically as an anti-flu therapy [ ] . oseltamivir is a prodrug that is readily absorbed by the gastrointestinal tract and is converted by hepatic esterases to the active compound (oseltamivir carboxylate). zanamivir has poor oral bioavailability and is currently available as a dry powder mixed with lactose. moreover, laninamivir and peramivir were also approved in north asia recently. laninamivir has excellent in vitro activity against wild type, as well as oseltamivir-resistant, influenza viruses currently circulating [ ] . additionally, peramivir is another nai that differs structurally from other inhibitors through novel substitutions that result in multiple binding interactions with the active site and allows the antiviral to be active against nai-resistant viruses [ ] . non-enveloped viruses, such as the picornavirus (picornaviridae family) and human papillomavirus (hpv) (papillomaviridae family), interact with their functional receptors through viral capsid proteins. picornaviruses are typical non-enveloped viruses, and some members, including enterovirus (ev ) and human rhinoviruses (hrvs), are responsible for causing severe human infection diseases. the non-enveloped capsids of picornaviruses are icosahedral structures comprising copies of viral structural proteins vp - [ , ] . vp - each adopt a b-barrel configuration and are arranged with icosahedral symmetry such that vp surrounds the -fold axes and vp and vp alternate around the -and -fold axes [ ] . although the receptor-binding sites on the surface of picornavirus capsids are not conserved [ ] , these sites have been used to discover inhibitors that block virus-receptor interactions. for example, the canyon structure on the surface of the hrv capsid serves to bind to the hrv receptor, and the soluble portion of the intercellular adhesion molecule- (icam- ) [ ] and numerous compounds that compete with the putative hrv receptor binding site have been shown to bind in a nearby hydrophobic pocket to inhibit virus attachment to the receptor [ ] . however, none of these compounds have been clinical successes to date. after attaching to host cells, a virus will release its genome into the cytoplasm through endocytosis or direct membrane fusion. because this viral entry is one of the key early steps in the viral life cycle (figure ), entry inhibitors have been successfully developed for antiviral therapies. as an enveloped virus, hiv- uses gp to facilitate its entry process after gp is activated by the binding of gp to the receptor. the extracellular portion of gp contains two heptad repeat domains (hr and hr ) separated by a loop region and a hydrophobic fusion peptide (fp) at the n terminus. during the fusion process, the fp of gp is inserted into the host cell membrane, and hr adopts a triple-stranded coiled-coil structure, forming a meta-stable prefusion intermediate. hr subsequently folds into the hydrophobic grooves of the hr coiled-coil to form a stable six-helix bundle that juxtaposes the viral and cellular membranes for fusion ( figure c ). fusion inhibitors are designed to block the conformational changes that are required for membrane fusion. the t peptide (enfuvirtide), which is a peptidic mimic of hr and acts by competitively binding to hr , is the first and the only clinically approved fusion inhibitor [ , ] . t can inhibit a broad range of hiv strains at the nanomolar level, but the poor bioavailability of the drug (it has a plasma half-life of h) make the clinical application of this drug difficult [ , ] . to overcome this pharmacokinetic disadvantage, a series of modified peptides including cp m [ ] , sifuvirtide [ ] , and t [ ] have been generated to stabilize the helical structure of the hr -like peptide by incorporating intrahelical salt bridges between the helix turns. in particular, the plasma half-life of sifuvirtide is -fold greater than that of t [ ] . moreover, chemical modifications including the pegylation [ ] , glycosylation [ ] , and more have also been introduced to improve the pharmacokinetics of hr -based fusion inhibitors. alternatively, a subset of bioavailable small molecule fusion inhibitors have been developed targeting the hr binding pocket on the hr trimer [ ] [ ] [ ] or other undefined regions [ ] . a similar strategy has been successfully used against many viruses that use a class i fusion mechanism, including influenza virus, ebola virus (filoviridae family), and severe acute respiratory syndrome coronavirus (sars-cov) (coronaviridae family) [ ] , but this approach is rare in antiviral development targeting viruses with a class ii/iii fusion mechanism. however, the difficulty in production and delivery means that none of these small molecule fusion inhibitors can be approved for clinical use. non-enveloped viruses use a different strategy for virus entry. after attaching to their receptors, a non-enveloped virus releases its genome into the host cell through a conformational shift of its capsid protein(s). for example, the capsid of ev harbors copies of a hydrophobic 'pocket factor', a natural lipid (sphingosine), at the base of the canyon in the capsid protein vp [ ] (figure ). the expulsion of sphingosine following the binding of the virus to its receptor triggers the opening of the capsid to release the viral genome. pleconaril [ ] and bta are two examples of hydrophobic compounds that can replace the natural pocket factor and inhibit picornaviral uncoating by generating resistance to the expulsion of pocket factor [ ] [ ] [ ] . using the skeletons of pleconaril and related molecules, a novel class of imidazolidinones has been further synthesized with significant anti-ev activity (ic in the range of . - mm) [ ] . most viruses encode one or several proteases that play crucial roles in the viral life cycle. the viral proteases carry out the proteolysis of a polyprotein precursor and release functional viral proteins, allowing them to function correctly and individually in replication/transcription and maturation [ , ] . viral proteases also effectively protect viral proteins by modulating host cell pathways, including ubiquitination and isgylation [ ] [ ] [ ] [ ] [ ] [ ] . in contrast to the diversity of viral protease functions and structures, the catalytic active site of viral proteases generates stringent substrate specificity in protein cleavage. synthetic substrate peptides, which can be designed according to the natural substrates of individual viral proteases, usually generate high-affinity binding and thus provide potent candidates for further drug discovery. one of the great successes is the hiv- protease inhibitors (pis). there are ten pis currently approved to treat hiv- infection: amprenavir (apv), atazanavir (atz), darunavir (tmc ), fosamprenavir (lexiva), indinavir (idv), lopinavir (lpv), nelfinavir (nfv), ritonavir (rtv), saquinavir (sqv), and tipranavir (tpv) [ ] . all hiv- pis share relatively similar chemical structures derived from its natural peptidic substrate and, therefore, the cross-resistance to pis occurs at the active site of hiv- protease [ ] . as a result, pis are commonly used as the combination with other anti-hiv drugs to avoid drug resistance. because most of the pharmaceutical disadvantages have been overcome, pis have become the most potent types of antiviral drugs. current progress in generating antiviral pis has been systematically reviewed elsewhere [ , ] . almost all viruses encode polymerases in the central steps of replication and transcription. thus, polymerases are becoming the most attractive and suitable targets for antiviral development. based on the function and structure of viral polymerases, there are two major types of polymerase inhibitors: (i) nucleoside and nucleotide substrate analogs and (ii) allosteric inhibitors. nucleoside/ nucleotide analogs play a dominant role in antiviral drugs targeting viral polymerases. nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids. the disadvantage of nucleoside analogs is that the initial phosphorylation step, that is, production of the monophosphorylated form, required for activation to a triphosphate may not correctly occur in the host cell [ ] . therefore, monophosphate nucleotide analogs were developed as polymerase inhibitors to avoid this problem. to date, most of the approved antiviral drugs for anti-hiv therapy utilize this mechanism, including zidovudine (azt, , and others. the same strategy was also successfully used in the development of antivirals against a wide range of viruses, including cytomegalovirus (cmv) [ ] and herpes simplex virus (hsv) [ ] (herpesviridae family), hepatitis b virus (hbv) (hepadnaviridae family) [ ] , and hcv [ ] . during the course of a polymerase cycle, the relative orientation of the polymerase domains undergoes a slight shift and this shift causes the conformational change of a specific site, the allosteric site, in the viral polymerase. therefore, compounds that bind to the allosteric site could conceivably block the structural movement of polymerase domains and thus inhibit the function of viral polymerases. antiviral inhibitors that work by this mechanism are known as 'allosteric inhibitors'. the allosteric inhibitors of hiv reverse transcriptase (rt) are also known as nonnucleoside reverse transcriptase inhibitors (nnrtis). nnrtis and the hydrophobic binding site on hiv rt were first identified by screening compound libraries against hiv- rt combined with structural biological analysis [ ] [ ] [ ] [ ] . the binding of nnrti to hiv- rt prevents the flexibility and movement of rt required for the elongation of the nucleic acid. to date, there are five nnrtis, that is, nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine, that have been approved by the fda for clinical use to treat hiv- infection. furthermore, the development of nnrtis against other viral polymerases, such as that of hcv, is ongoing, and other polymerases are likely to have suitable sites for allosteric inhibitors [ ] . integrase is an enzyme that helps the retrovirus to facilitate the incorporation of proviral dna into the host cell genome and catalyzes a vital function in viral replication. inhibitors of integrase represent the newest class of figure ) . raltegravir, an integrase strand transfer inhibitor (insti), was the first generation drug of this class to be approved and is a potent and welltolerated antiviral agent [ ] . dolutegravir is the most advanced second generation insti, and it possess good tolerability, once-daily dosing with no need for a pharmacological enhancer, and relatively little cross-resistance with raltegravir [ ] . another insti, elvitegravir, was recently approved for the treatment of hiv infection as part of a fixed dose combination known as stribild [ ] . furthermore, inhibitors of the lens epithelium-derived growth factor (ledgf)/p binding site of integrase (led-gins) have also been developed, but these are still in a very early stage. each of these drugs contributes a new benefit to the class and will extend the treatment options for patients with hiv- infection. the terminus of the genomic rna in a subset of rna viruses requires a cap structure, which can be directly taken from the mrna of host cells, as in influenza virus, or synthesized by viral enzymes, as in flavivirus and coronavirus. in the latter cases, a cap structure is generated by a series of enzymes and attached to the first nucleotide of the genome rna via a - triphosphate linker. for example, the genome of flavivirus is capped by a type cap structure (m gpppam), which is methylated at the n- position of the guanine (m g) and on the oh of the ribose of the first nucleotide (am) of the genome rna [ ] . three enzymatic steps are required to form the cap structure, including an rna triphosphatase (ns ), a guanylyl transferase (gtase), and a methyltransferase (mtase), provided by the n terminus of ns protein. based on the structure of mtase and its complex with different substrates, three key ligand-binding sites were identified ( figure ). the binding site for s-adenosyl methionine (sam), which acts as the methyl donor, is located at the core domain of mtase, whereas the binding site for the receiver gtp molecule and the guanine moiety of the rna cap is formed by the core domain and the n-terminal extension. moreover, there is another site that binds rna between the sam and gtp pockets; this site is known to be a second, lower affinity binding site that is formed by the core domain and by a channel between two helices of the nterminal extension. because these sites are crucial for mtase activity and virus replication, all are valid sites for the design of dvtas (figure ) . ribavirin was first shown to inhibit the binding of the rna cap to dengue virus (denv) mtase by competitively binding to the gtp/rna cap pocket [ ] . through structure-based drug design, aurintricarboxylic acid was found to inhibit the -o activity of denv mtase with an ic of mm by binding to the lower affinity site [ ] . another inhibitor, sinefungin, which is an analog of sam with the methylated sulfur of sam replaced by a carbon and amine, can inhibit flavivirus activity with an ic as low as . mm [ ] . recently, the compound bg has been discovered using high-throughput screening (hts) methods as an in vitro inhibitor of the guanylyltransferase activity of denv mtase (and thus denv replication) [ ] . however, these inhibitors must be used at a high concentration to outcompete the high-affinity endogenous ligand, and it is necessary to solve this problem before this mechanism can be clinically exploited. some viruses utilize a viral helicase to separate one strand of dna or rna from the complementary strand in a process driven by adenosine triphosphate (atp) hydrolysis. the most widely studied viral helicase is the flaviviral helicase, which is the helicase domain of nonstructural protein (ns ) encoded by hcv. sars-cov (nsp protein), simian virus (sv ) (polyomaviridae family) (tag protein), and hpv (e protein) are also known to encode helicases for their replication [ ] . the inhibitors of helicase-catalyzed atp hydrolysis are the most straightforward class, and several nucleotide and nucleobase analogs have been discovered targeting this mechanism. biphenyls and triphenylmethanes have been studied as inhibitors of sv tag [ ] , hpv e [ ] , and hcv ns helicase [ ] . biphenyls and biphenysulfonacetic acid were found to inhibit hpv growth, an inhibitor of hpv e -catalyzed atp hydrolysis with an ic value > mm [ ] . compounds similar to triclocarban (cid ) and bisphenol a (bpa; cid ) were found to inhibit the helicase activity of sv tag. based on the complex structure of the hcv ns helicase domain and soluble blue ht (pdb code zjo), triphenylmethanes, a more potent triphenylmethane [ ] , and aurintricarboxylic acid (ata) [ ] were developed to inhibit the helicase activity of hcv ns . the nucleic acid binding site of helicase is also a promising site for the discovery of antiviral compounds that directly inhibit helicase-catalyzed unwinding [ ] . for example, many dna-binding pharmacophores, such as anthracyclines, acridones, tropolones, and amidinoanthracyclines, have been optimized as hcv helicase inhibitors [ ] . although there are numerous efforts that have been launched to find antivirals targeting viral helicase, little progress had been made to push them into clinical usage, and these projects have met great challenges on cytotoxicity, bioavailability, and pharmacokinetic properties [ ] . replication and transcription complex blockers following viral entry, viral proteins, together with a number of host cell factors, assemble a viral replication and transcription complex (rtc) that is responsible for the production of the viral genome or other nucleic acids [ ] . therefore, reagents that can efficiently block the formation of the viral rtc could conceivably inhibit viral proliferation. for example, once hcv enters the host cell, the genome of hcv will work as mrnas to produce viral proteins and form an rtc in which the host factors account for viral replication and transcription. ns a is a membrane-associated nonstructural phosphoprotein, and it is believed that ns a has no enzymatic activity but plays a critical role in regulating the formation of the hcv rtc. gao et al. identified a potent hcv inhibitor, bms , that targets ns a and produced few side effects in a phase i clinical study [ ] . although the exact mechanism by which bms exerts its effects is yet to be defined, the resistance profile reveals that inhibitor sensitivity maps to the n terminus of domain of ns a. this inhibitor is further shown to block the hyperphosphorylation of ns a, which is believed to play an essential role in the viral life cycle. this work, for the first time, proved the concept that small molecules targeting a non-traditional viral protein without any known enzymatic activity can also have profound antiviral effects with considerable promise for the treatment of hcv infection. during the replication of ev and other picornaviruses, polymerase (also named d pol ), b (also named vpg), and protease (also named c pro ) participate in the formation of viral rtc, together with host polya-binding protein (pabp- ) and polyc-binding protein (pcbp- ) [ ] . unlike many other viruses, the replication of the picornavirus genome is initiated by the end of genome covalently linked to vpg through a so-called vpg uridylylation process [ ] . although vpg-binding sites vary in picornaviruses [ , , , ] , the binding of vpg to d pol is known to be critical for vpg uridylylation and virus replication. in our recent study, we demonstrated that a ten amino acid peptide of vpg can effectively inhibit the vpg uridylylation process with an ec as low as nm (z. lou et al., unpublished). these results shed light on discovering future indvtas. throughout the life cycle of a negative sense single-stranded rna (ssrna) virus, the genome length rna is encapsidated by a virally encoded nucleoprotein (np), instead of a naked rna, and associated with rdrp (polymerase complex) to form a stable ribonucleoprotein (rnp) complex, which is responsible for virus replication, transcription, and assembly [ ] . during this process, np can protect the rna against exogenous nucleases or the innate immune system in the host cell. based on the crystallographic achievements regarding the ssrna virus rnp complex [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] over the past few years, great progress has been made in this new putative antiviral strategy. kao et al. first identified a compound, nucleozin, that triggers the aggregation of and inhibits the nuclear accumulation of np; this compound can inhibit the replication of influenza virus with a nanomolar median effective concentration (ec ) [ ] . in a parallel effort, gerritz et al. discovered a series of influenza replication inhibitors and showed that they interfere with np-dependent processes via the formation of higher order np trends in pharmacological sciences february , vol. , no. oligomers with an ec of nm [ ] . notably, the structure of the np in complex with a representative compound from this class of inhibitors revealed that two molecules of an inhibitor in an antiparallel orientation lock two adjacent np protomers. this unexpected quaternary complex explained the viral inhibition via ligand-induced formation of stable np oligomers [ ] . in addition to inhibiting np, the disruption of the polymerase complex of influenza virus has been proposed for antiviral strategies. based on the complex structure of the pa c-terminal domain (pa c ) and the first amino acids of pb [ ] , a subset of modifications on n-terminal peptide of pb was shown to diminish the binding affinity of pa and pb , inhibit polymerase activity, and attenuate the replication of influenza virus [ ] [ ] [ ] . moreover, the structure of sftsv np in complex with suramin, an antiviral inhibitor, revealed that the blockers bind to the rna-binding cavity and can attenuate sftsv replication; this indicated that targeting rnp formation may be a new therapeutic antiviral approach [ , ] . because both the polymerase complex and np show significant conservation between different influenza viruses, these results demonstrated that targeting the formation of viral rnp is a valid approach to the development of small molecule therapies against serious antiviral resistance to currently available drugs, such as adamantanes or neuraminidase inhibitors. other key interactions between viral proteins and/or host factors play essential roles in virus entry, replication, and maturation. a very interesting example is the interaction between hpv e and e protein, helping e helicase to tether to the hpv origin of replication. this interaction can therefore be used to guide the development of antivirals treating hpv infection. the disruption of the hpv e -e interaction was first facilitated by a very potent inhibitor, chembl . this inhibitor can effectively diminish the interaction between hpv e and e and thus inhibit hpv proliferation with an ec of nm [ ] . although a few indvtas have been discovered for antiviral therapy, their mechanisms of working are still required to be further investigated. we cannot conclude the possibility that indvtas may have additional functions (or may be the major function) and the category of antivirals will be refined according to further knowledge. viruses utilize many host factors for their efficient proliferation. the correct functions of these host factors are crucial for virus replication and, therefore, compounds that regulate the function of host factors can be introduced as antiviral agents. next we discuss two host factors that are involved in broad spectrum virus infection. apart from the acquired immune response, host cells mount a number of defenses, such as the innate immune response, to virus infection. interferon (ifn) is one of the most crucial molecules in the innate immune response and acts as the primary switch for initiating antiviral immunity in vertebrates. upon being infected by a virus, host cells produce and secrete type i (mainly ifn-a and ifn-b) and type iii (ifn-l) ifns. these secreted ifns interact with the membrane-anchored ifn receptors (ifnars), mainly ifnar and infar , and subsequently stimulate and upregulate the expression of hundreds of ifn-stimulated genes (isgs) to inhibit the replication of viruses [ ] . among these isgs, ifn-inducible transmembrane (ifitm) proteins restrict the entry of influenza virus, west nile virus (wnv), and denv. additionally, mx (myxovirus resistance) gtpases can inhibit the correct function of viral nucleocapsids and polymerases, such as influenza virus [ ] . due to the significance of ifn in the host cells to restrict viral infections, ifn is used in certain instances as a primary antiviral therapy, particularly in the absence of an effective antiviral or vaccination strategy. the use of ifn for antiviral therapy was first developed for treating hbv infection [ ] and subsequently applied to treat hcv in combination with ribavirin [ ] . ifn-b [ ] and ifn-g [ ] have also been evaluated for use in anti-hcv treatment. to further improve the therapeutic efficacy of ifn-a therapy, several options are being investigated. some clinical benefits were observed in pilot studies with ofloxacin [ , ] and an immunomodulatory peptide, a -thymosin [ ] . in particular, peg-modified ifn-a [ ] combined with ribavirin is now standard treatment for hcv infection. cyclophilins (cyps) are key cellular factors that function in numerous cellular processes, including transcriptional regulation, the immune response, protein secretion, and mitochondrial function [ , ] . cyclophilin a (cypa) is a key member of the cyp family and was first identified as a mediator of the immunosuppressive function of cyclosporin a (csa) through the formation of a csa-cypa complex. this complex binds to and inhibits the function of the protein phosphatase calcineurin [ ] , which normally functions to dephosphorylate nf-at, a transcription factor important for t cell activation. cypa is also known to play critical roles in the proliferation of viruses, including hiv- , influenza virus, hcv, vesicular stomatitis virus (vsv), vaccinia virus, sars-cov, rotavirus (rv), and hpv [ , ] , by interacting with viral proteins or facilitating ifn-b production [ , [ ] [ ] [ ] [ ] . cypa was first revealed to be incorporated into hiv- virions by interacting with the capsid protein (ca), and an interaction between newly synthesized hiv- ca and cypa is required for hiv- to induce dendritic cell maturation [ , ] . cypa also interacts with other hiv- proteins, such as vpr and p , to regulate hiv infection [ , , [ ] [ ] [ ] [ ] . several lines of evidence indicate that cypa and cyclophilin b (cypb) [ ] function in the replication of hcv by either increasing the affinity of hcv polymerase ns b for viral rna to enhance hcv replication [ ] or binding to the hcv ns a protein to aid viral replication [ ] . the discovery of cypa inhibitors as antiviral agents started with the immunosuppressive drug csa that inhibits hcv [ ] and hiv [ ] . on the basis of these results, cypa antagonists, which are often derived from review trends in pharmacological sciences february , vol. , no. csa, have been developed, including alisporivir (debio- ), nim , and scy ; these compounds lack immunosuppressive effects but retain high-affinity cypa binding and show very good antiviral effects against hiv or hcv infection [ ] [ ] [ ] . hiv- co-receptors antagonists hiv- co-receptor antagonists that block the interactions between hiv- and ccr and/or cxcr have also been introduced to the anti-hiv efforts, and a few of these have been successful. for instance, ccr antagonists that potently inhibit hiv- replication and have good pharmaceutical properties, including aplaviroc [ ] , maraviroc (the only clinically used anti-hiv drug as a co-receptor antagonist) [ ] , vicriviroc [ ] , and cenicriviroc [ , ] , have been successfully advanced to phase ii/iii clinical trials or approved for clinical usage in the past years. however, the precise mechanisms of these co-receptor antagonists are still not clear. recently, the crystal structures of ccr [ ] and cxcr [ ] have been reported. these two structures provided the atomic information of the ligand-binding pocket and the sites for co-receptor oligomerization. further structural study on the complex of ccr and cxcr with their antagonists will promote the investigation into the inhibitory mechanisms of these inhibitors and help us to increase their anti-hiv efficacy. at present, diverse antiviral drugs are clinically approved or in the later stages of clinical trials. most are based on the conserved mechanisms described in this review, in particular through targeting of viral polymerases and proteases. however, the majority of drug-resistant infection cases have been reported with the usage of antivirals based on this strategy. the requirement for new antiviral drugs to treat chronic infectious diseases and the emergence of more efficient new viruses serve as catalysts for research to find additional targets and mechanisms for antiviral development. a few novel strategies have been introduced for antiviral research, including inhibitors of viral mtase and helicase, blockages of viral rtc formation (e.g., nucleozin to influenza), and host factor antagonists or agonists. however, protease and polymerase inhibitors still occupy a dominant place among antivirals. this is because we still do not have very precise knowledge on the mechanisms underlying alternative strategies, and this requires further investigations on their mechanism before these strategies can be widely used for antiviral development, in particular those targeting drug-resistant viral infections. in our opinion, protease and polymerase inhibitors will still be the first, and probably the major, choice in the development of therapies against emerging novel viruses. in addition, we must consider another important aspect for antiviral development. for some viruses, currently available drugs can effectively eliminate the virus in the host, but genetic components are left behind. thus, the host still suffers from the infection because the virus can integrate its genetics into the host cell. for example, treatment with the combination of inf and adefovir (or other antivirals) can effectively, if not completely, reduce hbv titer in human liver. however, the covalently closed circular dna (cccdna) cannot be removed and still exists in the nuclei of infected liver cells, where it continuously coordinates the expression of hbv antigens. therefore, new mechanisms and strategies to completely remove viral components integrated in host cells, as well as to kill the virus itself, are required in future antiviral development. molecular architecture of the uncleaved hiv- envelope glycoprotein trimer elicitation of hiv- -neutralizing antibodies against the cd -binding site biological and pharmacokinetic properties of a novel immunoglobulin-cd fusion protein recombinant soluble cd therapy in patients 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tightly binds to human cyclophilin a cyclophilin a inhibits rotavirus replication by facilitating host ifn-i production binding of the human immunodeficiency virus type gag polyprotein to cyclophilin a is mediated by the central region of capsid and requires gag dimerization cyclophilin a interacts with hiv- vpr and is required for its functional expression structural characterization of the hiv- vpr n terminus: evidence of cis/trans-proline isomerism hiv- p -another viral interaction partner to the host cellular protein cyclophilin a the host-pathogen interaction of human cyclophilin a and hiv- vpr requires specific n-terminal and novel c-terminal domains cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes critical role of cyclophilin a and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns a protein the effect of cyclosporine a on infection of susceptible cells by human immunodeficiency virus type debio , a cyclophilin binding molecule, is highly efficient in clearing hepatitis c virus (hcv) repliconcontaining cells when used alone or in combination with specifically targeted antiviral therapy for hcv (stat-c) inhibitor scy- , a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis c virus rna replication in vitro inhibition of human immunodeficiency virus type replication in human cells by debio- , a novel cyclophilin binding agent antiviral activity and safety of , a novel ccr antagonist, during short-term monotherapy in hiv-infected adults maraviroc versus efavirenz, both in combination with zidovudine-lamivudine, for the treatment of antiretroviral-naive subjects with ccr -tropic hiv- infection vicriviroc plus optimized background therapy for treatment-experienced subjects with ccr hiv- infection: final results of two randomized phase iii trials pharmacokinetics and pharmacodynamics of tbr- , a novel ccr antagonist, in hiv- -infected, antiretroviral treatment-experienced, ccr antagonist-naive patients safety, efficacy, and pharmacokinetics of tbr- , a ccr /ccr antagonist, in hiv- -infected, treatmentexperienced, ccr antagonist-naive subjects structure of the ccr chemokine receptor-hiv entry inhibitor maraviroc complex structures of the cxcr chemokine gpcr with small-molecule and cyclic peptide antagonists parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses structure of a v -containing hiv- gp core a sensor-adaptor mechanism for enterovirus uncoating from structures of ev this work was supported by the ministry of science and technology of china ' ' project (grant numbers cb and cb ) and the national natural science foundation of china (grant numbers , , , , and ). key: cord- -elom nx authors: yip, tsz-fung; selim, aisha sami mohammed; lian, ida; lee, suki man-yan title: advancements in host-based interventions for influenza treatment date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: elom nx influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. keywords: host factors, influenza, cytokines, metabolism, immunomodulation introduction influenza remains a source of public health concern. influenza a virus (iav) has been the cause of historical noxious pandemics, such as the spanish flu h n , asian flu h n , hong kong h n flu , and more recently the pandemic of h n (swine flu). influenza also causes seasonal epidemics and outbreaks with high morbidity and mortality rates such as the h n outbreak in india ( , ) . the error-prone nature of the viral rna polymerase (rdrp) and virus' capacity for genetic re-assortment (antigenic drift and shift) result in the viral components' susceptibility to mutations, allowing the viruses to evade the immune system and increases their resistance to control strategies. currently, influenza vaccination and two classes of antiviral drugs-m ion channel blockers (amantadine and rimantadine) and neuraminidase (na) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. the use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups ( ) . the disadvantages of using the conventional antiviral drugs have also been a concern. significant levels of resistance to both classes of drugs have been repeatedly reported ( , ) . high level of resistance (up to %) to m blockers has been reported in h n virus strain in american isolates ( ) . resistance has also been reported in h n virus ( ) . iav resistance to na inhibitors has also become an increasingly prevalent concern, with the recent highly fatal outbreak of influenza a(h n )pdm in india associated with oseltamivir drug resistance ( , ) . in addition, a large cluster of influenza a(h n )pdm viruses in japan was found to have increased oseltamivir and peramivir drug resistance ( ) . there is an urgent need to search for alternative targets to treat influenza virus infections, including non-viral targets such as host cellular factors; which are promising as viruses rely on the host machinery for replication. while host immune response is intended to confer a degree of protection against the infection, an impaired or exaggerated host immune response could be detrimental-iav h n and h n virus infection was reported to exaggerate aberrant cytokine release, resulting in a cytokine storm that caused accelerated host death ( ) ( ) ( ) . many recent studies have focused on the investigation of targeting host factors to control virus replication as well as modulate immune response, which we have previously evaluated ( ) . in this review, we will discuss the latest studies (in the past years) on the investigation of novel host-based approaches with potential for influenza treatment. the replication cycle of iav can be grossly divided into four different stages: ( ) entry, ( ) genome nuclear import, ( ) replication and protein synthesis, and ( ) genome nuclear export, apical transport, assembly, and budding. as an obligate intracellular pathogen, iavs are heavily dependent on host machinery for replication and propagation. to this extent, studies employing genome-wide rna interference (rnai) to screen for host factors involved in iav replication cycle have been performed ( , ) and an increasing number of approaches targeting these host factors to control iav replication have been investigated. entry of iav into the host cell is divided into several steps ( , ) . first, hemagglutinin (ha) on the surface of iav binds to the terminal α-sialic acid on the host cell receptor. this induces the internalization of the viral particle by clathrin-dependent, caveolin-, and clathrin-independent endocytosis ( ) . macropinocytosis was revealed as an alternative entry pathway for iav ( ) , which subsequently enters the canonical endocytic pathway ( , ) . the vesicle-containing viral particle forms an early endosome (also known as sorting endosome), which matures into a late endosome as the endocytic pathway progresses. a gradual decrease in intraluminal ph from ph . to . , mediated by v-atpase proton pump ( ) , takes place as the endosome matures ( , ) . this ph drop in the endosomal lumen induces a conformational change in ha, which is activated by proteolytic cleavage to generate ha and ha from precursor molecule ha ( , ) . this conformational change triggers the fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the cytoplasm. acidification of the endosome causes the subsequent acidi fication of viral lumen via the iav m proton channel ( ) , which in turn promotes the dissociation of m layer from both the viral envelope ( ) and the viral ribonucleoprotein (vrnp) complex ( ) . interestingly, a sharp decrease in ph from neutral to an acidic ph of . as utilized by acid bypass has been observed to be sub-optimal for viral replication. it is hence proposed that a gradual decrease in endosomal ph is necessary for sequential reduction in viral stiffness, dissociation of m from the np in the vrnp complex, destabilization of m layer from the viral envelope, and the eventual conformational change of the ha for the release of viral genome and proteins to the cytoplasm from late endosome ( ) . proteolytic cleavage of ha to ha /ha is an important step in iav replication. this cleavage relocates ha , converting previously uncleaved ha to a metastable conformation that induces membrane fusion at acidic ph ( ) . inefficient cleavage and activation of ha leads to low infectivity ( ) . as identified proteins encoded by the viral genome do not possess proteolytic properties, the virus is dependent on host protease for the cleavage of ha. this provides a potential target to control iav infection. ha is commonly cleaved by trypsin-like proteases at the single arginine residue at position . human airway epithelium serine proteases hat and tmprss were identified as the host factors for cleavage at this residue ( ) . aprotinin, purified from bovine lung ( ) , is a protease inhibitor with a long history of clinical use as an antifibrinolytic agent in cardiac surgery ( ) . its potential as an anti-iav drug has been recognized for over a decade ( ) and has been shown to reduce the infectivity of a broad spectrum of iav strains ( , ) both in vitro ( ) and in vivo ( ) . once withdrawn from the western drug market due to its association with mortality ( ), aprotinin has been approved as a locally administered, small-particle aerosol drug for the treatment of iav infection in russia ( ) . however, side-effects associated with the systemic administration of aprotinin raises the need for an alternative protease inhibitor for use in treatment of iav infections. camostat, a serine protease inhibitor, was reported to demonstrate anti-iav potential in mice dating back to ( ) , but little to no research has been conducted to develop it into an anti-iav treatment. it was revisited and proven to be one of the most efficient serine protease inhibitors for the inhibition of iav replication in primary human tracheal epithelial cells in vitro when tested compounds were used at similar molarities ( ) . at present, camostat is widely administered for the treatment of liver fibrosis, chronic pancreatitis, and cancer ( , ) , making it a highly promising candidate for drug repurposing. despite the lack of association between camostat and increased mortality (as with aprotinin), reports of camostat potentially inducing acute eosinophilic pneumonia ( ) warrants the need for careful consideration and further research into the repositioning of drugs from the same class. highly pathogenic iav, such as the h and h subtypes, are reported to have ha cleavage sites rich in basic residues ( ) . the polybasic nature of the cleavage sites provides multiple targets for a broad spectrum of proteases, including the more ubiquitously expressed intracellular proteases such as furin ( ) . this increased protease spectrum could be utilized by these viruses for the activation of ha prior to viral budding, allowing for evasion of potential inhibition by exogenously administered serine protease inhibitors. furthermore, an in vivo study utilizing mice treated with a single protease inhibitor prior to infection with h virus bearing a polybasic cleavage site showed poor efficacy despite good results were obtained for infection with h n virus bearing single cleavage site ( ) , suggesting strain specificity in using serine protease inhibitors to treat iav infections. endosomal acidification is required for the release of iav genome (in the form of a vrnp complex) into the cytoplasm ( ) . research has shown that an increase in endosomal ph during the early phases of infection could inhibit iav infection in vitro ( ) , bringing to light the possibility of controlling iav infection through the prevention of endosomal acidification. the v-atpase inhibitor bafilomycin a , when used at high concentrations ( - nm) has been proven to inhibit iav replication through the efficient suppression of v-atpase ( , ) . however, prominent cytotoxicity to host cells was also observed at such concentrations ( ) . interestingly, lower concentrations ( . nm) of bafilomycin a lack inhibitory effects on v-atpase attenuated iav replication due to disruption of endosomal trafficking. thus, bafilomycin a is suggested to exert its antiviral function via distinct mechanisms at differing concentrations. diphyllin, isolated from the plant cleistanthus collinus, is a natural compound able to induce a v-atpase inhibitory effect ( ) . in contrast to bafilomycin a , diphyllin is well-tolerated in vitro without inducing obvious cytotoxic effects ( ) . most notably, diphyllin is found to effectively inhibit replication of viral strains resistant to amantadine and/or oseltamivir ( ) . since drug resistance to these widely administered antivirals is of major public health concern ( ), diphyllin is regarded as a promising antiviral against drug-resistant iav strains. the release of iav genomic material during replication requires the fusion of the endosomal membrane with the viral envelope. since cholesterol plays a major role in controlling the fluidity of the lipid bilayer in cells, it is hence suspected to have a role in the infection cycle of iav. interferon-induced transmembrane proteins (ifitms) are proteins expressed in many vertebrates (including humans) and are found on the plasma membrane, the membranes of early and late endosomes, as well as on lysosomes ( , ) . while humans express ifitm , ifitm , ifitm , ifitm , and ifitm , only ifitm , , and are both immune-related as well as interferon (ifn)-inducible ( ) , and have been observed to restrict the replication of different viruses, including iav ( ) . studies suggest that ifitms limit viral infection by reducing membrane fluidity and hence restrict the hemifusion (the mixing of lipid bilayer without the release of viral content) of viral and endosomal membranes ( ) , probably via the disruption of cholesterol homeostasis of late endosomes, where viral fusion and genome release conventionally take place ( ) . a recent study using rnai also demonstrated that cholesterol homeostasis can be regulated via acid phosphatase (acp )-mediated niemann-pick c activity and impaired the membrane fusion of iav and influenza b virus (ibv) ( ) , further suggesting the importance of controlling cholesterol homeostasis in the release of viral genome to cytoplasm. on the contrary, later studies suggest that ifitm exerts its antiviral activity in a cholesterol-independent manner, showing that an increase in cholesterol composition of late endosomal membranes fail to inhibit viral membrane fusion ( ) . in addition, studies suggested the accumulation of cholesterol level in the late endosome does not inhibit the iav genome release into cytoplasm ( , ) . with the modulation of cholesterol levels in host endosomal membrane as a mean to inhibit iav host cell entry is still under debate, further studies are required before clear conclusions can be drawn. by comparing the mirna profiles of the iav-permissive hek t cells and (less permissive) hela cells, mirna- a has been identified as a negative regulator for iav infection via the inhibition of archain (arcn , also known as δ-copi) ( ). arcn is a subunit of the copi complex that is required for intracellular trafficking and endosome function ( ) , depletion of which has been reported to inhibit iav infection ( ) . despite impaired iav internalization caused by arcn depletion via sirna ( , ), it was not able to recapitulate through acute inhibition of copi complex by pharmaceutical means ( ) . it is hypothesized that the long-term (lasting days) perturbation on arcn by rnai affected the general endosomal trafficking network, a phenomena which cannot be recapitulated by acute pharmaceutical inhibition to block iav infection ( ) . the potential of targeting arcn for iav treatment deserves further investigation, despite the favorable results from rnai studies. nuclear import of vrnp complexes from the cytoplasm following fusion of the viral and the endosomal membrane is required for replication to take place ( ) . an early study suggested that vrnp complexes could be transported to the periphery of the nucleus ( ), while recent studies report that vrnp complexes utilize the importin-α-importin-β (impα-impβ ) system for nuclear import ( , ) and lacking of importin-α , in an importin-α knockout mouse model were found to be resistant to iav infection ( ) . ivermectin has long been clinically administered for the treatment of parasitosis ( ) , but has recently come to attention as a potential inhibitor of impα/β ( ) . ivermectin inhibition of impα/β has shown to inhibit the replication of rna viruses such as dengue virus and hiv- ( ) . ivermectin was recently tested for the inhibition of iav in vitro, with nuclear import of vrnp complex (of both wild-type and antiviral mxa escape mutant) efficiently inhibited ( ) . given ivermectin's longstanding record of clinical applications and fda-approved status, repurposing of this drug for the treatment of iav should be considered, especially while under threat of pandemic iav outbreak. following the import of the vrnp complex into the nucleus of the host cell, rdrp uses the vrna as a template to synthesize mrna or crna. synthesized crna remains in the nucleus for new vrna generation, while mrna is exported out of the nucleus for translation. viral protein products are either transported to the cell surface via golgi (in case of ha and na) or imported back into the nucleus to bind with vrna, forming new vrnp complex ( ) . numerous host factors are involved in this process and hence could be possible targets for therapeutic intervention. out of the eight genome segments of iav, the m and ns segments are well known for undergoing splicing to generate at least two different mrnas per individual segment ( , ) . cdc -like kinase (clk ) is a kinase which regulates alternative splicing of pre-mrna ( ) . inhibition of clk by the chemical tg or knockdown of clk is shown to cause a decrease in m mrna generation and disrupt downstream m protein expression, prominently reduced iav propagation ( ) . clypearin and corilagin were both found to be potent anti-iav compounds, with a higher therapeutic index than tg in vitro ( ) . clypearin is isolated from herbs used by chinese medicine practitioners for treating respiratory tract diseases during replication, viral mrna is exported from the nucleus to cytoplasm, where protein synthesis takes place. human rna polymerase ii activity is found to be correlated with iav replication through the inhibition of nuclear export of certain viral mrnas, such as m mrna ( ) . cyclosporine a (csa) is a fda-approved drug with immunomodulatory functions ( ) that has been found to have an anti-iav effect in both cyclophilin a (cypa)-dependent and -independent manners ( ) . the cypa-dependent effect was found to correlate with nuclear export of vrnp complex (see targeting nuclear export complex). the cypa-independent effect caused inhibition of host rna polymerase ii. csa is a prospective drug candidate for treatment of iav infections with a relatively high barrier for development of intrinsic drug resistance, as opposed to commonly used antivirals ( ) . nuclear rna export factor (nxf ) is a host factor that has been identified to be involved in the nuclear export of iav mrna. the knockdown of nxf in hek t cells revealed prominent viral mrna nuclear retention in host cell nucleus ( ) . protectin d (pd ), an endogenously produced lipid in the respiratory tract, has been identified to have potent anti-inflammatory and antiviral effects ( ) . pd production was notably found to be reduced in the lungs of iav-infected mice. therapeutic administration of pd was shown to significantly reduce iav mrna expression, lower lung viral titer, as well as improve survival of iav-infected mice. mechanistic studies revealed attenuated cytoplasmic translocation of viral mrna with such treatment. a decrease in recruitment of viral transcripts to nxf was observed while nuclear export of host rna remained largely unaffected, suggesting a role of pd in regulating nxf in nuclear export of viral rna. natural pd expression in the human airway makes this an ideal candidate for novel therapeutics in the treatment of iav infection. the eukaryotic initiation factor- a (eif a) family plays an important role in protein translation ( , ) . eif a impairment has been proven to be related to antiviral activity in a broad spectrum of rna viruses in vitro ( ) , with inhibition of iav mrna translation ( ) . the eif a inhibitors, silvestrol and pateamine a were demonstrated to arrest viral protein synthesis, thus blocking viral genome replication in vitro ( ) . although both silvestrol and pateamine a caused high cytotoxicity at the concentration required effective for iav inhibition, drugs targeting mrna translation for various diseases have been approved by fda or are under active development ( ) . as such, inhibition of iav infections by disrupting mrna translation may well be a therapeutic approach in the future. post-translational modifications during protein maturation ensure proper function of proteins, with proteins of iav no exception. nitazoxanide, a fda-licensed drug used to treat enteritis, was found to be effective in controlling iav infection by interfering with ha n-glycosylation as well as intracellular trafficking in host cell and eventually led to a reduction in viral budding ( ) . despite the mechanism of nitazoxanide being presently unknown, its ability to inhibit replication of numerous viruses [iav, respiratory syncytial virus, coronavirus, hepatitis b virus, and many others ( ) ] suggests that it may act on host machinery. the drug has also been proven in vitro to inhibit the propagation of many circulating strains of human iav, including those resistant to oseltamivir or zanamivir ( ) . nitazoxanide has a high barrier of resistance to iav ( ) and other viral strains resistance to neuraminidase inhibitors ( ), making it a very promising therapeutic target for iav treatment. the drug is currently under phase iii clinical trials ( ). in the later stage of viral replication, viral rnas of iav packed with rdrp and np (known as vrnp complexes) are exported from the nucleus ( ), assembled ( ) , and transported to the plasma membrane [apical in polarized cells ( ) ] for budding. novel targets for influenza treatment frontiers in immunology | www.frontiersin.org july | volume | article exportin (xpo , also known as crm ) is well known for its function in the nuclear export of protein ( ) and rna, including viral rna ( ) . similar to hiv ( , ) , iav viral rna does not directly bind to xpo but is instead held together by several viral proteins. the viral nuclear export protein (nep, or previously known as ns ) and the vrnp complex have been proposed as the nuclear export complex ( ) . cellular xpo has been proven to be crucial in the nuclear export of the vrnp complex, with early studies using leptomycin b (lmb), a potent xpo inhibitor, revealing that in vitro inhibition of xpo led to nuclear retention of vrnp complex ( , ) . however, lmb was deemed unsuitable for development as a potential drug in the phase i clinical trial due to observed cytotoxic effects ( ) . verdinexor (also known as kpt- ) is a new bioavailable selective inhibitor of xpo . it has been shown to be effective against different strains of iavs both in vitro and in vivo as prophylactic and therapeutic treatments ( , ) . it is worth mentioning that delayed administration of verdinexor at day post-infection was still deemed beneficial, with reduced viral load in vivo ( ) . this suggests a prolonged therapeutic time window when compared to the mainstay antiviral drugs such as oseltamivir, where recommended administration is at the early stage of infection (within h of symptom onset) ( ) . currently, verdinexor has passed the phase i clinical study trials, suggesting that it does not pose severe cytotoxic effects as lmb does. in addition, a recent report demonstrated that a new drug, dp -e , which binds and inhibits the function of xpo , can suppress iav replication in vitro ( ) further strengthens the concept of iav intervention by targeting xpo . viral m protein is crucial in assisting the nuclear export of vrnp complex. it was commonly suggested that m protein links vrnp complex to viral nuclear export protein nep which interacts with xpo for nuclear export ( ) . thus, viral m protein may serve as a target to inhibit nuclear export of vrnp. as previously mentioned (see inhibition of mrna export), csa inhibits iav replication via both cypa-dependent and -independent mechanisms. a recent study using a transgenic mice overexpressing cypa showed greater resistance to iav challenge ( ) . in the cypa-dependent mechanism, csa enhances the binding of cypa to m protein ( ) , increases the self-association of m , and hinders m nuclear import ( ) . csa also promotes the cypa-dependent degradation of viral m protein ( , ) . csa seems to be a promising drug to inhibit the nuclear export of vrnp complex by inhibiting viral m protein stability and function. recently, cd , a tetraspanin (defined by four transmembrane domains with conserved residues) that is expressed abundantly in lungs and interacts with integrins has been implicated in the regulation of iav replication in vitro and in vivo ( ) . knockdown of cd in primary human nasal epithelial cells resulted in the nuclear retention of host xpo , viral np, nep, and m proteins, with an increased survival rate observed in iavinfected cd knockout mice. co-immunoprecipitation assays suggest that cd interacts with viral np, m , and nep proteins ( ) ; however, the exact domains involved in interaction and the mechanism of cd function in nuclear export remain unclear. given that a small molecule inhibitor for cd is now under development ( ) , more data revealing the role of cd in iav infection and subsequent use in targeting cd as anti-iav therapy is anticipated. during iav infection, raf/mek/erk signaling cascade is activated, while the inhibition of mek by u , probably mediated via myosin (light chain) ( ), a known motor protein, impairs the nuclear export of vrnp complexes ( ) . suppressing iav replication by inhibition of raf/mek/erk signaling cascade has been illustrated both in vivo ( ) and in vitro ( ) . the replication of ibv ( ) as well as borna disease virus ( ) was shown to be inhibited by u , suggesting the versatility of this approach in controlling infection by different viruses. despite being effective when administered locally to lungs via aerosol, u has little effect when administered orally ( ) . another mek inhibitor, ci- (also known as pd ) was shown to have high potency against iav in vitro ( ). ci- has completed phase ii clinical trials as an anti-tumor drug, with the application of ci- as a potential anti-iav drug candidate recently revisited. unlike u , ci- is orally bioavailable and oral administration of ci- at h post-infection protected % of the iav-infected mice, while the oseltamivir-treated group experienced a % death rate ( ) . oseltamivir is known to be effective only when administered in the early stages of iav infection. this suggests the potential use of ci- as an agent used in iav treatment due to its potentially longer therapeutic time window than mainstay antivirals. formyl peptide receptor (fpr ) located at the host cell surface was identified as an erk stimulator ( ) . antagonizing fpr promoted the survival of iav-infected mice ( ) . furthermore, fpr antagonists have been described to possess antiviral activity against not only iav but also ibv infection ( ) , promoting the idea that antagonizing fpr to suppress raf/mek/erk signaling cascade could potentially be a novel approach for the treatment of a broad spectrum of influenza viruses. after the nuclear export of the vrnp complexes, host cell's intracellular transport mechanism is required to deliver vrnp complexes to the host plasma membrane for the assembly of viral rnas and proteins at the final stage of viral replication. among the various vesicular compartments found in a cell, the rab a + endosomes are known to recycle endocytosed membrane proteins and lipids to the plasma membrane for membrane homeostasis ( ) , a property utilized by many rna viruses, including iav ( , ( ) ( ) ( ) . iav progeny virus production was found to be significantly reduced in rab a + knockdown human cell lines ( ) . furthermore, vrnp complex plasma membrane transport perturbation was observed in rab a knockdown cells ( , ) ; in cells expressing deletion mutant of rab family interacting proteins ( ) ; as well as cells treated with chemicals to interfere microtubule ( ) . direct interaction of vrnp complex with rab a has also been verified ( , ) , demonstrating the dependence of vrna complex transport on rab a + vesicles and the microtubule network during viral replication. since rab a proteins do not confer any mobile properties to the vesicle, molecular motors such as kinesins are required for the active transportation of vesicles through cytoskeletons. kif a, a kinesin- family member, was recently identified as a molecular motor for plasma membrane transportation of vrnp-loaded rab a + vesicles ( ) . kif a knockdown was found to reduce progeny virus production. overexpression of a mutant form of kif a lacking in motor capacity resulted in disruption of the plasma membrane distribution of vrnp complex during later stages of infection. this data suggest that the apical transport of viral components via rab a or kif a could potentially serve as therapeutic targets against iav infection. further examination is merited. tubulin acetylation and deacetylation affects microtubule stability ( ) . histone deacetylase (hdac ) was found to deacetylate α-tubulin, one of the subunits of microtubule ( ) . a study has demonstrated that hdac is involved in iav replication ( ) . inhibition of hdac by tubacin or knockdown of hdac gene resulted in an increase of progeny virus production with vrnp complex redistributed toward the periphery of infected cells. in addition, transportation of ha to the plasma membrane for viral budding was also found to be inhibited by hdac . this data suggests that activation of hdac by its stimulant could be a potential approach to anti-iav therapy, despite hdac stimulants still being under development. while several studies have suggested iav transmission between cells through apical membranes ( ) and intercellular connections ( ) , virus budding from cell membranes remains the major route for transmission of viruses to uninfected cells. na is responsible for the cleavage of sialic acid to prevent the interaction between ha and the host cell during viral budding. besides, viral na, viral ha, m as well as m , are also suggested to play an important role in the initiation of the budding process ( , ) . in section "controlling cholesterol homeostasis, " we discussed the involvement of host cholesterol in viral membrane fusion and viral genome release to cytoplasm. recent studies have demonstrated that host cholesterol may also play an important role in viral budding. it was demonstrated that overexpression of annexin a (anxa ), a phospholipid binding protein, could lead to a decrease in cholesterol levels within the golgi apparatus and plasma membrane ( ), ultimately causing a reduction in egression of progeny virion from infected cells ( ) . this reduction could be reversed by the addition of exogenous cholesterol ( ) . similar to anxa overexpression, addition of a hydrophobic polyamine, u a, could reduce cholesterol level in plasma membr ane, also inhibited viral replication ( ) . since iav is assumed to bud from lipid rafts (cholesterol-rich plasma membrane domains) ( ) , it was demonstrated that anxa overexpression or u a treatment could hinder progeny virus production by lowering the cholesterol content in the plasma membrane. this hypothesis was strengthened through recent studies resolving the cholesterol-binding site of viral m protein, suggesting that iav m clustering (which provides membrane curvature for scission) is mediated by cholesterol ( ) . a recent report utilizing two different fda-approved cholesterol-lowering drugs, gemfibrozil and lovastatin, stated that there was reduction in stability and infectivity of progeny virus compared to that replicating within cholesterol-sufficient host cells ( ) . taken together, this data suggests that controlling cellular cholesterol content would be an effective alternative with drugs available for repurposing iav treatment. further in vivo works are needed to confirm this hypothesis. the gi-type g-protein coupled receptor α -adrenergic receptors (α -ars) have been recently identified as a key host factor involved in iav replication ( ) . apical transport of the viral protein ha is inhibited by low intracellular camp level after stimulating the α -ar-mediated signaling. in vitro stimulation of α -ar by its agonist clonidine inhibits iav replication. therapeutic administration of clonidine reduced pulmonary edema and improved survival rate of iav-infected mice. development of a new antiviral targeting the α -ar-mediated signaling seems promising and deserves further investigation. although targeting host factors for viral interventions generally provides a better resistance barrier, emergence of resistance may still arise ( ) . therefore, combined use of interventions targeting both virus and host factors have been recommended to reduce opportunities for viral development of resistance. one such example would be the combined administration of na inhibitor (oseltamivir) alongside an anti-host factor [such as v-atpase inhibitor diphyllin ( ) , ha maturation inhibitor nitazoxanide ( ) , fpr antagonists ( ) , and xpo inhibitor verdinexor ( ) ]. while further direct assessment for the ease of emergence of escape mutants between single and combinatory use of drugs is required, the synergistic effects of a combined, multi-drug approach observed thus far highly suggest an increased effectiveness over a single-drug approach. table summarizes novel host targets regulating iav replication. compared to rnai, small molecular chemicals remain the best choice as drug candidates due to their fast acting and easy-todeliver properties. although small molecular chemicals targeting certain host factors aforementioned have yet to be developed, their rnai-identified involvement in the iav replication cycle provide leads for the development of new iav interventions. the immune system aims to protect the host from infection and clear the pathogen once an infection occurs. in addition, the complex networks formed between the host physiology and the immune system co-operatively shape the disease outcome; modulations on the networks could alleviate disease severity in iav infections. the immunological responses elicited by iav infection has been reviewed in detail ( ) ( ) ( ) . at the initial stage of iav infection, the respiratory epithelial cells are the primary target for infection. once the infection is initiated, the recognition of infection is accomplished via the detection of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) (see toll-like receptors), and lead to the expression and secretion of different cytokines and chemokines, such as il- , il- , tumor necrosis factor (tnf)-α, and ccl as well as type i and iii ifns. as sentinel cells, alveolar macrophages could also be infected, inducing cytokines and is the main source of type i ifns ( , ) . type i ifns are known inducer for the upregulation of death receptor , which is the receptor for tnfrelated apoptosis-inducing ligand (trail), in lung pneumocytes ( ) . il- and ccl produced by both epithelial cells and macrophages act as chemoattractants for neutrophils and monocytes, respectively. neutrophils are one of the earliest immune cells being recruited to the site of infection ( ) with transmigration of neutrophils carry out by adhesion molecules, such as cd a, cd b, and cd ( ) . in addition to the antiviral activity of neutrophil-released reactive oxygen species (ros), defensin and pentraxin ( ) , uptaking iav by neutrophils could also help in controlling viral propagation as these cells do not support replication of iav ( ) . besides controlling viral replication, neutrophils also play an important role in guiding the migration of iav-specific cd + t-cells in the infection site by secreting and leaving a trail of cxcl ( ) . infiltrated monocytes will, however, differentiate into macrophages or dendritic cells (dcs). the monocytes-derived macrophages are reported to be a permissive host for iav production ( ) , sustaining inflammation by producing cytokines in a magnitude larger than that of the resident alveolar macrophages. the monocyte-derived dc as well as the resident airway cd c low b + plasmacytoid dc (pdc) and two types of conventional dcs (cd + cd b low and cd − cd b hi ) acquire the antigen of the invading pathogen through either direct infection or up-taking infected dead cells ( ) . in the presence of type i ifns, dcs mature when encountering pamps from invading pathogen ( ) . depending on the sub-cellular localization of the antigen, cytosolic and endosomal antigen will be loaded onto major histocompatibility complex (mhc) class i and ii molecules respectively ( ) . once mature, dcs migrate from the infection site to the draining lymph nodes via the interaction of ccr and ccl /ccl ( , ) for antigen presentation via mhc class i and ii to naïve cd + and cd + t-cells, respectively ( ) ( ) ( ) ( ) . interestingly, monocytesderived dcs that engulfed the infected dead cells are poor antigen presenters for cd + t-cells and require the transfer of intact mhc class i/peptide complex to lymph node-resident cd α + dcs which are the most efficient antigen-presenting cells to cd + t-cells ( ) . in addition to antigen presentation, pdc are well known for their high ability in type i ifns production to limit viral propagation ( ) . within the lymph node, naïve cd + t-cells are activated by the dcs, differentiate and clonal expand into cytotoxic t-lymphocytes (ctls) with the aid of various cytokines, including ifn-γ, il- , type i ifns, and il ( , ) , and the help from activated cd + t helper cells ( ). differentiated ctls downregulate their lymph node homing receptor ccr and upregulate ccr and cxcr for the migration to the site of infection. within the site of infection, ctls control viral replication by targeting and inducing apoptosis of virus-infected cells via the secretion of perforin and granzymes as well as the ligation of death receptors on the infected cells by tnf, fas ligand, and trail. on the other hand, cd + t-cells are activated by the presentation of mhc class ii/ antigen complex by dcs, with co-stimulatory receptors such as cd expressed on the t-cells and the ligand for cd (cd and cd ) expressed on dcs playing an important role ( ). activation of cd + t-cells lead to differentiation into different effector cells subsets, including the classical th and th , and the more recently identified regulatory t cells, follicular t helper cells, th , and th subsets ( ). th cells regulate to the differentiation of ctls as mentioned whereas th cells contributes to the activation of b-cells through cd l. within the pregerminal center of the lymph node, the follicular t helper cells interact with antigen-primed b-cells and promote their proliferation. antigen-primed b-cells differentiates into plasmablast and undergo antibody class-switching in the germinal center ( ) . detailed functions of regulatory t cells, follicular t cells, th , and th cells are discussed elsewhere ( , ). plasmablasts enter the blood-stream, are recruited to the inflamed tissue, and terminally differentiate into plasma b cells which specialize in the production of antibody for pathogen neutralization, opsonization, and antibody-dependent cell-mediated cytotoxicity, etc. memory t-and b-cells are also developed during the maturation process, and has been discussed and reviewed elsewhere ( ) ( ) ( ) ( ) . a schematic diagram showing a summary of the immune response after iav infection has been illustrated in figure . the yin and yang theory is always used to describe the importance in balancing the host immune response. in the light of this theory, the treatment strategy aims to suppress the overwhelming activation of the host immune response and in reverse to compensate any unfavorable suppression. although adaptive immune responses are important in viral clearance, the immediate innate immunity play an important role in the early control of an infection, and conversely, is a major factor for disease severity due to immunopathology. dysregulated immune responses caused by viral infections have been implicated in severe disease development ( , ) , such as acute lung injury (ali). ali in its most severe form, known as acute respiratory distress syndrome (ards), is reported to be the most prevalent cause of mortality in iav-infected patients ( ) . studies suggested that iav strains could be associated with either over-activating (human infection by avian h n and h n ) ( , ) or suppressing (h n , h n ) ( ) immune response. recent history has seen the outbreak of iav pandemics of varying severity takes place at the cost of millions of lives. one such example would be the deadly spanish flu of , which claimed the lives of - million of the million people infected worldwide. the pathological examination of lung sections from mice infected with reconstituted iav virus revealed necrotizing bronchiolitis and severe alveolitis in tissue, with neutrophils observed as the predominant inflammatory cell type present ( ) , suggesting neutrophil involvement in the pathogenesis of iav infection. the majority of immune cells in blood circulation are neutrophils; of which they are among the first innate immune cells recruited to the site of infection ( ) . neutrophils characteristically control microbial infections by generating bactericidal ( ) neutrophil extracellular traps (nets), consisting of granule proteins, histones, and decondensed chromatin ( ) . both protective and destructive role of neutrophils in iav infections have been described. the contrasting role of neutrophils could be explained by factors such as viral strain and viral dose used in different experimental setup, etc. the protective role of neutrophils was observed when mice infected with a low, non-lethal dose of iav h n strain hkx displayed neutrophil-mediated viral clearance via phagocytosis ( , ) . depletion of neutrophils has found to enhance viral load in the iav-infected animals ( ) . on the contrary, this protective nature is disputed due to the association of neutrophil-generated nets. extensive net formation was observed in mice infected with pr , an iav strain highly pathogenic to mice ( ) . histones and myeloperoxidase within the net induce cell death of lung epithelium and endothelium ( ) , leading to the loss of integrity of the alveolarcapillary barrier, a characteristic of ali. yet, while histones have been shown to suppress iav replication in vitro ( ) , in vivo study demonstrated that there was increase in lung inflammation and damage in iav-infected mice treated with histones ( ) . interestingly, co-treatment of lethally infected mice with anti-histone antibody and oseltamivir resulted in an increase in animal survival when compared to infected mice groups treated solely with oseltamivir ( ) . in agreement with the in vitro and in vivo data, it has been reported that net produced by cultured neutrophils from patient with h n and severe h n infection increased alveolar epithelial cell permeability ( ) leading to ali. more importantly, plasma net level positively correlated with the disease severity index (including higher acute physiology and chronic health evaluation ii score) and multiple organ dysfunction syndrome ( ) , further demonstrating the detrimental role of net in the pathogenesis of severe iav infections. studies have demonstrated the involvement of superoxide dismutase and myeloperoxidase in netosis, the formation of net ( ) . the presence of anti-myeloperoxidase antibody as well as the superoxide dismutase inhibitor (detc) significantly reduced netosis. finally, tetrahydroisoquinolines ( ) and a panpeptidylarginine deiminase (pad) inhibitor, named cl-amidine ( ) have been suggested to inhibit netosis. despite it has been reported that during iav h n infection, pad knockout mice displayed only slight improvement in weight loss and a slight prolonged but no end-point survival advantage was observed compared to wt mice ( ) , based on the extensive findings presented above, targeting net to prevent ali in the severe case of iav infection, including the highly pathogenic avian iav, remain promising and may warrant further investigation. innate lymphoid cells are cells of lymphoid lineages that do not express antigen-specific b-or t-cell receptors ( ) . similar to t-helper cells, they are classified into subsets by their ability to produce type (th ), type (th ), and type (th and th ) cytokines. previous studies confirmed the involvement of ilcs of group linage (ilc ) in iav infection and airway inflammation ( , ) . on the positive side, during the recovery phase of iav infection, ilc expresses amphiregulin which promote airway epithelium repair ( , ) , thus facilitating the recovery of the infected lung. on the other hand, in response to il- produced by macrophages, dcs, and nkt cells, ilc secretes il- and il- and induce airway hyper-responsiveness. recruitment of eosinophils by il- to the lung also mediates airway inflammation ( ) . since eosinophilia is a characteristic of allergic asthma and influenza is a major cause for morbidity and mortality in asthma patients ( ) , it will be of particular interest to investigate the role of ilc in iav infection, particularly in asthma patients. ilc s have been initially described as immature nk cells residing in the liver and share many phenotypic similarities with nk cells ( ) . it was recently appreciated that tissue-resident ilc s other than the previously recognized nk cells are the major early source of the antiviral ifn-γ at the primary site of various viral infection, including iav ( ) . interestingly, ifn-γ was found to suppress ilc activity and reduce il production which exacerbates disease severity during influenza a(h n ) pdm infection ( ) . this data may highlight a link between ilc and ilc and suggesting ilc can suppress ilc activity via ifn-γ production during iav infection. with ilcs finally identified, functions of these cells and their role in immune response to tumors and pathogen infections have been massively investigated in recent years. type i ifns, prostaglandin i , corticosteroids, and testosterone have been reported to suppress ilc activity ( , ) . in addition to il- , the epithelial cytokines il- , thymic stromal lymphopoietin, as well as the lipid mediator prostaglandin d were found to activate ilc ( ) . the therapeutic potential of these ilc activators and suppressors is yet to be deduced. with more and more studies demonstrating the involvement of ilc in iav infection, the interplay between different ilc subtypes in iav infection would, therefore, be an interesting area to explore and modulate the ilc activity may be a future approach to combat iav infection. reactive oxygen species, generated by specialized enzymes such as nadph oxidases, are released during iav infection ( ) . the nadph oxidase family consists of enzymes containing different catalytic subunit named nox - and dual oxidase (duox) and . ros have been reported to display both beneficial (limiting viral replication) and detrimental (promoting ali) effects in the course of iav infection. interestingly, the protective or destructive effect of ros is dependent on the enzyme of which the ros is generated ( ) . dual oxidase and are found to be host-protective ( , ) . in vitro, ros generated by nuclear duox indirectly regulates the splicing of iav mrnas via the nuclear speckle-associated splicing complex ( ) . in addition to altering viral mrna splicing, ros generated by doux has been attributed to the production of ifn-λ, an important anti-iav ifn. in response to iav infection, increased viral mrna replication was observed when duox was silenced in vitro ( ) . increased viral replication was also observed in mice with doux silenced ( ) , further depicting the protective role of doux in iav infection. unlike doux, nox activation could be harmful to host. iav infection was reported to induce nox -dependent endosomal ros production ( ) . ros could target the conserved cys on toll-like receptor (tlr) , and inhibit tlr -mediated type i ifn expression during a mild iav h n infection in vivo ( ) . iav-infected mice treated with specific nox inhibitor, cholestanol-conjugated gp ds-tat, were found to have reduction in endosomal ros production, restored tlr activity, and displayed a decreased viral load ( ) . in addition to nox , nox dependent ros production has also been reported to activate mapk/erk signaling ( ) , enhancing the export of vrnp complex, thus increasing viral replication (see targeting the raf/ mek/erk pathway). nox knockdown resulted in a reduction of viral replication in vitro ( ) . targeting the different nadph oxidase isoforms, instead of scavenging ros should be considered as the therapeutic approach for iav infection, as doux-mediated ros production is beneficial ( , ) , while nox and nox are harmful during iav infections ( , ) . finally, ns (not to be confused with iav ns protein) has been demonstrated to be a nox inhibitor, which could inhibit the activity of nox , nox , and nox . a study demonstrated that ns suppresses iav-induced nox and significantly inhibits iav virus replication ( ) . besides cholestanolconjugated gp ds-tat and ns aforementioned, apocynin, a phagocytic nox inhibitor as well as ros scavenger ( ) ( ) ( ) , has been demonstrated to ameliorate hyper upregulation of cytokines induced by iav infection through socs and socs in vitro ( ) and reduce peri-bronchial inflammation and viral titer in vivo ( ) . interestingly, ebselen, another nox inhibitor and glutathione peroxidase mimetic, could reduce inflammatory status measured in bronchoalveolar lavage fluid (balf) of mice pre-exposed to cigarette smoke and subsequently infected with iav ( ) . taken together, these reports highlight the potential use of nadph oxidases inhibitors and ros scavengers to treat iav infections. dysregulated cytokine production has been associated with the elevated mortality rate observed in severe iav infections ( , ) . as such, the immunomodulation of cytokines are regarded as promising therapeutic tactics. recent advancements developed with this approach will be highlighted in the following section. tumor necrosis factor has two main functions during viral infection-it activates nf-κb, inducing the expression of cytokines responsible for the host immune response; and induces apoptosis through activation of a signaling cascade involving tradd, fadd, and caspase , , , and ( ) ( ) ( ) . tnf is known to be highly upregulated in iav-infected hosts, especially in hosts infected with highly pathogenic iav ( , ) . however, it is both protective and counter-protective functions associated with tnf that makes it a target in the treatment of iav. the protective role of tnf is observed during infection by low pathogenic iav, where extrinsically derived tnf is responsible for attenuating tissue-damaging cd + t-cell response ( ) . in addition to recruiting monocytic cells to the infection site, cd + t-cells response was observed to deteriorate lung pathology ( ) and damage healthy, non-infected lung epithelial cells ( ) upon iav infection. furthermore, tnf deficiency has been associated with an increased detection of il- and il- in balf ( ) , which promote the survival of and proliferation of cd + t-cells ( , ) and subsequent tissue damage. exacerbated lung pathology caused by the upregulation of the monocyte chemoattractant protein- was observed in tnf −/− mice infected with sub-lethal dose of iav ( ) . in addition, decreased cd + t-cell contraction due to enhanced expression of the anti-apoptotic protein bcl- was observed in sub-lethally iav-infected tnfdeficient mice when compared to wt mice ( ) . as a whole, there is substantial evidence supporting the protective role of tnf in iav infection. on the other hand, the correlation of tnf with pulmonary edema has been well-documented ( ) . tnf has been observed to stimulate the expression of cxcl in alveolar epithelial cells in a transgenic mice model resembling extensive iav infection in lung tissue, causing alveolar damage, lung edema, and hemorrhage ( ) . in addition to lung edema, tnf has also been reported to correlate with iav-associated encephalopathy ( , ) . however, it is notable that despite iav-associated encephalopathy, direct invasion of the central nervous system is rare ( ) , suggesting that iav-associated encephalopathy could instead be a result of peripheral infection. furthermore, tnf has been shown to increase the permeability of the blood-brain barrier (bbb) ( , ) , contributing to neural damage ( ) . these studies further support an anti-tnf approach as a potential therapy for severe iav infection. at present, etanercept, an anti-tnf drug administered in the treatment of rheumatoid arthritis, is the only tnf inhibitor (or even tnf directed treatment) tested for iav treatment. etanercept has been shown to protect against the in vivo lethal infection of mice with a highly virulent, mouse-adapted iav strain ( ) , with observations made of an increased survival rate with decreased morbidity, expression of the proinflammatory cytokine il- , lung injury, and edema ( ) . the protective role of il- was demonstrated in mice challenged with sub-lethal iav infection. il- -deficient mice displayed exacerbated pulmonary damage ( , ) and lung injury due to an observed decline in the survival of alveolar type ii cells and alveolar epithelial cells ( ) . iav suppresses the anti-apoptotic mcl- and bcl-xl expression, causing cell death of neutrophils which are critical in viral clearance ( ) . addition of il- restored the expression of mcl- and bcl-xl in vitro and is considered as the underlying mechanism for the observed survival advantage of wt mice over il- knockout mice during mild iav infection. il- has also been shown to induce the proliferation of lung il- + regulatory t cells and il- , which act to limit excessive proliferation of cd + t-cells and subsequent cd + -inflicted damage. this would hence prevent the tissue damage observed in lung immunopathology ( ) . despite the apparent protective role of il- , high levels of il- in serum or cerebrospinal fluid have been reported in severe neurologically complicated iav cases, with il- used as a marker for prognosis ( - , , ) . the role of il- in regulation of bbb permeability was reported ( ) , with potentially detrimental neurological complications. as such, the suppression of hyper-induced il- as a form of therapy in severe iav infection should be considered. one such option is the anti-il antibodybased drug tocilizumab, which is currently administered clinically for the treatment of rheumatoid arthritis. however, study on the usage of this drug to treat hyper upregulation of il- due to severe iav infection has yet to be conducted. on the other hand, in a case of h n virus-induced ards, the use of an extracorporeal cytokine hemoadsorption device to remove cytokines including tnf and il- from the bloodstream ( ) has showed beneficial to the patient ( ) . more research is required to confirm whether the removal or neutralization of il- could be a potential therapy for severe iav infections. the activation of cd + t-cell is crucial for viral clearance. it should, however, be tightly regulated to limit cd + t-cell inflicted host cell damage. il- mediates il- induction ( ) . il- acts to suppress cd + t-cells and reduce morbidity through il- and regulatory t-cells ( ) . much like other immunomodulatory approaches, the timing for applying il- should be carefully assessed. compared to placebo-treated iavinfected group, early administration of il- to iav-infected mice in fact led to poorer viral clearance, increased morbidity, and deteriorated lung histopathology, while il- administration during the recovery phase ( - days post-infection) accelerated recovery and improve lung immunopathology ( ) . notably, il- could also suppress th responses and increases susceptibility to secondary s. aureus infection ( ) . therefore, co-administration of antibiotics should be considered when utilizing il- as potential iav treatment. both type i and iii ifns have antiviral properties, with viruses counteract ifns to gain an advantage for their propagation. the iav viral protein ns inhibits the production of ifns by antagonizing irf- , a key transcriptional factor for ifns. this prevents the processing of cellular pre-mrnas (including those for ifns) and directly interacts with retinoic acid-inducible gene (rig)-i receptors, which are critical in innate sensing, to suppress ifn production during infection ( , ) . in addition to inhibiting ifn expression, the induction of socs inhibits ifns signaling by suppressing cytokine signaling has been documented ( ) . the recognition of ′ triphosphate on viral rna by rig-i receptor is shown to induce the expression of socs , which in turn represses type i ifns expression ( ) . due to ifns being a key contributor to antiviral immune response, an impairment of type i or iii ifn production may cause the escalation of otherwise mildly pathogenic iav infection into a life-threatening one ( ) . while type i ifn has been demonstrated to inhibit iav replication in vitro ( ) ; the in vivo administration of type i ifn in animal models only displayed effectiveness in a prophylactic capacity. a lowered viral titer was detected in the nasal wash of test animals. however, host susceptibility to iav infection remained unchanged ( ) . notably, this protective effect is only conferred by an optimal dose of type i ifn of low to moderate amounts ( - units per mice daily); with higher dosages ( , - , units per mice daily) shown to increase morbidity ( ) . in addition, clinical trials demonstrated that prophylac tic administration of type i ifn reduced disease severity and lowered susceptibility to iav in males and participants aged or above ( ) . despite relatively successful results seen in the prophylactic use of ifns, its therapeutic use is of greater clinical relevance. mice treated with type i ifn post-iav infection showed a successful reduction in lung iav titer but displayed increased morbidity and mortality in comparison to vehicle-treated mice ( ) . a possible explanation for this phenomenon is the induction of excessive inflammatory response and trail-dr -mediated epithelial cell death by type i ifn ( ) , which accounts for the observed lung pathology in iav-infected animals treated with type i ifn ( ) . in addition, downregulation of γδ t-cells by type i ifn has been correlated with increased susceptibility to secondary s. pneumoniae infection ( ) , further arguing against the potential use of type i ifns for the treatment of iav infection. in comparison to type i ifns, the administration of type iii ifns may provide advantages in the control of iav replication ( , , ) without the risk of previously reported type i ifns-mediated immunopathologic side-effects ( , , ) . however, a recent study aiming to stimulate ifns signaling through the systematic administration of rig-i ligand post-iav infection demonstrated that type i, but not type iii ifns signaling is important in conferring protection during fatal iav infection in vivo ( ) . though, this study did not measure the production of type i and iii ifns as well as any changes in viral load with respect to ifnar or ifnlr knockout. in addition, while human immune cells are not primary targets in iav infection, they could be susceptible to iav and become efficient host cells for virus replication. they are reported to possess a subpar response to type iii ifns ( ) ; leading to the preliminary conclusion that solely using type iii ifn as treatment may not be feasible. as such, reports suggesting the use of type iii ifns over type i ifns as a front-line therapeutic agent to counter iav infections may require further investigation. the inhibition of cox- by selective inhibitors, nimesulide and celecoxib, was previously demonstrated to suppress the hyper upregulation of pro-inflammatory cytokines induced by highly pathogenic avian iav ( ) ( ) ( ) . in addition, the use of zanamivir in tandem with a specific cox- inhibitor was shown to increase the survival rate of mice lethally infected with avian h n iav, when compared to mice treated solely with zanamivir ( ) . activated cox- regulates downstream prostaglandin production. one such example is pge , a major type of prostaglandin recently demonstrated to play an important role during iav infection. pge was significantly upregulated in response to iav infection, leading to the inhibition of antiviral type i ifn production in macrophages and the subsequent increase in virus replication ( ) . the use of chemicals ah and gw x to antagonize pge downstream signaling molecules ep and ep respectively, was shown to induce antiviral type i ifn production. the in vivo treatment of mice lethally challenged iav with both ep and ep antagonists significantly improved the survival rate. a recent study demonstrated the ability of a modified tcm decoction to reduce peg production and subsequent morbidity in mice lethally challenged with iav. improved lung pathology was observed ( ) . the long history of clinical tcm use supports the clinical feasibility of peg inhibition as an option to treat severe iav infections. pattern recognition receptors on host cells sense specific pamps present on the viral surface or generated during replication. prrs can be broadly divided into two classes by their function or location. when defined by location, prrs are classified into groups-membrane-bound (tlrs and c-type lectin receptors), cytosolic (rig-i-like and nod-like receptors), and secreted (collectins and pentraxins) ( ) . significant research has been conducted on prrs with regards to iav infection. tlrs and rig-i receptors have been extensively studied for their major roles in eliciting host immune responses (cytokine and ifn expression) during iav infection ( ) ( ) ( ) . rig-i receptors have been investigated for their functional relevance to iav infection and targeting these receptors as a form of iav treatment has been extensively reviewed ( ) ( ) ( ) . this section will cover recent research on tlrs and the targeting of different tlrs to treat iav infection. humans have been identified to express tlr - , while mice have been identified to express functional tlr - as well as tlr - ( ) . most tlrs-with the exception of tlr utilize myd as an adaptor protein during signal transduction. tlr utilizes trif as an adaptor. tlr is known for its ability to utilize either myd or trif, with the choice of adaptor dependent on its sub-cellular location ( ) . different tlrs, such as tlr , , and ( ) as well as tlr , tlr , and most recently tlr ( ) , have been revealed to play a role in the orchestration of host immune responses contributing to iav pathogenesis. with tlr being an exception ( ) ( ) ( ) , tlr activation largely causes the release of pro-inflammatory cytokines, with hypercytokinemia leading to ali as a major cause of mortality in severe iav infections. in addition to dysregulated cytokine release, excessive production of ros has been associated with ali development. in fact, lung injury during severe pulmonary infections, such as iav and sars, could be caused by oxidative stress ( ) . iav infection activates nadph oxidase that subsequently produces oxidized papc, an endogenous phospholipid. the oxidized papc serves as an agonist for tlr , activating a tlr -trif-traf -nf-κb signaling cascade to eventually trigger the release of il- , ultimately inducing the onset of ali. in addition to oxidized papc, the induction of endogenous protein s a upon intracellular prr ddx recognition of iav subsequently induces the activation of tlr , further contributing to iav-induced mortality ( ) . since tlr has been proven to be important in ali induction (and hence iav-related mortality), manipulating the stimulation and antagonism of tlr could potentially reduce the severity of iav infections. eritoran (e ) is a specific tlr antagonist initially purposed for the treatment of sepsis, but a failed a phase iii clinical trial due to improved patient care in the placebo group prevented its eventual use in sepsis treatment ( ) . in vivo administration of eritoran in mice lethally infected with iav resulted in improved clinical score, lung pathology results, and reduced viral titer. delayed administration of eritoran, at day after infection beyond the recommended therapeutic time window (within h after the first display of clinical symptom) for use of oseltamivir ( ), also demonstrated a significant benefit to infected mice compared to non-treated group, suggesting a prolonged therapeutic time window for iav treatment when compared to mainstay antiviral drug treatment. a newer and structurally simpler specific tlr antagonist, fp ( ), alongside a newly developed decoy peptide r that has been shown to disrupt tlr , , , and signaling via tirap, has been shown to protect mice from lethal iav infection ( ) . these results support the potential use of tlr antagonism as a means to treat severe iav infection. the suppression of other tlr signaling pathways-such as blocking tlr -mediated signaling through the use of an anti-tlr antibody, significantly protected against lethality when administered on day and post-iav infection ( ) . a study also demonstrated that h n -infected tlr knockout mice had better survival than h n -infected wild-type mice, which is evident through the significantly faster regaining of body weight post-infection, lower viral titer in the lung, and fewer pathological changes in the lung ( ) . an increasing number of tlr antagonists are now under development ( , ) , alongside several other agents also shown to have effects on tlrs. polysaccharides isolated from r. isatidis, a traditional chinese medicinal herb used to treat iav infection, have recently been shown to inhibit pro-inflammatory cytokines such as il- and ccl- in vitro by down-regulating upstream tlr expression ( ) . menk, an endogenous protein expressed in the adrenal medulla, was shown to both prophylactically and therapeutically increase the survival rate while reducing viral-caused lung pathology and viral titer in mice lethally challenged with iav ( ) . this was determined to be caused by the downregulation of tlr . these results suggest the potential of down-regulating tlr expression in the treatment of iav infection. the above-mentioned data suggest modulation of tlr signaling or expression as a promising approach in treating severe influenza disease and deserves immediate investigation. table summarizes new immunomodulatory approaches to combat iav infections. it is well documented that patients with diabetes mellitus have a greater tendency to develop severe iav infection than healthy patients ( ) . hyperglycemia increases susceptibility of the host to iav infection via viral uptake, through the promotion of v-atpase assembly ( ) and immunosuppression ( ) . in addition, viruses rely on host metabolism to perform essential functions during replication ( ) ( ) ( ) ( ) . these processes exert a large energy demand on the host within a very short period of time ( ) ; energy of which is supplied by and is dependent on host metabolism. iav viruses have been reported to modify the metabolic state of the host. for example, increased c-myc-dependent glycolysis and glutaminolysis has been demonstrated in infected cells ( ) . the changes in glucose and glutamine metabolism were reversed upon the addition of bez , which inhibited the iav-mediated c-myc induction. administration of bez days prior to infection and up to days post-infection was shown to decrease lung viral titer and improve the survival rate in iav-infected mice. small molecules such as clotrimazole and α-mangostin that target lipid metabolism have also been demonstrated to suppress iav replication in vitro ( ) . in addition to being important for generating energy and biosynthesis, recent research demonstrates that cellular metabolism affects immune cell function. dysregulated immune responses observed in many diseases are associated with specific metabolic configurations. viruses, influenza inclusive ( ) , were found to induce drastic alterations in metabolic levels and programs ( ) . macrophages in infected hosts were observed to have marked differences in the krebs cycle, a key metabolic pathway. this is of significance due to the role of macrophages, which are immune cells critical in the pathogenesis of many inflammatory diseases ( , , ) . in activated macrophages, succinate, a krebs cycle intermediate, was found to possess inflammatory signal. accumulation of succinate generates ros, leading to subsequent activation of hypoxia-inducible factor α and the induction of cytokines such as il- β ( ) . a recent study identified the ability of itaconate, another krebs cycle-derived metabolite, to block the production of inflammatory factors. this prevented inflammation, protecting mice from lethal levels of inflammation that can occur during infection ( ) . this data suggest the critical roles of krebs cycle intermediates in regulating cytokine profiles and inflammation. metabolites generated by innate immune cells in distinct configurations could have different roles beyond that of bioenergetics, with functions in signaling regulation, transcription, and orchestrating innate immune responses. despite the lack of research conducted thus far on the application of immunometabolic approaches to influenza treatment, the prospect of manipulating immune responses by modulating immune cell metabolic state is promising. further research should focus on the identification of metabolites for modulation of immune cell function with substantial improvement of therapeutic strategies to treat iav disease. latest advancements in high-throughput technologies, e.g., meta bolomics is a useful approach to systematically investigate the changes of metabolic mechanisms during iav infections. identification of important metabolites involved during iav infection should be a new approach by modulating the host metabolism for interventions. multiple host-based intervention strategies against influenza have been developed or are under development. while approaches targeting host machinery required for virus replication seem to be promising thus far, additional research is needed to determine the effect of modulating host immune response on influenza treatment. this is increasingly important, since targeted host factors may play distinct roles in response to infection by different influenza viral strains ( ) , making the management of influenza through solely targeting a single specific host factor is difficult. host-based interventions offer obvious advantages over conventional antivirals, such as a higher barrier to drug resistance ( , , ) due to greater genetic stability of host factors than the mutation-prone nature of viral components. in addition, administration feasibility is a key factor to consider the usage of drugs. the mainstays of antivirals for iav infections, the na inhibitors, and m blockers, are recommended to be administered within h of symptom onset for optimal antiviral activity. this short treatment window may not be fully fulfilled in a clinical setting. novel host-based interventions were reported to have therapeutic time windows longer than this conventional timeframe ( , , , ) , even up to days post-infection ( ) , providing a clear clinical advantage over na inhibitors and m blockers. in addition, hypercytokinemia and ards could contribute to disease severity and mortality in instances of severe influenza infection, with virustargeting antivirals providing little to no alleviation of such complications. since host immune response is indispensable in host defense against invading pathogens, the use of immune-modulators to suppress detrimental effects while retaining beneficial protection of the host remains challenging. the timing and dosage of medication administration would be critical in determining the drug effectiveness in influenza treatment. targeting virus-induced metabolic changes to restore host normal metabolism may be a new direction to combat influenza disease. further research in the immunometabolism field, along side studies on modulating immune response to infectious disease by altering host metabolic processes; would create a new direction for future research and is expected to yield significant discoveries that may provide new therapeutic options in the treatment of iav infections. smyl conceptualized the work. il and asms drafted some review sections and tfy and smyl wrote the manuscript. influenza a(h n )pdm outbreak detected in inter-seasonal months during the surveillance of influenza-like illness in pune avian influenza a (h n ) virus infections in humans 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influenza highly pathogenic avian influenza a h n and pandemic h n virus infections have different phenotypes in toll-like receptor knockout mice inhibition of toll-like receptor signaling as a promising therapy for inflammatory diseases: a journey from molecular to nano therapeutics small-molecule inhibitors of the tlr /dsrna complex radix isatidis polysaccharides inhibit influenza a virus and influenza a virus-induced inflammation via suppression of host tlr signaling in vitro novel effect of methionine enkephalin against influenza a virus infection through inhibiting tlr -myd -traf -nf-kappab p signaling pathway influenza virus and glycemic variability in diabetes: a killer combination? front microbiol glycolytic control of vacuolar-type atpase activity: a mechanism to regulate influenza viral infection metabolism goes viral dynamics of the cellular metabolome during human cytomegalovirus infection saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny stealing the keys to the kitchen: viral manipulation of the host cell metabolic network viral activation of cellular metabolism targeting metabolic reprogramming by influenza infection for therapeutic intervention metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling krebs cycle rewired for macrophage and dendritic cell effector functions succinate is an inflammatory signal that induces il- beta through hif- alpha itaconate is an anti-inflammatory metabolite that activates nrf via alkylation of keap key: cord- -wpxhk pf authors: jaume, francesca; valls-mateus, meritxell; mullol, joaquim title: common cold and acute rhinosinusitis: up-to-date management in date: - - journal: curr allergy asthma rep doi: . /s - - - sha: doc_id: cord_uid: wpxhk pf purpose of review: the purposes of the review are as follows: ( ) to define acute rhinosinusitis (ars) and their phenotypes, ( ) to highlight the ars management according to international guidelines, ( ) to compare the physicians’ management with the ars guideline recommendations, and ( ) to report ars socioeconomic burden. recent findings: bacterial and non-bacterial ars have similar symptoms, although they can be discriminated by using a combination of specific signs and symptoms. the prescription of antibiotics should be limited to clearly suspected bacterial ars. there is an overuse of diagnosis tools and treatment prescriptions. the total cost per ars episode in europe is over € . summary: ars is mainly an inflammatory disease triggered by viral infection, and few cases end up developing bacterial infection. in most of the cases, it is a self-resolving disease which diagnosis is mainly clinical and the treatment symptomatic. the incidence of complications is low and independent of antibiotic use. there is a high socioeconomic burden associated to ars. acute rhinosinusitis (ars) is an inflammatory disease affecting the nose and paranasal sinuses with duration up to weeks. the main trigger cause is a viral infection (common cold) that can be prolonged on time (post-viral) and, in a small number of patients, may develop a bacterial infection. it is important to discriminate the different phenotypes of ars to understand the diagnostic and therapeutic requirements in every individual case [ ••]. ars has a significant impact on quality of life [ ••], although it usually is a self-resolving disease and the incidence of chronicity or complications is very low. despite this, both primary care physicians and ent specialists abuse diagnostic tools and overuse drug prescriptions [ •]. the aim of the present article is to review the incidence of ars, discuss its etiology (inflammation versus infection), describe ars different phenotypes, and analyze the recommendations of international guidelines for its management. furthermore, we will highlight the use and abuse of diagnostic tools and prescribed medications, while exploring the similarities and differences between children and adult disease. when there are two or more nasal symptoms, one of which should be either nasal congestion/blockage/obstruction or rhinorrhea (anterior or post-nasal drip), while the others could be either facial pain/pressure or reduction/loss of smell, lasting up to weeks. in children, ars should be considered when there are two or more of the following symptoms: nasal blockage/congestion, discolored nasal discharge, and cough. although it is important to note that there are other infectious etiologies (bacteria, fungi) of this illness, the most common is caused by viruses. the disease may present in three main clinical phenotypes: viral ars or common cold when the episode lasts up to days and post-viral ars when symptoms persist longer than days or worsens after days. bacterial ars is defined by the presence of three or more of the following clinical findings: fever (≥ °c), severe local pain, double sickening, unilateral disease (with discolored mucus), or elevation of c-reactive protein (crp)/erythrocyte sedimentation ratio (esr) in blood test ( fig. ) american guidelines (icar) note similar definitions and symptoms but stratify to ars, when symptoms last up to weeks, and sub-acute rhinosinusitis when the duration is between and weeks. like the european guidelines, they consider viral ars when the disease duration is less than days [ ••]. the prevalence of ars in the general population is variable depending on different studies, noted to be between % and % [ , ] . viral ars or common cold has a very high incidence, presenting two to five episodes per person a year [ ] . in children, this incidence could be up to four times higher [ ] , with urtis being one of the main causes of primary care consultations [ •]. post-viral ars is less common, with an incidence of about episodes per inhabitants a year [ ] in adults (iceland) with a lower frequency in pediatric populations and differences noted among different age groups ( cases per , in ≤ years old, - cases per , in - years old, and cases per , in - years old) [ ] . in a recent study in germany, the incidence was found to be . episodes per inhabitants per year [ ••]. classically, the incidence of bacterial ars is estimated to be . - % of all ars viral infections, although recent studies have suggested it to be higher. the rate of positive cultures is about % in patients with clinical suspicion of bacterial ars [ •]. many predisposing factors for ars have been described: environmental dampness, anatomical factors (particularly in recurrent ars episodes [ ]), mucocilliary impairment, smoking, as well as anxiety and depression [ ••]. there is also a higher incidence of episodes during the cold months fig. definition of ars phenotype bases on epos consensus. the duration of symptoms is used to differentiate viral ars (common cold) from post-viral ars, which is considered when the symptoms persist longer than days or worsen after days. bacterial ars should be suspected at any time when the presence of three or more of the sings or symptoms related to bacterial ars are found (especially in patients with crs at baseline [ •]). on the other hand, laryngo-pharyngeal reflux was not found to be a clear underlying factor [ ••]. one of the most interesting and controversial predisposing factors to develop ars is allergic rhinitis (ar). the incidence of ars in patients with ar was reported to be . times higher than in non-allergic rhinitis [ ars is mainly an inflammatory disease of the nose and paranasal sinuses. usually, a viral infection triggers the inflammatory cascade in the context of common cold. in few cases, this inflammatory condition of the mucosa may facilitate a bacterial infection [ ••]. as a result, three different ars phenotypes are described: viral, post-viral, and bacterial ars. but it should be noted that these entities often overlap, and their symptoms are very similar. regarding viral ars, the rhinovirus has been found to be the cause of % of the common cold episodes [ ] although other viruses such as adenovirus, coronavirus, influenza virus, and even sars-cov- virus (responsible for the recent covid- pandemics) could also be involved [ ] . typically, the common cold has a duration of up to to days. when the symptoms persist after the viral disease (over days), the clinical process is called post-viral ars [ ••]. in a few number of cases ( . - % of all common colds), this inflammatory condition may lead into a bacterial infection [ ] . although ars seems to have similar pathophysiology in children and adults, viral ars incidence is however greater in children, while post-viral ars incidence is more common in adults [ ••]. the symptomatology may also differ. while in adults cough is considered a secondary symptom (except for sars-cov- virus), in children, it is one of the main common symptoms [ ] being strongly considered in the diagnostic protocols [ ••, ••]. on the other hand, posterior rhinorrhea and hyposmia, which are cardinal symptoms in adult, are not in children, likely because it is not easy for the young child to describe or acknowledge them. as in adults, no complementary tests are needed to diagnose ars and its treatment, if there is no suspicion of bacterial origin or complication, should be strictly symptomatic [ ••]. a detailed description of medical therapy in children and adults is provided later in the treatment section. according to epos and american guidelines [ ••, ••], ars diagnosis is strictly based on the sudden onset of ≥ nasal symptoms (nasal congestion/obstruction, rhinorrhea, facial pressure, or loss of smell). this diagnosis may be supported by endoscopic findings (mucosal edema or rhinorrhea), but they are not necessary in a primary care consultation. although the use of imaging tests is not recommended, except in complicated cases [ ••, ], a clear overuse of diagnostic tools has been found, where physicians recommended plain x-ray or ct scan in % and % of post-viral ars episodes and even in % and % of viral (common cold) episodes, respectively [ •]. the most difficult issue is to correctly diagnose bacterial ars (abrs). although a rather invasive technique, the gold standard to diagnose abrs is the antral puncture and culture [ ] . the culture of middle meatus secretions obtained under endoscopic visualization has been demonstrated to have similar specificity and sensibility [ •]. however, a culture result takes a few days, and is not useful in the acute situation. some authors have considered the presence of opacification in the sinuses in x-ray or ct can predict a bacterial origin; however, it has been clearly demonstrated that this is not specific for abrs. as a matter of fact, the majority of patients with common cold present with opacification due to mucous in the paranasal sinuses [ ••]. for this reason, recent studies have been trying to find biochemical markers or specific symptoms that could help to differentiate bacterial from non-bacterial ars. regarding symptoms, unilateral facial or dental pain has been identified as a good predictor of bacterial ars [ ], but with limited evidence. dental pain in the superior jaw has been recently identified to be the symptom more strongly related to bacterial ars [ ••]. classically, the purulence of nasal discharge has also been considered a sign of bacterial infection, but a recent work by ebell et al. [ ••] has showed however that discolored discharge may be also present in post-viral and even in viral cases, thus invalidating the previous correlation with abrs. a body temperature ≥ °c has also been associated with a high risk of bacterial infection , in a recent systematic review, noted that an elevation of c-reactive protein (crp) and/or erythrocyte sedimentation rate (esr) support the diagnosis of abrs, but with poor sensitivity and without being considered a diagnostic marker of the disease. with that said, it does seem clear that the presence of low rates of crp provides evidence against the use of antibiotics [ ] . the epos consensus [ ••] recommends the use of a combination of signs and symptoms to determine the probability of bacterial origin and defines abrs when or more of the following five criteria are present: discolored discharge with unilateral predominance, severe local pain, fever ≥ °c, double sickening, or elevation of crp/esr. these epos criteria have been demonstrated to have better specificity than idsa (infectious diseases society of america) criteria for diagnosing bacterial ars [ •]. the idsa guideline considers bacterial ars when there are ≥ of the following criteria: symptoms lasting for more than days with no improvement, severe symptoms from the onset (fever ≥ °c or discolored discharge from the beginning and during - days), or double sickening after - days [ ] . to summarize, in normal situations (apart from virus epidemics or pandemics), there is no need for complementary diagnostic tools to diagnose viral or post-viral ars, while a blood test to determine crp/esr may be helpful when abrs is suspected. the first step in common cold and ars management is the prevention of viral infection mainly reinforcing the use of hygiene rules such as hand washing. in special epidemic situations, such as the covid- pandemics, more strict recommendations such as social distancing, facemask and eye guards, as well as home confinement may be required [ ] . the updated management of viral (common cold) and postviral ars, and abrs according to epos , is summarized in table . the highlights for medical therapy, according to phenotypes and different guidelines [ ••, ••], include the following: -recommended therapy (mainly symptomatic): paracetamol; non-steroidal anti-inflammatory drugs (nsaids); second-generation antihistamines with short-term benefit in reducing symptoms the first days [ •]; nasal decongestants with small effect in nasal congestion in adults [ ] ; combination of analgesics and nasal decongestants [ ] ; ipratropium bromide for reducing rhinorrhea [ ] ; probiotics; zinc when administered the first h after the onset of symptoms [ , •] ; nasal saline irrigations [ ] ; vitamin c, in selected patients with suspected deficit or with high levels of physical activity [ ] ; and some herbal medicines (bno , cineole, and andrographis paniculata sha- ) [ ••]. -not recommended therapy: antibiotics [ ] , intranasal corticosteroids (incs) [ ] , heated humidified air [ • ], echinea products [ ] , homeopathy products [ • ]. -preventive therapy recommended: probiotics, with slight benefit but low-quality evidence [ ] , practice of moderate and regular exercise [ ] . -recommended therapy: symptomatic treatment; incs, although the beneficial effect in symptoms is clear, as ars is a self-limited disease, consider the need of their use depending on the severity of symptoms [ ••]; sinfrontal, a homeopathy product with slight benefit but low evidence [ ] ; and some herbal compounds such as cyclamen europaeum, which improves some symptoms but with low evidence [ ] , pelagorium sidoides [ ] , and bno , mainly for nasal congestion [ ] . - the challenge in discriminating bacterial from nonbacterial ars often leads to an over-diagnosis of abrs, which result in an overuse of diagnostic testing and early unnecessary prescription of antibiotics. in a study from the uk, % of the consultations for rhinosinusitis resulted in antibiotic prescription, while only % were deemed appropriate [ ••] . the same was true in the netherlands where % of the interviewed primary care physicians chose an antibiotic as treatment for a patient with moderate severe acute rhinosinusitis [ ] . in a study from spain, even when abrs patients were excluded, the use of antibiotics was found to be around % in patients with common cold and % in those with post-viral ars [ •]. the american rhinosinusitis guidelines highlight the fact that although effective in adults, the actual benefit of antibiotics is small, needing to treat between and patients to get individual to improve [ ••]. the overuse of antibiotics has also been associated with an increment of antibiotic resistance, which is directly related to increased morbidity and mortality due to resistant bacterial infections [ , •] . so, once again, in spite of the clinical suspicion of abrs, the decision to treat a patient with antibiotics should be made on an individual basis. in order to help to decrease the inappropriate use of antibiotics for ars, published studies emphasize the importance of physician communication skills on the use of antibiotics, responsible justification, and peer comparison and training of physicians, to help according to epos recommendations, one should rule out a complication when a patient presents with one or more of the following signs and/or symptoms: periorbital edema/erythema, displaced globe, double vision, ophtalmoplegia, reduced visual acuity, severe headache, frontal swelling, signs of sepsis, or other neurological signs [ ••]. regarding diagnosis of complications, the accuracy of a clinical diagnosis is estimated to be around % and the accuracy of ct % [ ] . mri is, however, considered the "gold standard," as it is more sensitive than ct scan. when available, mri should be the imaging modality of choice, having the additional diagnostic value to exclude or confirm cavernous sinus thrombosis and soft tissue involvement [ , ] . according to epos guidelines, the main indications for surgical intervention in orbital complications of abrs are evidence of subperiosteal or intraorbital abscess in ct scan or mri (exception for small volume abscesses). subperiosteal abscess in children is not an absolute indication for immediate surgical intervention. conservative measures can be safe and effective if appropriately used. a reduced visual acuity, loss of color vision, affected afferent pupillary reflex, or inability to assess vision, however, are indications for urgent surgery. when conservative treatment is chosen, progression or no improvement in orbital signs (diplopia, ophthalmoplegia, proptosis, swelling, chemosis) or in the general condition (fever, infection parameters), after h of intravenous antibiotic treatment is also an indicator of the need for emergency surgery [ ••]. endocranial complications of abrs are usually associated with fronto-ethmoidal or sphenoid rhinosinusitis [ ] and include epidural or subdural empyema, brain abscess, meningitis, cerebritis, and superior sagittal and cavernous sinus thrombosis. they may present with specific central nervous system signs, such as nausea and/or vomiting, neck stiffness, and altered mental stat, or non-specific symptoms and signs (high fever, headache, reduced consciousness), or can even be silent [ ] . the pathogens most commonly isolated are streptococcus and staphylococcus species including methicillin-resistant (mrsa) and anaerobes [ ] . the recommended treatment involves neurosurgical drainage procedures and endoscopic drainage of the paranasal sinuses (most often the frontal sinus) [ •]. despite the fact that ars is usually a self-limited, with low risk of further morbidity, it presents a considerable burden to public health [ ••], being an important cause of work absenteeism [ ] . as reported above, a significant overuse of diagnostic tests and medications has been reported in multiple countries [ •, , ], with very few studies addressing the economic impact of ars. in the s, the cost of ars reached us$ million per year in the usa [ ] . in europe, a total cost of~€ per ars episode was recently examined, with the major cost ( %) attributable to indirect costs [ ••]. recent data from spain demonstrated that direct costs of ars where greater in postviral (~€ ) than viral (€ ) ars episodes, and not surprisingly, severe cases resulted in greater direct cost [ ] , with the main driver of direct cost attributable to medical visits [ , ] . as the economic costs are quite large, there is a clear unmet need with further research needed to optimize appropriate testing and therapy. concerning medical visits, health education should be improved and encouraged, teaching the public that ars is a self-limited and non-complicated disease, which usually only requires symptomatic treatment, while medical consultations should be restricted to severe or complicated cases. on the other hand, decreasing the costs related to diagnosis and treatment are directly linked to medical management. svensson et al. showed that the cost of treating ars with topical corticosteroid was much lower compared with the use of amoxicillin [ ] . regarding the costs related to antibiotic use, cramer et al. reported a dramatic decrease in costs when t guideline recommendations were followed, compared with when they were not (us$ vs. us$ million per year) [ ••] . therefore, knowledge of up-to-date guidelines and scientific recommendations is strongly recommended for both primary care physicians and specialists in order to avoid the overuse of diagnostic tools and prescription of unnecessary medications, especially antibiotics, in the management of ars [ • ]. & the first and most important rule for common cold and ars management is the prevention of viral infection through hygiene behavior such as hand washing. in special epidemic situations such as the covid- pandemics, more strict recommendations such as social distance and home confinement may be required. & post-viral ars is mainly an inflammatory disease, which usually begins as a viral infection (common cold, urti, or viral ars), but may persist longer than - days or worsen after days. some rare cases (less than %) may develop bacterial ars. & the incidence of viral ars is very high ( - episodes/ person/year) while post-viral ars has an incidence about episodes per people a year. & the most common ars symptoms are nasal blockage or congestion and anterior or posterior rhinorrhea. in adults, facial pain or pressure and loss of smell are also cardinal symptoms, while in children, cough is more relevant. & the diagnosis is clinical, based on the sudden onset of nasal symptoms (nasal blockage/obstruction, nasal discharge/rhinorrhea, hyposmia, and facial pain/pressure), and there is no need for complementary tests. & the distinction between bacterial and non-bacterial ars remains a diagnostic challenge. the presence of a fever, unilateral focality, local pain, and elevation of cpr/ers seems to be the best way to predict bacterial ars. & in special situations, such as the covid- pandemics, a sudden severe loss of smell (anosmia), even with the absence of other nasal or general symptoms (dry cough, fever), should be considered a symptom of suspicion while the definitive diagnosis should be specific by using a pcr test for the sars-cov- virus. & viral ars treatment should be symptomatic (analgesic, nsaids). some herbal compounds or minerals like zinc may also help. & intranasal corticoids have proven to be useful in post-viral ars, but, being a self-resolving disease, its use should be individualized. antibiotics, mucolytic, and antihistamines have not demonstrated any benefit in patients with postviral ars. & antibiotics have only shown some effect in bacterial ars, although there is a high rate of resolution even without their use. therefore, individual considerations, taking into account the adverse effects and increased drug resistances, have to be made before prescribing antibiotics. & complications are very uncommon, and their incidence is not dependent on the antibiotic use. orbital complications are common in children while intracranial complications are less frequent. the presence of ophthalmological or neurological symptoms should raise the suspicion of a complication and imaging tests should be obtained. therapeutic management of ars complications includes hospital admission and intravenous antibiotics and often requires surgery (orl and/or neurosurgery). & the economic burden of ars is incredibly high due to the large number of medical visits, the misuse of diagnostic testing, and the overuse of medications, as well as for the high indirect costs. disseminating the concept of ars being a mild and self-resolving disease among patients and physicians remains an unmet need that is required to reduce the high costs of this illness. papers of particular interest, published recently, have been highlighted as: hand hygiene and the novel coronavirus pandemic: the role of healthcare workers cochrane database syst rev. :cd this systematic review stated that antihistamines have a limited but significant short-term (days one and two of treatment), but not in the mid to long-term nasal decongestants in monotherapy for the common cold oral antihistamine-decongestant-analgesic combinations for the common cold intranasal ipratropium bromide for the common cold zinc for the common cold zinc acetate lozenges may improve the recovery rate of common cold patients: an individual patient data meta-analysis saline nasal irrigation for acute upper respiratory tract infections vitamin c for preventing and treating the common cold antibiotics for the common cold and acute purulent rhinitis corticosteroids for the common cold ): cd this recent cochrane review advice against the use of heated echinacea for preventing and treating the common cold homeopathic medicinal products for preventing and treating acute respiratory tract infections in children cochrane commentary: probiotics for prevention of acute upper respiratory infection the effect of exercise on prevention of the common cold: a meta-analysis of randomized controlled trial studies efficacy of a complex homeopathic medication (sinfrontal) in patients with acute maxillary sinusitis: a prospective, randomized, double-blind, placebo-controlled, multicenter clinical trial cyclamen europaeum nasal spray, a novel phytotherapeutic product for the management of acute rhinosinusitis: a randomized double-blind, placebo-controlled trial pelargonium sidoides extract for acute respiratory tract infections placebo-controlled, randomized doubleblind clinical trial with sinupret sugar coated tablets on the basis of a therapy with antibiotics and decongestant nasal drops in acute sinusitis in this work, patients with bacterial ars were randomized in two groups both using antibiotics, steroids, and nasal saline but in the study group, sodium hyaluronate was added to nasal saline solution. symptom vas showed statistically significant differences between the two groups, in particular for smell in this study, the proportions of consultations, concentrated on acute presentations in patients without relevant comorbidities, resulting in an antibiotic prescription were established. the prescribing proportions were then compared with previously established 'ideal' proportions by condition. for rhinosinusitis, a huge difference between real ( % of cases) vs ideal ( %) prescriptions was found management of rhinosinusitis in dutch general practice burden study group. mortality and hospital stay associated with resistant staphylococcus aureus and escherichia coli bacteremia: estimating the burden of antibiotic resistance in europe : this study analyzed the effect of drug resistant gram-negative bacteria in the uk. over a period of years, cumulative estimates indicate , cases of resistant gram-negative bacteremia presentation and treatment of acute maxillary sinusitis in general practice: a french observational study this is a retrospective cohort study among patients hospitalized due to ars complication, where the use or not of antibiotics previous admission was recorded. they concluded that antibiotic treatment of ars in general practice does the pathogenesis of orbital complications in acute sinusitis this is a retrospective study analyzing patients with ars complications. the main findings were: st ) crs is very common in adults with orbital complications of rhinosinusitis, with previous sinus surgery and orbital wall dehiscence being noticeably common; nd ) older patients are at risk for more severe complications most preschool children hospitalized for acute rhinosinusitis had orbital complications, more common in the youngest and among boys the authors analyze pediatric patients hospitalized with ars complications, showing that most children hospitalized for ars had orbital complications, this being more common in boys and < years old the role of computed tomography and magnetic resonance imaging in patients with sinusitis with complications imaging findings of the orbital and intracranial complications of acute bacterial rhinosinusitis intracranial complications of sinusitis in children and adolescents and their outcomes pediatric sinogenic epidural and subdural empyema: the role of endoscopic sinus surgery the complications of sinusitis in a tertiary care hospital: types, patient characteristics, and outcomes the significance of streptococcus anginosus group in intracranial complications of pediatric rhinosinusitis this study investigates the role of endoscopic surgery in patients with intracranial complications and conclude that patients who underwent ess prior to a neurosurgical procedure had significantly less risk of needing a neurosurgical intervention contemporary assessment of the disease burden of sinusitis. the economic burden and symptom manifestations of chronic rhinosinusitis first line management of sinusitis: a national problem? direct costs of acute rhinosinusitis in spain-a prospective and observational study (prosinus) costeffectiveness of mometasone furoate nasal spray in the treatment of acute rhinosinusitis this study compared the costs of antibiotic prescription for ars in the uk, with the ideal cost when following guideline recommendations. study results reported that current us antibiotic prescriptions for ars account for an estimated this is a review of the most recent recommendations and guidelines for different ent infections, including ars, which concludes that there is a need of continuous medical education and changes in physicians publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -al v b x authors: crotty, matthew p.; meyers, shelby; hampton, nicholas; bledsoe, stephanie; ritchie, david j.; buller, richard s.; storch, gregory a.; kollef, marin h.; micek, scott t. title: impact of antibacterials on subsequent resistance and clinical outcomes in adult patients with viral pneumonia: an opportunity for stewardship date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: al v b x introduction: respiratory viruses are increasingly recognized as significant etiologies of pneumonia among hospitalized patients. advanced technologies using multiplex molecular assays and polymerase-chain reaction increase the ability to identify viral pathogens and may ultimately impact antibacterial use. method: this was a single-center retrospective cohort study to evaluate the impact of antibacterials in viral pneumonia on clinical outcomes and subsequent multidrug-resistant organism (mdro) infections/colonization. patients admitted from march to november with positive respiratory viral panels (rvp) and radiographic findings of pneumonia were included. patients transferred from an outside hospital or not still hospitalized hours after the rvp report date were excluded. patients were categorized based on exposure to systemic antibacterials: less than days representing short-course therapy and to days being long-course therapy. results: a total of patients (long-course, n = ; short-course, n = ; mixed bacterial-viral infection, n = ) were included with most being immunocompromised ( . %) with active malignancy the primary etiology ( . %). rhinovirus/enterovirus ( %), influenza ( %), and parainfluenza ( . %) were the viruses most commonly identified. a total of different systemic antibacterials were used as empiric therapy in the patients with pure viral infection for a total of days-of-therapy. vancomycin ( . %), cefepime ( . %), azithromycin ( . %), meropenem ( . %), and linezolid ( . %) were most frequently used. in-hospital mortality did not differ between patients with viral pneumonia in the short-course and long-course groups. subsequent infection/colonization with a mdro was more frequent in the long-course group compared to the short-course group ( . vs . %; p = . ). conclusion: this study found that long-course antibacterial use in the setting of viral pneumonia had no impact on clinical outcomes but increased the incidence of subsequent mdro infection/colonization. interactions between viral and bacterial respiratory pathogens have been recognized dating back to the influenza pandemic [ ] . bacterial pneumonia is a wellrecognized serious complication of influenza infections and coinfections are commonplace [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . respiratory syncytial virus (rsv), parainfluenza viruses, rhinoviruses, and adenoviruses have also been linked to bacterial coinfections in humans [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . animal studies have suggested synergism between bacterial pathogens and other respiratory viruses [ , ] . the relationship between viral and bacterial respiratory infections creates a difficult situation for clinicians determining the appropriate use of antimicrobials as they treat hospitalized patients with pneumonia while also trying to minimize the development and selection of resistant organisms. respiratory viruses are increasingly recognized as the primary etiology of pneumonia among patients requiring hospitalization ( . - % of pneumonia cases) [ , ] . advanced technologies using multiplex molecular assays and pcr improve the diagnostic ability to identify viral pathogens in a timely manner and may impact the use of antibacterials in patients with no bacterial infection identified. several studies have investigated the impact of respiratory viral pathogen identification on antibacterial exposure [ ] [ ] [ ] [ ] . decreased antibiotic use was observed in two pediatric studies assessing the impact of rapid viral diagnostic tests for respiratory tract infections; however, these results were not mirrored in similar adult studies [ ] [ ] [ ] . these studies all used immunofluorescent staining as the primary diagnostic technology. to our knowledge, only one study using pcr-based respiratory virus detection has been reported and found no change in antibacterial use with improved diagnoses for lower respiratory tract infections [ ] . broad-spectrum antibacterial exposure increases the risk of subsequent infections with multidrug-resistant organisms (mdros) and leads to a vicious cycle of empiric broad-spectrum antibacterials to combat increasingly resistant organisms [ ] . we and others have previously shown that patients with culture-negative pneumonia frequently receive treatment with broadspectrum antibiotics, usually in excess of - days of therapy despite lack of evidence for a bacterial etiology of infection [ , ] . it is important to recognize that these studies were performed prior to the availability of rapid viral diagnostics which may have influenced how antibiotics were used during those study periods. use of new diagnostic technologies for respiratory virus detection could decrease unnecessary antibacterial exposures and subsequent mdro infections. this study aimed to describe the use of continued empiric antibacterials in patients with known viral pneumonia and to determine the impact of such therapies on subsequent bacterial infections/colonization and clinical outcomes. this was a single-center, retrospective cohort study of patients with a positive respiratory virus panel (rvp) at barnes-jewish hospital (bjh) (a -bed urban academic medical center in st. louis, mo, usa) between march and november . the study protocol was approved by the bjh, washington university, and st. louis college of pharmacy institutional review boards. consecutive patients who were ≥ years of age and admitted to bjh for ≥ hours were assessed for study inclusion. patients were identified through a query of an internal database which tracks respiratory viruses. patient admissions in which a respiratory virus was identified by filmarray ® respiratory panel (farp) assay (biofire diagnostics, salt lake city, ut, usa) were screened for inclusion in this study. included patients had to meet the study definition of viral pneumonia. patient admissions were excluded if rhinovirus or enterovirus was identified by nasopharyngeal (np) swab alone but could also be included if identified from lower respiratory tract specimens. additionally, patients were excluded if there was a virus identified by rvp within the previous days or if a bacterial pathogen was identified by the respiratory panel. patients who were transferred from an outside hospital and those who were discharged/died or were made comfort care less than hours after the index rvp report date were also excluded to better evaluate continued empiric antibacterial use in this population. the farp assay is a multiplexed nucleic acid test capable of simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids. viral pneumonia was defined as identification of a respiratory virus by farp and a new or progressive radiographic infiltrate within hours of the index rvp. short-course antibacterial administration was defined as treatment with systemic antibacterials for less than days while long-course therapy was defined as - days of antibiotics after the index rvp report date with no bacterial infection identified at any point during the admission. mdros were defined as methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococci (vre), or a bacterium exhibiting in vitro resistance to at least one drug in ≥ classes of antibacterials for which the organism is not intrinsically resistant [ ] . subsequent isolation of a mdro was defined as isolation of a study-defined mdro or a positive epidemiologic screen (mrsa np swab; vre stool specimen) ≥ days after the report date of the index rvp. positive epidemiologic screens for mrsa or vre were only considered to be subsequent colonization of an mdro if the patient had at least one screen in the previous days and all screening tests were negative prior to the index rvp. immunocompromised status was defined as a diagnosis of human immunodeficiency virus (hiv), active malignancy (stem cell transplant or receiving chemotherapy), solid organ transplant, or currently on immunosuppressive therapy (prednisone mg/day for at least days or equivalent). information regarding the farp including time of collection/report, type of specimen, patient location at time of collection, and resulting findings were obtained from an internal database. additionally, all available aerobic and anaerobic bacterial cultures were evaluated based on electronic medical record (emr) query. in vitro susceptibilities of isolated bacterial pathogens were evaluated as reported per institutional practices. urine legionella antigen, direct-fluorescent antibody for pneumocystis jiroveci, and clostridium difficile toxin assay were also evaluated. data collected to describe patient groups included demographic information, comorbid conditions, and clinical outcomes. charlson's comorbidity index was used as a summative score of underlying disease states [ ] . antibacterial exposures were calculated using emr orders for systemic antibacterials. days of therapy (dot) and dot normalized per patient-days (dot/ pd) were calculated as described previously [ ] . emr queries were used to acquire patient information where possible. manual chart review was used to validate and supplement all emr queries. the primary endpoint was subsequent isolation of a mdro ≥ days after the index rvp report date. inhospital mortality and readmission at , , and days were secondary endpoints. dichotomous variables were compared using the chisquare test or fisher's exact test as appropriate. continuous variables were compared using the mann-whitney u test. all tests were two-tailed and p < . was considered significant. univariate analyses were performed to compare the group that received long-course antibacterials and the group that received short-course antibacterial therapy. kaplan-meier survival analysis was used to compare risk of in-hospital mortality between comparator groups while censoring for patient discharge. all statistical analyses were performed using spss software (ibm spss statistics, version . ; chicago, il, usa). a total of consecutive patients (long course, n = ; short course, n = ; mixed bacterial-viral infection, n = ) were included in this study (fig. ). demographic and clinical characteristics are summarized in table . the median age was years and . % were male. a majority of patients in the cohort were deemed to be immunocompromised ( . %), with active malignancy the most common etiology ( . %). only of patients had been admitted within the days prior to the index admission. figure provides a breakdown of the subgroup of patients screened in the emergency department setting according to the presence of pure viral and mixed infections. rhinovirus/enterovirus ( . %), influenza ( . %), and parainfluenza ( . %) were the viruses most commonly identified in the cohort ( table ). of the total patients, had multiple respiratory viruses identified by rvp: rsv ( of ), rhinovirus/enterovirus ( of ), influenza ( of ), human metapneumovirus ( of ), parainfluenza ( of ), coronavirus ( of ), and adenovirus ( of ). specimens resulting positive for a respiratory virus in the cohort included np swabs ( . %), bronchoalveolar lavage ( . %), tracheal aspirates ( . %), bronchial washes ( . %), and sputum samples ( . %). the rvp identified a respiratory virus from multiple specimens in patients. among the patients with multiple positive specimens, the most common were bronchoalveolar lavage ( of patients) and tracheal aspirates ( of patients). no significant differences in comorbidities between groups were identified ( table ) . patients with mixed viral-bacterial infection had statistically greater apa-che ii scores and were more likely to require vasopressor support compared with patients with pure viral infection. there was no significant difference in the number of immunocompromised patients between groups, although numerically more patients who received long-course antibacterials had an active malignancy or solid organ transplant compared with patients receiving short-course therapy. virus types identified by farp assay were similar in all three patient groups ( table ). the bacterial coinfecting organisms identified in patients with mixed viral-bacterial infection are presented in table . respiratory coinfection with a bacterial pathogen was most common, with s. aureus, streptococcus pneumoniae, and pseudomonas aeruginosa accounting for the most frequent respiratory coinfecting bacteria. a total of different systemic antibacterials were used as empiric treatment in patients with viral pneumonia without bacterial coinfection for a total of dot. vancomycin ( . %), cefepime ( . %), azithromycin ( . %), meropenem ( . %), and linezolid ( . %) were the most frequently used empiric antibacterials in patients with viral pneumonia without bacterial coinfection (fig. ) . the most common regimens used in viral pneumonia without bacterial coinfection were vancomycin plus cefepime ( . %) and vancomycin plus meropenem ( . %). a total of ( . %) patients with viral pneumonia without bacterial coinfection received empiric mrsa coverage with vancomycin or linezolid. empiric antibacterial therapy was continued for a median of . days (interquartile range, . - . days) in viral pneumonia without bacterial coinfection, with most ( %) being days on intravenous antibacterials. total antibacterial exposure differed between the longcourse and short-course groups at and dot/ pd, respectively ( the number of patients with subsequent mdro colonization or infection was not significantly different between groups (table ). however, in instances of subsequent infection or colonization, where a single patient could have more than one organism, there was a higher rate of mdro identification among isolates from the group that received long-course antibacterials compared with the group receiving short-course therapy ( . vs. . %; p = . ) ( table ) in-hospital mortality was statistically higher for the mixed-infection group compared with the long-course therapy group (table ) . kaplan-meier survival analysis showed that the mixed-infection group had the lowest overall survival, but these differences were not statistically icu admit was determined to be oncology related if the patient fit the study definition for active malignancy *statistically significant difference (p < . ) between long-course and mixed-infection groups **statistically significant difference (p < . ) between short-course and mixed-infection groups bmi body mass index, cci charlson's comorbidity index, copd chronic obstructive pulmonary disease, esrd end-stage renal disease significant (fig. ) . icu mortality was also significantly higher for patients in the mixed-infection group compared with the long-course therapy group. patients receiving long-course therapy or those with mixed infection had statistically longer icu length of stay compared with patients receiving short-course therapy. hospital readmission rates were similar between groups at , , and days after index hospitalization discharge. this study compared a cohort of patients with viral pneumonia and mixed viral-bacterial infection based on exposure to continued empiric antibacterials after respiratory virus identification. more of the subsequent infecting or colonizing bacterial isolates from the group with pure viral pneumonia who received continued long-course antibacterials were defined as mdros compared with the short-course group (p = . ). these findings suggest that more prolonged exposure to broad-spectrum antibacterials in patients with viral pneumonia may have promoted resistance in these patients. no benefit of continued empiric antibacterials for patients with pure viral pneumonia was seen in this study. the risk of bacterial coinfection in the setting of viral pneumonia, especially with influenza, creates a challenging situation for clinicians. the potential detrimental impact of not treating a bacterial pathogen weighs data expressed as number (% of total) or median (interquartile range) los length of stay, mdro multidrug-resistant organism, mrsa methicillin-resistant staphylococcus aureus, vre vancomycin-resistant enterococci *statistically significant difference (p < . ) between short-course and long-course groups **statistically significant difference (p < . ) between long-course and mixed-infection groups ***statistically significant difference (p < . ) between short-course and mixed-infection groups heavily on the decision process and downstream effects of such therapies may be disregarded. our findings of similar clinical outcomes between patients with pure viral pneumonia who received long-course antibacterials after virus recognition and those who did not may suggest opportunity for de-escalation of empiric antibacterial therapy when viral pneumonia is identified. a previous randomized controlled trial by oosterheert et al. [ ] evaluated implementation of real-time pcr rapid diagnostics for respiratory pathogen identification. they found increased diagnostic yield with the assay but no difference in antibiotic use, and hypothesized that reluctance to change treatment based on testing results may have inhibited cost-effectiveness from being demonstrated. in our study, systemic antibacterials were discontinued following identification of a respiratory virus by rvp for several patients; however, whether virus identification directly led to discontinuation of antibacterials cannot be determined. the willingness of prescribers to de-escalate and stop antibacterials in this setting may suggest increased recognition of the role of viral pathogens in pneumonia. additionally, the expanded panel of viruses detected may have factored into how results were perceived, as prescribers may have been more likely to attribute pneumonia to newly detectable viruses such as human metapneumovirus. however, it is not possible to definitively determine the rationale for stopping antibacterial therapy. timely antibiotic administration is crucial for treating hospitalized patients with suspected pneumonia [ ] . antimicrobial de-escalation attempts to balance the use of these essential drugs up front with the emergence of resistance [ ] . the optimal strategy for de-escalation of antibacterials in the setting of viral pneumonia without an identified bacterial coinfection is unclear. our study found no difference in clinical outcomes based on antibiotic duration of therapy in patients with viral pneumonia despite significantly different total antibacterial exposure (dot/ pd) between groups. byington et al. [ ] found previously that improved diagnostic technologies enhancing detection of respiratory viruses decreased antibacterial use at a children's hospital. the authors concluded that improved diagnostics are an important tool in decreasing unnecessary antibacterial prescribing. our study similarly illustrated the potential impact of respiratory virus diagnostics on antibacterial use in an adult population. c. difficile infection is a major cause of morbidity and mortality in us hospitals and has been directly linked to exposure to broad-spectrum antibiotics [ , ] . in a cohort of hospitalized adult patients, shiley et al. [ ] found that significantly more patients who continued to receive antibacterials after diagnosis of a viral respiratory tract infection developed c. difficile infection. one patient in our study who was treated with long-course antibacterials after identification of a respiratory virus also developed c. difficile infection. strategies to best limit the use of unneeded antibacterials are important to curtail against the growing issues of c. difficile and resistance, and may be aided by de-escalation approaches using enhanced viral diagnostic technologies. limitations of this study should be noted. first, this was a small retrospective cohort study of patients at a single institution and may not be representative of all settings. it is important to note that bjh is a regional specialty referral hospital and not a community hospital. this accounts for the case mix with a high prevalence of immunosuppressed patients and the low prevalence of narrow spectrum empiric antibiotic utilization. the small number of patients meeting inclusion criteria did not allow for definitive conclusions to be made regarding group comparison as a lack of statistically significant differences being found could be due to the lack of sample size. second, patients were determined to have viral pneumonia based on virus identification and radiographic findings but other markers of illness, such as white blood cell count and fever, were not considered and the retrospective nature of the study did not allow evaluation of what drove continuation of antibacterials in some patients but not others. moreover, we did not attempt to identify risk factors associated with pure viral pneumonia. third, although coinfecting bacterial pathogens were not identified in patients with pure viral pneumonia, it is impossible to prove that they were not present. receipt of antibacterials prior to obtaining bacterial cultures could have limited the diagnostic yield of bacterial cultures in some cases and yield from bacterial cultures is not perfect. finally, all of the viral pneumonia cases occurred in a -month period. viral epidemiology during this time may not be representative of all seasons. influenza h n p was the primary influenza virus identified in our study ( %). incidence rates of bacterial coinfection and coinfecting organisms may differ from year to year and from virus type to virus type, which may hinder application of de-escalation strategies using the results of this study. it is not possible to directly link the development of subsequent mdro infections/colonization and c. difficile infection seen in our study to the continued empiric antibacterials administered. all of the patients included in the cohort received antibacterials at some point during their index hospitalization and infection control measures were not directly assessed in these patients. additionally, hospitalization itself probably increases the risk of these patients being colonized with mdros. use of cephalosporins and vancomycin, two of the most commonly administered empiric agents in our study, have been implicated as increasing the prevalence of vre, the most commonly identified subsequent mdro in this study [ , ] . decreasing exposure to broad-spectrum antibacterials such as third-generation and fourth-generation cephalosporins and vancomycin would be expected to lessen the incidence of vre and other mdros as was seen in this study, but the risk of development and transmission of resistance in the hospital cannot be completely eliminated. antibacterials are extraordinarily important in the treatment of many hospitalized patients and their use is often warranted. decreasing unnecessary use may help curb acquirement of resistant organism in healthcare settings but even appropriate use can lead to the development of resistance. only through multifaceted efforts of infection control and antimicrobial stewardship can the spread of mdros between patients, clinicians, workers, and visitors be diminished. this study highlights the potential benefits of improved diagnostics for respiratory viruses, primarily the potential for decreased antibacterial exposure and thus decreased selective pressure for resistant bacterial isolates. antibacterial exposure applies selective pressure and promotes colonization/infection by resistant organisms including mrsa and vre [ , ] . halting this process is essential to maintain effective therapeutic options in the future and may be aided by discontinuation of antibacterials in cases of viral pneumonia. in our study, patients with viral pneumonia exposed to long-course antibacterials had more occurrences of subsequent infection or colonization with mdro isolates. in contrast, the number of patients with subsequent mdro infection or colonization was not different between groups although this may be due to the small number of patients in each group. no differences in clinical outcomes, including in-hospital mortality and readmission rates, were observed between patient groups. in the setting of viral pneumonia and no coinfecting bacterial pathogens, discontinuation of antibacterials is reasonable in many if not most cases, and may allow for decreased overall antibacterial use. enhanced diagnostic technologies can potentially be incorporated into antimicrobial stewardship practices to allow for de-escalation of unnecessary antibacterials. these findings warrant further investigation to determine the applicability of an antibacterial deescalation approach in the setting of viral pneumonia. in this single-center retrospective cohort, patients with viral pneumonia who continued to be treated with systemic antibacterials days after virus identification were more likely to have a subsequent infection or colonization with a mdro than were patients in whom systemic antibacterials were stopped. in-hospital mortality based on kaplan-meier survival analysis and readmission rates were not different between groups based on 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meta-analysis vancomycin-resistant enterococcal infections submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www authors' contributions mpc and stm conceived the study. mpc, stm, djr, mhk, rsb, and gas participated in the study design. nh and sb performed the electronic medical record queries. mpc and sm conducted data collection and managed the data, including quality control. mpc and stm analyzed the data and interpreted the results. mpc, stm, and djr drafted the manuscript, and all authors contributed substantially to its revision. all authors read and approved the manuscript. key: cord- -zh wzza authors: magleby, reed; westblade, lars f; trzebucki, alex; simon, matthew s; rajan, mangala; park, joel; goyal, parag; safford, monika m; satlin, michael j title: impact of sars-cov- viral load on risk of intubation and mortality among hospitalized patients with coronavirus disease date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: zh wzza background: patients hospitalized with coronavirus disease (covid- ) frequently require mechanical ventilation and have high mortality rates, but the impact of viral burden on these outcomes is unknown. methods: we conducted a retrospective cohort study of patients hospitalized with covid- from march to april , at two hospitals in new york city. sars-cov- viral load was assessed using cycle threshold (ct) values from a reverse transcription-polymerase chain reaction assay applied to nasopharyngeal swab samples. we compared patient characteristics and outcomes among patients with high, medium, and low admission viral loads and assessed whether viral load was independently associated with risk of intubation and in-hospital mortality. results: we evaluated patients with covid- . higher viral load was associated with increased age, comorbidities, smoking status, and recent chemotherapy. in-hospital mortality was . % with a high viral load (ct< ; n= ), . % with a medium viral load (ct - ; n= ), and . % with a low viral load (ct> ; n= ; p< . ). the risk of intubation was also higher in patients with a high viral load ( . %), compared to those with a medium ( . %) or low viral load ( . %; p< . ). high viral load was independently associated with mortality (adjusted odds ratio [aor] . ; % confidence interval [ci]: . - . ; p< . ) and intubation (aor . ; % ci: . - . ; p< . ) in multivariate models. conclusions: admission sars-cov- viral load among hospitalized patients with covid- independently correlates with the risk of intubation and in-hospital mortality. providing this information to clinicians could potentially be used to guide patient care. severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel pathogen that has rapidly caused a devastating pandemic of coronavirus disease (covid- ) . as of june , , sars-cov- had infected more than million people and killed more than , people throughout the world [ ] . although the majority of patients who develop covid- have mild presentations [ ] , - % of patients who are hospitalized require mechanical ventilation and up to % of hospitalized patients die [ ] [ ] [ ] [ ] [ ] . investigations of risk factors for intubation and mortality with covid- in hospitalized patients have largely focused on patient characteristics, such as older age, obesity, and comorbidities, as well as presenting symptoms and laboratory parameters [ , [ ] [ ] [ ] . in contrast, the impact of sars-cov- viral load on clinical outcomes in hospitalized patients has not been thoroughly investigated. in two studies of hospitalized patients in china, those with severe presentations of covid- had higher viral loads than those with mild presentations, but the impact of sars-cov- viral load on the risk of intubation or death was not evaluated [ , ] . the current standard-of-care test to diagnose covid- is to collect a nasopharyngeal (np) swab and use a reverse transcription-polymerase chain reaction (rt-pcr) assay to detect sars-cov- rna [ ] . these rt-pcr assays only report to clinicians whether sars-cov- is detected or not detected. however, these assays also contain quantitative information on cycle threshold (ct) values that are inversely correlated with viral load and are not reported clinically. we hypothesized that assessing sars-cov- viral load by analyzing ct values from an initial np swab sample could be a clinically valuable tool to identify patients at highest risk of intubation and death and provide insights into the pathogenesis of covid- . we therefore conducted this retrospective analysis of sars-cov- viral loads on admission, clinical presentations, and outcomes at two affiliated new york city hospitals using a high-throughput rt-pcr assay. m a n u s c r i p t this retrospective observational study consisted of all patients who were hospitalized at newyork-presbyterian hospital/weill cornell medical center and affiliated lower manhattan hospital and had a np swab sample collected and analyzed for sars-cov- by the cobas rt-pcr system (roche molecular systems, inc., branchburg, nj) between march , and april , . the predominant np swab collection and transport kits used were the bd universal viral transport system (becton, dickinson and company, franklin lakes, nj) and the universal transport medium (hardy diagnostics, santa maria, ca). patients who did not have an np swab sample collected and analyzed within one day of hospital admission or whose sample was analyzed on a different diagnostic platform or at a different institution were excluded. the policy during the study period was to only perform sars-cov- tests in patients who were thought to require hospital admission; however, some patients who were tested were subsequently discharged from the emergency department (ed) without hospital admission. the cobas sars-cov- rt-pcr test received emergency use authorization approval by the united states food and drug administration and was performed according to the manufacturer's instructions [ ] . this assay amplifies two different targets within the sars-cov- genome: orf ab, a sars-cov- -specific target and the e gene, a pan-sarbecovirus target that is present in sars-cov- and sars-cov, but not in seasonal coronaviruses or a c c e p t e d m a n u s c r i p t middle east respiratory syndrome-cov. for routine clinical care, results are classified as detected if either the orf ab or e gene is detected, or not detected if neither target is detected. however, the instrument also generates a ct value for each target that correlates inversely with quantitative viral load and is not released to clinicians. the ct value represents the number of replication cycles required for sufficient gene amplification to produce a fluorescent signal that crosses a predefined threshold. for this study, we reviewed ct values for both gene targets for all initial sars-cov- tests that were performed on np swab samples that were collected from study subjects for routine clinical care within one day of hospital admission. we separated the ct values for the sars-cov- -specific target (orf ab) into terciles based upon the quantitative values. we then designated high viral load samples as the lowest ct tercile, medium viral load samples as the middle tercile, and low viral load samples as the highest tercile. specimens that were designated positive for sars-cov- , but for which only the e gene was detected, were designated low viral load samples. data were retrospectively abstracted manually from the electronic medical record using a quality-controlled protocol and entered into a redcap database [ ] . all data collectors were trained and a random re-sampling of data previously showed high interrater reliability (mean cohen's kappa of . ) [ ] . data included demographics, comorbidities, social characteristics, selected outpatient medications on admission, presenting symptoms on arrival to the hospital, oxygen supplementation required within three hours of presentation, laboratory parameters, chest radiograph findings, concurrent bloodstream infections, in-hospital complications, and inhospital mortality. clinical data after hospital discharge were not consistently available, and thus a c c e p t e d m a n u s c r i p t only outcomes that occurred during the hospital admission were analyzed. the study was approved by the institutional review board (# - ) at weill cornell medicine with a waiver of informed consent. we compared baseline characteristics and outcomes of hospitalized patients with covid- who had high, medium, and low initial viral loads using the non-parametric nptrend command in stata (statacorp, college station, tx) that tests for trend across ordered groups. continuous variables were represented with medians and interquartile ranges (iqr) and categorical variables were represented as proportions. a two-sided p value of ≤ . was used to designate statistical significance. the risk of in-hospital intubation and death was also compared across eight different numerical ct value ranges. we also constructed cox proportional hazard models to compare the cumulative risks of intubation and death during the inpatient admission among patients with high, medium, and low viral loads. we then identified baseline factors that were associated with in-hospital mortality and intubation using univariate logistic regression models. all variables that were statistically significantly associated with each outcome were then entered into separate multivariate logistic regression models. adjusted odds ratios of mortality and intubation were calculated for each of these variables with % confidence intervals (ci). analyses were conducted using stata, version . . m a n u s c r i p t a total of np swab samples were available for analysis from unique hospitalized patients who met the study inclusion criteria ( figure ). ct values for the orf ab locus ranged the median age of patients with high, medium, and low viral loads was , , and years, respectively (p< . ; table ). in addition to older age, patients with higher viral loads were more likely to have coronary artery disease, congestive heart failure, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease (copd), chronic kidney disease, and active cancer. they were also more likely to be a former or current smoker or have received recent chemotherapy. patients with high viral loads had a median of days from symptom onset until hospital admission, compared to and days for patients with medium and low viral loads, respectively (p< . ). patients with higher viral loads were also more likely to a c c e p t e d m a n u s c r i p t require oxygen by a non-rebreather, high-flow nasal cannula, or mechanical ventilation within three hours of presentation to the ed, but were less likely to present with fever, nausea, or vomiting. lymphopenia, anemia, and thrombocytopenia were more common among patients with higher viral loads; whereas, alanine aminotransferase elevations were less common. there were no differences in chest x-ray findings among patients with high, medium, or low viral loads. there were also no differences in viral loads among different racial or ethnic categories or between patients who did and did not use angiotensin-converting enzyme inhibitors (aceis), angiotensin ii receptor blockers (arbs), or hydroxychloroquine. the last day of study follow-up was june , . by that day, . % of patients had died during their admission, . % had been discharged alive, . % had been transferred to another hospital, and . % were still hospitalized. the risk of intubation and death increased with higher viral loads. in-hospital mortality was . % in patients with a high viral load, compared to . % in patients with a medium viral load, and . % in patients with a low viral load (p< . ; table ). the risk of intubation was . % in patients with a high viral load, compared to . % and . % in patients with a medium or low viral load, respectively (p< . ; table ). these associations were also observed in time-based analyses (figure ) , where compared to a low viral load, a high viral load was associated with a hazard ratio (hr) of in a multivariate model that adjusted for age, race, coronary artery disease, congestive heart failure, cerebrovascular disease, hypertension, copd, days of symptoms prior to admission, symptoms upon presentation, initial chest x-ray findings, and level of oxygen support within three hours of arrival to the ed (table ) , having a high viral load was independently associated with increased risk of in-hospital mortality (adjusted odds ratio [aor] . ; % ci: . - . ; p< . ) compared to having a low viral load. the risk of in-hospital mortality was also higher in patients with a medium viral load compared to a low viral load, but this association was not statistically significant (aor . ; % ci: . - . ; p= . ). compared to those with a low viral load, having a high viral load was also independently associated with increased risk of intubation (aor . ; % ci: . - . ; p< . ); whereas, the risk of intubation associated with a medium viral load did not reach statistical significance (aor . ; % ci: . - . ; p= . ). patients with higher viral loads were also more likely to develop myocardial infarction, congestive heart failure, and acute kidney injury requiring hemodialysis (table ) . this study demonstrated that patients who were admitted to the hospital with high sars-cov- viral loads, as assessed by ct values of np swab samples, were more likely to be intubated or die during their hospitalization. this association persisted even when adjusting for age, comorbidities, presenting symptoms, chest radiography findings, and degree of presenting hypoxia. while prior studies indicated that viral load correlates with severity of covid- presentation [ , ] , our study of a larger cohort of hospitalized patients adds to this knowledge a c c e p t e d m a n u s c r i p t base by identifying that admission viral load has important prognostic implications. reporting sars-cov- viral load based on ct values from admission np swab samples could therefore help identify patients who are at highest risk of adverse outcomes and who therefore may benefit from more intensive monitoring. identifying high viral load patients could also be helpful for allocating scare therapeutic interventions such as antiviral agents (e.g., remdesivir) [ ] . our findings also suggest that stratification or adjustment for baseline viral load would benefit the design of clinical trials of antiviral agents for covid- . it is also possible that viral load could be used along with other factors, such as age, comorbidities, and severity of symptoms and hypoxia to decide upon the need for hospital admission. however, additional studies that evaluate viral loads and clinical outcomes among all patients who present to the ed are warranted prior to pursuing this strategy clinically. older age and the presence of comorbidities such as hypertension, coronary artery disease, congestive heart failure, copd, and cancer are known to be associated with worse outcomes in covid- [ , , , ] . such patients may have decreased cardiopulmonary reserve and thus are less likely to tolerate the physiologic insults caused by covid- . our findings suggest these patients also have higher sars-cov- viral loads when they present to the hospital, which may contribute to the worse outcomes observed in these patients. reasons for higher viral loads specifically in these populations are not well understood and warrant further investigation. given that sars-cov- uses the angiotensin-converting enzyme receptor (ace ) for entry into host cells [ ] , there have been concerns that use of aceis and arbs may upregulate ace expression and lead to increased viral proliferation into host cells [ ] . although patients with hypertension and congestive heart failure were more likely to have higher viral loads, use of aceis and arbs was not associated with higher viral load. our findings are consistent with those of observational studies that have not demonstrated worse outcomes in a c c e p t e d m a n u s c r i p t patients who use aceis or arbs [ ] [ ] [ ] and support the recommendations of professional societies of not discontinuing these medications in the setting of covid- [ ] . another notable finding from this study is that there were no differences in admission sars-cov- viral loads or outcomes among different racial or ethnic groups. in the u.s., hispanic and black communities have been disproportionately affected by covid- , with a greater proportion of deaths among these patients than what would be expected based on their population proportions [ ] [ ] [ ] . our finding that admission viral loads were not different among race and ethnicity groups suggests that these disparities are not to related to viral load, but instead may be related to comorbid illnesses and non-biological factors such as social determinants of health. this further underscores the importance of studies that examine the impact of social determinants of health on outcomes during the covid- pandemic. we also found that patients with higher viral loads were more likely to develop myocardial infarction, congestive heart failure, and acute kidney injury. it is unclear whether these associations were from chance, were related to increased hypoxia in heart and kidney tissue, or were related to increased viral infection of these organs. a recent autopsy study demonstrated that sars-cov- frequently directly infects both the heart and kidney [ ] and kidney injury and myocardial injury are commonly reported complications of severe covid- [ , ] . additional studies are warranted to assess the relationship between viral loads in np swab samples, disease burden in the heart and kidney, and clinical outcomes. ct values as the cobas assay used in this study [ , ] . thus, we suspect that our findings may also be applicable to other diagnostic platforms. we encourage others to evaluate the relationship between clinical outcomes and ct values using other diagnostic platforms and other patient populations. another potential role for reporting sars-cov- viral loads through ct values is to guide the use of isolation precautions, given that viral load correlates with infectivity [ ] [ ] [ ] . our study did not assess this potential use of ct values, but we believe this is an important area for future investigation. another limitation is that our study was retrospective and relied on data that were documented in the electronic medical record, and thus could have misclassified patient characteristics or outcomes. however, our data abstraction process utilized a standardized protocol and our queries identified high interrater reliability for data collection. lastly, we focused on in-hospital mortality, and did not capture deaths that occurred after discharge from the hospital. in conclusion, we found that admission sars-cov- viral loads, as determined by ct values that are generated with standard-of-care diagnostic assays, are independently associated with intubation and death among hospitalized patients with covid- . these a c c e p t e d m a n u s c r i p t p values were calculated using the non-parametric nptrend command in stata, version . , that tests for trend across ordered groups. this variable was not assessed in all participants. the denominator is listed next to the variable. ast elevation indicates a value > units/l. alt elevation indicates a value > units/l. a c c e p t e d m a n u s c r i p t covid- ): situation report - characteristics of and important lessons from 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association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease (covid- ) addresses concerns re: using raas antagonists in covid- covid- fatalities tracker/nysdohcovid- tracker-fatalities?% aembed=yes&% atoolbar=no&% atabs=n. accessed on chicago's coronavirus disparity: black chicagoans are dying at nearly six times the rate of white residents, data show. chicago tribune hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease -covid-net, states multiorgan and renal tropism of sars-cov- association of cardiac injury and mortality in hospitalized patients with covid- in wuhan, china renal involvement and early prognosis in patients with covid- pneumonia comparison of two high-throughput reverse transcription-polymerase chain reaction systems for detection of severe acute respiratory syndrome coronavirus comparison of cepheid xpert xpress and abbott id now to roche cobas for the rapid detection of sars-cov- temporal dynamics in viral shedding and transmissibility of covid- predicting infectious sars-cov- from diagnostic samples viral rna load as determined by cell culture as a management tool for discharge of sars-cov- patients from infectious diseases wards key: cord- -e yfo authors: rainwater-lovett, kaitlin; rodriguez-barraquer, isabel; moss, william j. title: viral epidemiology: tracking viruses with smartphones and social media date: - - journal: viral pathogenesis doi: . /b - - - - . - sha: doc_id: cord_uid: e yfo the science of epidemiology has been developed over the last years, using traditional methods to describe the distribution of diseases by person, place, and time. however, in the last several decades, a new set of technologies has become available, based on the methods of computer sciences, systems biology, and the extraordinary powers of the internet. technological and analytical advances can enhance traditional epidemiological methods to study the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. social media are increasingly used to detect the emergence and geographic spread of viral disease outbreaks. large-scale population movement can be estimated using satellite imagery and mobile phone use, and fine-scale population movement can be tracked using global positioning system loggers, allowing estimation of transmission pathways and contact patterns at different spatial scales. advances in genomic sequencing and bioinformatics permit more accurate determination of viral evolution and the construction of transmission networks, also at different spatial and temporal scales. phylodynamics links evolutionary and epidemiological processes to better understand viral transmission patterns. more complex and realistic mathematical models of virus transmission within human and animal populations, including detailed agent-based models, are increasingly used to predict transmission patterns and the impact of control interventions such as vaccination and quarantine. in this chapter, we will briefly review traditional epidemiological methods and then describe the new technologies with some examples of their application. the science of epidemiology has been developed over the last years, using traditional methods to describe the distribution of diseases by person, place, and time. however, in the last several decades, a new set of technologies has become available, based on the methods of computer sciences, systems biology, and the extraordinary powers of the internet. technological and analytical advances can enhance traditional epidemiological methods to study the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. social media are increasingly used to detect the emergence and geographic spread of viral disease outbreaks. large-scale population movement can be estimated using satellite imagery and mobile phone use, and fine-scale population movement can be tracked using global positioning system (gps) loggers, allowing estimation of transmission pathways and contact patterns at different spatial scales. advances in genomic sequencing and bioinformatics permit more accurate determination of viral evolution and the construction of transmission networks, also at different spatial and temporal scales. phylodynamics links evolutionary and epidemiological processes to better understand viral transmission patterns. more complex and realistic mathematical models of virus transmission within human and animal populations, including detailed agent-based models, are increasingly used to predict transmission patterns and the impact of control interventions such as vaccination and quarantine. in this chapter, we will briefly review traditional epidemiological methods and then describe the new technologies with some examples of their application. insight into the epidemiology of viral infections long preceded the recognition and characterization of viruses as communicable agents of disease in humans and animals, extending at least as far back as the treatise of abu becr (rhazes) on measles and smallpox in the tenth century. successful efforts to alter the epidemiology of viral infections can be traced to the practice of variolation, the deliberate inoculation of infectious material from persons with smallpox (see chapter on the history of viral pathogenesis). documented use of variolation dates to the fifteenth century in china. edward jenner greatly improved the practice of variolation in using the less-virulent cowpox virus, establishing the field of vaccinology. an early example of rigorous epidemiological study prior to the discovery of viruses was the work of the danish physician peter panum who investigated an outbreak of measles on the faroe islands in . through careful documentation of clinical cases and contact histories, panum provided evidence of the contagious nature of measles, accurate measurement of the incubation period, and demonstration of the long-term protective immunity conferred by measles. the discovery of viruses as "filterable agents" in the late-nineteenth and early twentieth centuries greatly enhanced the study of viral epidemiology, allowing the characterization of infected individuals, risk factors for infection and disease, and transmission pathways. traditional epidemiological methods measure the distribution of viral infections, diseases, and associated risk factors in populations in terms of person, place, and time using standard measures of disease frequency, study designs, and approaches to causal inference. populations are often defined in terms of target and study populations, and individuals within study populations in terms of exposure and outcome status. the purpose of much traditional epidemiological research is to quantify the strength of association between exposures and outcomes by comparing characteristics of groups of individuals. exposures or risk factors include demographic, social, genetic, and environmental factors, and outcomes include infection or disease. in viral epidemiology, infection status is determined using diagnostic methods to detect viral proteins or nucleic acids, and serologic assays to measure immunologic markers of exposure to viral antigens. infection status can be defined as acute, chronic, or latent. standard measures of disease frequency include incidence, the number of new cases per period of observation (e.g., person-years), and prevalence, the number of all cases in a defined population and time period. prevalence is a function of both incidence and duration of infection and can increase despite declining incidence, as observed with the introduction of antiretroviral therapy for human immunodeficiency virus (hiv) infection in the united states. although the number of new cases of hiv infection declined, the prevalence of hiv infection increased as treated individuals survived longer. commonly used study designs include, l cross-sectional studies in which individuals are sampled or surveyed for exposure and disease status within a narrow time frame, l cohort studies in which exposed and unexposed individuals are observed over time for the onset of specified outcomes, l case-control studies in which those with and without the outcome (infection or disease) are compared on exposure status, and l clinical trials in which individuals are randomized to an exposure such as a vaccine or drug and observed for the onset of specified outcomes appropriate study design, rigorous adherence to study protocols, and statistical methods are used to address threats to causal inference (i.e., whether observed associations between exposure and outcome are causal), such as bias and confounding. much can be learned about the epidemiology of viral infections using such traditional methods and many examples could be cited to establish the importance of these approaches, including demonstration of the mode of transmission of viruses by mosquitoes (e.g., yellow fever and west nile viruses), the causal relationship between maternal viral infection and fetal abnormalities (e.g., rubella virus and cytomegalovirus), and the role of viruses in the etiology of cancer (e.g., epstein-barr and human papilloma viruses). the epidemiology of communicable infectious diseases is distinguishable from the epidemiology of noncommunicable diseases in that the former must account for "dependent happenings." this term was introduced by ronald ross to capture the fact that infectious agents are transmitted between individuals or from a common source. traditional epidemiological and statistical methods often assume disease events in a population are independent of one another. in infectious disease epidemiology, individuals are defined in terms of susceptible, exposed, infectious, and recovered or immune. key characteristics of viral infections that determine the frequency and timing of transmission, and thus the epidemiology, include the mode of transmission (e.g., respiratory, gastrointestinal, sexual, bloodborne, and vector-borne), whether infection is transient or persistent, and whether immunity is short or long lasting. temporal changes in the transmission dynamics of viral infection can be displayed with epidemic curves, by plotting the number or incidence of new infections over time to demonstrate outbreaks, seasonality, and the response to interventions. key metrics in infectious disease epidemiology that capture the dependent nature of communicable diseases include: ( ) the latent period, the average time from infection to the onset of infectiousness; ( ) the infectious period, the average duration of infectiousness; ( ) the generation time, the average period between infection in one individual and transmission to another; and ( ) the basic reproductive number (r ), the average number of new infections initiated by a single infectious individual in a completely susceptible population over the course of that individual's infectious period. if r is larger than one, the number of infected individuals and hence the size of the outbreak will increase. if r is smaller than one, each infectious individual infects on average less than one other individual and the number of infected individuals will decrease and the outbreak ceases. the reproductive number (r) is a function not only of characteristics of the viral pathogen (e.g., mode of transmission), but also the social contact network within which it is transmitted and changes over time in response to a decreasing number of susceptible individuals and control interventions. an important concept related to the interdependence of transmission events is herd immunity, the protection of susceptible individuals against infection in populations with a high proportion of immune individuals because of the low probability of an infectious individual coming in contact with a susceptible individual. the concepts and methods of infectious disease epidemiology provide the tools to understand changes in temporal and spatial patterns of viral infections and the impact of interventions. traditional epidemiological methods provide powerful analytical approaches to measure associations between exposures (risk factors) and outcomes (infection or disease). recent technological advances enhance these methods and permit novel approaches to investigate the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. expanded access to the internet and social media has revolutionized outbreak detection and viral disease surveillance by providing novel sources of data in real time (chunara, ) . traditional epidemiologic surveillance systems rely on standardized case definitions, with individual cases typically classified as suspected, probable, or confirmed based on the level of evidence. confirmed cases require laboratory evidence of viral infection. surveillance systems are either active or passive. active surveillance involves the purposeful search for cases within populations whereas passive surveillance relies on routine reporting of cases, typically by health care workers, health care facilities, and laboratories. data acquired through active surveillance are often of higher quality because of better adherence to standardized case definitions and completeness of case ascertainment but are more expensive and resource intensive. however, both active and passive surveillance are prone to delays in data reporting. the major advantage of using the internet and social media to monitor disease activity is that the signal can be detected without the lag associated with traditional surveillance systems. influenza is the most common viral infection for which the internet and social media have been used for disease surveillance because of its high incidence, wide geographic distribution, discrete seasonality, short symptomatic period, and relatively specific set of signs and symptoms. however, the internet and social media have several limitations compared to traditional active and passive surveillance systems and complement rather than replace these methods. these limitations include lack of specificity in the "diagnosis," and waxing and waning interest and attention in social media independent of disease frequency. in , the internet company google developed a webbased tool called google flu trends, for early detection of influenza outbreaks. google flu trends is based on the fact that millions of people use the google search engine each day to obtain health-related information (ginsberg, ). logs of user key words for pathogens, diseases, symptoms, and treatments, as well as information on user location contained in computer internet protocol (ip) addresses, allow temporal and spatial analyses of trends in search terms ( figure ). early results suggested that google flu trends detected regional outbreaks of influenza - days before conventional surveillance by the centers for disease control and prevention (carneiro, ). however, accurate prediction was not as reliable as initially thought, and google estimates did not closely match measured activity during the - influenza season. google now reevaluates estimates using data from traditional surveillance systems (specifically those of the centers for disease control and prevention) to refine model and parameter estimates. these refinements more accurately capture the start of the influenza season, the time of peak influenza virus transmission, and the severity of the influenza season. a similar approach, called google dengue trends, is used to track dengue virus infections by aggregating historical logs of anonymous online google search queries associated with dengue, using the methods developed for google flu trends. early observations suggest google queries are correlated with national-level dengue surveillance data, and this novel data source may have the potential to provide information faster than traditional surveillance systems ( figure ). other internet sources are being explored to enhance viral surveillance. wikipedia is a free, online encyclopedia written collaboratively by users and is one of the most commonly used internet resources since it was started in . as with google searches, the use of disease-specific queries to wikipedia are expected to correlate with disease activity. the number of times specific influenza-related wikipedia sites were accessed provided accurate estimates of influenza-like illnesses in the united states weeks earlier than standard surveillance systems and performed better than google flu trends (mciver, ) . similarly, social media data are being evaluated for surveillance purposes. twitter is a free social networking service that enables users to exchange text-based messages of up to characters known as tweets. as with google flu trends, the number of tweets related to influenza activity is correlated with the number of symptomatic individuals. several published studies reported correlations between twitter activity and reported influenza-like illnesses (chew, ; signorini, ; figure ). limitations to using social media, such as twitter, to monitor disease activity are illustrated by the ebola virus outbreak in west africa in early . despite the fact that ebola had not yet occurred in the united states, posts to twitter on ebola rose dramatically, likely in response to intense media coverage and fear. clearly, such tweets could not be interpreted to indicate ebola disease activity in the united states. studies reporting misleading associations, or the lack of correlation between social media and disease activity, are rarely published, providing a cautionary note. while initial efforts using data from the internet for viral disease surveillance offer promising results, concerns have been raised regarding the utility and robustness of these approaches (lazer, ) . integration into existing surveillance frameworks will be necessary to maximize the utility of these data streams. the internet allows rapid processing and communication of health-related information, including the aggregation and display of surveillance data for viral infections. traditional surveillance networks can be linked through the internet to allow rapid integration and dissemination of information. information on viral disease outbreaks available through internet postings of health care agencies such as the world health organization (who) and centers for disease control and prevention (cdc), as well as press reports and blogs, can provide data that are more current than traditional surveillance systems. information from these online sources can be made available to a large, global audience. several of the most commonly used surveillance sites report animal as well as human diseases (see sidebar and figure ). mapping spatial patterns of disease and relationships with environmental variables preceded the development of modern epidemiology. the classic example is john snow's hand-drawn map of london cholera cases of . however, routine mapping of health data only became commonplace in the s after desktop geographic information systems became widely available. combined with satellite imagery and remotely sensed environmental and ecological data, spatial mapping of viral infections is a powerful tool for surveillance and epidemiological research. spatial epidemiology is typically used to identify and monitor areas of differential risk. an early example was a large outbreak of st. louis encephalitis virus infection in houston, texas in . spatial analysis showed that the outbreak was concentrated in the city center, with lower incidence at the outskirts. further investigation revealed that the city center was associated with the lowest economic strata, unscreened windows, lack of air-conditioning and pools of standing water, factors facilitating virus transmission. investigation into the spatiotemporal dynamics of viral diseases at smaller spatial scales has become promed, the program for monitoring emerging diseases, is an internet-based reporting system established in that compiles information on outbreaks of infectious diseases affecting humans, animals, and food plants. promed relies on official announcements, media reports, and local observers, including the network of subscribers. a team of experts screen, review, and investigate reports before posting and often provide commentary. reports are distributed by email to direct subscribers and posted on the promed-mail web site. promed-mail currently reaches over , subscribers in at least countries. started in by epidemiologists and software developers at boston children's hospital, healthmap monitors disease outbreaks and provides real-time surveillance of emerging public health threats, including viral infections (figure ) . healthmap organizes and displays data on disease outbreaks and surveillance using an automated process. data sources include online news aggregators, eyewitness reports, expertcurated discussions and validated official reports. google flu trends http://www.google.org/flutrends google dengue trends http://www.google.org/denguetrends healthmap http://healthmap.org promed http://www.promedmail.org possible with increasing availability of global positioning systems devices and geocoding algorithms. such studies have revealed spatial heterogeneity in the local transmission of some directly (e.g., hiv and influenza) and indirectly (e.g., dengue and chikungunya) transmitted viruses. for example, clustering analyses of the residential locations of people with dengue in bangkok over a -year period showed evidence of localized transmission at distances less than km (salje, ; figure ). analyses of data from a large population-based cohort of hiv-infected persons in rakai district, uganda revealed strong within-household clustering of prevalent and incident hiv cases as well clustering of prevalent cases up to m (grabowski, ) . beyond descriptive applications, mapping spatiotemporal patterns of viral infections can provide fundamental insights into transmission dynamics at different spatial scales. traveling waves from large cities to small towns were shown to drive the spatiotemporal dynamics of measles in england and wales (xia, ) . the incidence of dengue hemorrhagic fever across thailand manifested as a traveling wave emanating from bangkok and moving radially at a speed of km/month (cummings, ) . insight into the spatial epidemiology of viral infections and associations with environmental risk factors can be greatly enhanced when information on the spatial location of cases is combined with remotely sensed environmental data (rodgers, ) . the spatial coordinates of cases can be overlaid on satellite imagery to demonstrate relationships with environmental features-such as bodies of water-and formally analyzed using spatial statistical techniques. satellite sensors that detect reflected visible or infrared radiation provide additional information on temperature, rainfall, humidity, and vegetation among other variables, which are particularly important for the transmission dynamics of vector-borne viral infections. satellite data for epidemiologic analyses are provided by a number of sources such as: ( ) earth-observing satellites with high spatial resolution ( - m) but low repeat frequencies such as ikonos and landsat satellites; ( ) oceanographic and atmospheric satellites such as modis and aster with lower spatial resolution ( . - km) that provide images of the earth surface twice a day; and ( ) geostationary weather satellites such as geos with large spatial resolution ( - km). the statistical relationships between cases and environmental risk factors can be used to construct risk maps. risk maps display the similarity of environmental features in unsampled locations to environmental features in locations where the disease is measured to be present or absent. spatial analysis of the initial cases of west nile virus infection in new york city in identified a significant spatial cluster (brownstein, ) . using models incorporating measures of vegetation cover from satellite imagery, the risk of west nile virus could be estimated throughout the city. a more recent risk map for west nile virus in suffolk county, new york, was generated with data on vector habitat, landscape, virus activity, and socioeconomic variables derived from publicly available data sets (rochlin, ; figure ). population movement plays a crucial role in the spread of viral infections. in the past, quantifying the contribution of movement to viral transmission dynamics at different spatial scales was challenging, due to limited data. as an early example, the impact of restrictions of animal movement on transmission of foot-and-mouth disease in was estimated, using detailed contact-tracing data from farms in the united kingdom (shirley, ) . however, such detailed data are rarely available for patterns of human movement. studies have attempted to model the impact of long-range human movement on the spread of viral diseases using measures such as distance between cities, commuting rates, and data on air travel. this approach has been used to explain regional and interregional spread of influenza viruses. data on air traffic volume, distance between areas, and population sizes have been invoked to describe and predict local and regional spread of chikungunya virus in the americas (tatem, ) . new technologies have greatly enhanced the capacity to study the impact of human movement on transmission dynamics of infectious diseases. data from mobile phones and gps loggers can be used to characterize individual movement patterns and the time spent in different locations (figure ) . individual movement patterns can be overlaid on risk maps to quantify movement to and from areas of high (sources) and low risk (sinks) as well as to estimate potential contact patterns. gps data loggers generated . million gps data points to track the fine-scale mobility patterns of residents from two neighborhoods in iquitos, peru, to better understand the epidemiology of viral infections (vazquez-prokopec, ) . most movement occurred within km of an individual's home. however, potential contacts between individuals were irregular and temporally unstructured, with fewer than half of the tracked participants having a regular, predictable routine. the investigators explored the potential impact of these temporally unstructured daily routines and contact patterns on the simulated spread of influenza virus. the projected outbreak size was % larger as a consequence of these unstructured contact patterns, in comparison to scenarios modeling temporally structured contacts. in addition to identifying individual and environmental characteristics associated with temporal and spatial patterns of viral infections, transmission networks are critical drivers of the dynamics of viral infections. analysis of transmission networks defines the host contact structure within which directly transmitted viral infections spread. network theory and analysis are complex subjects with a long history in mathematics and sociology, but have recently been adapted by infectious disease epidemiologists. the epidemiologic study of social networks is facilitated by unique study designs, including snowball sampling or respondent-driven sampling, in which study participants are asked to recruit additional participants among their social contacts. differing sexual contact patterns serve as an example of the importance of contact networks to the understanding of viral epidemiology. concurrent sexual partnerships amplify the spread of hiv compared with serial monogamy. this could partially explain the dramatic differences in the prevalence of hiv in different countries. social networks were shown to affect transmission of the h n influenza virus, and were responsible for cyclical patterns of transmission between schools, communities, and households. technological advances in quantifying contact patterns, with wearable sensors and the use of viral genetic signatures, have greatly enhanced the ability to understand complex transmission networks. self-reported contact histories and contact tracing are the traditional epidemiological methods to define transmission networks. contact tracing has a long history in public health, particularly in the control of sexually transmitted diseases and tuberculosis, and is critical to the control of outbreaks of viral infections such as the middle east respiratory syndrome coronavirus (mers-cov) and ebola virus. to better understand the nature of human contact patterns, sensor nodes or motes have been used to characterize the frequency and duration of contacts between individuals in settings such as schools and health-care facilities. these technologies offer opportunities to validate and complement data collected using questionnaires and contact diaries. as an example, investigators used wireless sensor network technology to obtain data on social contacts within m for high school students in the united states, enabling construction of the social network within which a respiratory pathogen could be transmitted (salathe, ) . the data revealed a high-density network with typical small-world properties, in which a small number of steps link any two individuals. computer simulations of the spread of an influenza-like virus on the weighted contact graph were in good agreement with absentee data collected during the influenza season. analysis of targeted immunization strategies suggested that contact network data can be employed to design targeted vaccination strategies that are significantly more effective than random vaccination. advances in nucleic acid sequencing and bioinformatics have led to major advances in viral epidemiology. population (sanger) sequencing has been the standard method for dna sequencing but is increasingly replaced by deep sequencing in which variants within a viral swarm are distinguished. sequencing allows for the detection of single nucleotide polymorphisms (snps) and nucleotide insertions or deletions ("indels"), analysis of synonymous and nonsynonymous mutations, and phylogenetic analysis (see chapter on virus evolution). sequencing techniques can be applied to both viral and host genomes. snps may be associated with changes in viral pathogenesis, virulence, or drug resistance. molecular techniques applied to pathogens also have been fundamental to the study of the animal origins of many viral infections including hiv and mers. phylogeographic approaches were used to trace the origins of the hiv pandemic to spillover events in central africa (sharp, ) . more recently, sequence data were used to track the animal reservoirs of mers-cov associated with the outbreaks (haagmans, ) , and to compare the ebola virus strain circulating in the west africa outbreak to strains from prior outbreaks (gire, ) . epidemiologic studies that probe host genomes can be either candidate gene studies or genome-wide association studies. the goal of these studies is to link specific changes with an increased risk of infection or disease. as an example, a small subset of individuals who failed to acquire hiv infection despite exposure, prompted studies to determine how these individuals differed from those who acquired infection. a -base-pair deletion in the human ccr gene, now referred to as ccr -delta , accounted for the resistance of these subjects. individuals who are ccr -delta homozygotes are protected against hiv infection by ccr tropic hiv strains, while heterozygotes have decreased disease severity. infectious disease epidemiologists are increasingly linking evolutionary, immunologic, and epidemiological processes, a field referred to as phylodynamics voltz, ) . because of the high mutation rates of viral pathogens, particularly rna viruses, evolutionary and epidemiological processes take place on a similar timescale (see chapter on virus evolution). according to this framework, phylodynamic processes that determine the degree of viral diversity are a function of host immune selective pressures and epidemiological patterns of transmission ( figure ). intrahost phylodynamic processes begin with molecular characteristics of the virus as well as the host's permissiveness and response to infection. for example, a single amino acid substitution in epstein-barr virus was shown to disrupt antigen presentation by specific human leukocyte antigen polymorphisms (liu, ) . this resulted in decreased t-cell receptor recognition and successful viral immune escape. the virus must also induce an "optimal" host immune response to maximize transmission to new hosts. if the virus induces a strong, proinflammatory immune response not balanced by the appropriate anti-inflammatory responses, the host may succumb to the overabundance of inflammation and cannot propagate viral transmission. alternatively, a virus that fails to stimulate an immune response may also replicate uninhibited, overwhelming, and killing the host prior to transmission. selective pressures maximize replication while sustaining transmission between hosts. interhost dynamics are affected by several factors including evolutionary pressures, timescales of infection, viral latent periods, and host population structures. typically, only a small number of virions are transmitted between hosts, creating a genetic bottleneck that limits viral diversity. a virus that mutates to cause highly pathogenic disease but is not transmitted cannot propagate its pathogenicity. cross-immunity between viral strains also precludes the replication of particular viral lineages. influenza vaccine strains require annual changes due to new circulating influenza strains that have escaped immune pressures through high mutation rates and gene re-assortment. the strong selection pressure of cross-immunity is reflected in the short branch lengths in a phylogenetic tree of influenza viruses isolated from infected individuals. thus, the selection of influenza strains for future vaccines is partly determined by cross-immunity to prior circulating strains, because influenza viral strains that circulated in the past may elicit immune protection against currently circulating strains. at the population level, phylodynamic methods have been used to estimate r for hiv and hepatitis c virus, for which reporting and surveillance data are often incomplete (volz, ) . phylodynamic and phylogeographic models also have been useful in reconstructing the spatial spread of viruses to reveal hidden patterns of transmission. for example, epidemiological and molecular studies of influenza virus transmission were compared at different spatial scales to highlight the similarities and differences between these data sources (viboud, ) . the findings were broadly consistent with large-scale studies of interregional or inter-hemispheric spread in temperate regions with multiple viral introductions resulting in epidemics followed by interepidemic periods driven by seasonal bottlenecks. however, at smaller spatial scalessuch as a country or community-epidemiological studies revealed spatially structured diffusion patterns that were not identified in molecular studies. phylogenetic analyses of gag and env genes were used to assess the spatial dynamics of hiv transmission in rural rakai district, uganda, using data from a cohort of , individuals residing in communities (grabowski, ) . of the phylogenetic clusters identified, almost half comprised two individuals sharing a household. among phylodynamics links evolutionary, immunologic, and epidemiologic processes to explain viral diversity, as shown here for equine influenza virus. for viral evolution, these processes take place on a similar timescale. within host mutations ( ) result from an interplay between optimization of viral shedding, immunologic selective pressures and host pathogenicity. transmission bottlenecks and host heterogeneity ( ) further determine the population genetic structure of the virus, which in turn influences and is determined by the epidemic dynamics. larger scale spatial dynamics at local, regional, and global levels ( ) the remaining clusters, almost three-quarters involved individuals living in different communities, suggesting transmission chains frequently extend beyond local communities in rural uganda. the timescale of infection is also important for viral diversity and transmission dynamics. some viruses are capable of initiating an acute infection that is cleared within days, while other viral infections are chronic and persist for a lifetime. the duration of infection impacts how quickly a virus must be transmitted and has implications for the infectious period and the potential to be transmitted to new hosts. viruses with long latent periods create interhost phylogenetic trees with longer branch lengths. the long duration between infection and transmission permits accumulation of viral changes through many rounds of viral replication before transmission to the next host. examples include hepatitis b virus, hepatitis c virus, and human immunodeficiency virus. availability of computational resources allows widespread use and development of classic approaches to the mathematical modeling of viral transmission dynamics, such as compartmental, metapopulation and network models, to address epidemiologic questions (see chapter on mathematical methods). these models have been used extensively in the study of viral dynamics and to explore the potential impact of control interventions. new sources of high-resolution spatial, temporal, and genetic data create opportunities for models that integrate these data with traditional epidemiological data. such analyses improve estimates of key transmission parameters and understanding of the mechanisms driving virus spread. agent-based models (also known as individual-based models) can now be run using desktop computers, and offer advantages over more traditional mathematical models. because each unit in a population is modeled explicitly in space and time and assigned specific attributes, agent-based models can reproduce the heterogeneity and complexity observed in the real world. more traditional compartmental, differential equation models often require simplifying assumptions that limit applicability. agent-based models have been used to study the spread of viruses in populations as well as the evolution of viruses within and across populations. while agent-based models are intuitive and easy to formulate, these models are often difficult to construct due to the large number of parameters necessary to describe the behavior and interaction between individual units. commercial frameworks that offer large computational power and intuitive user interphases have also become increasingly available. the global epidemic and mobility model (gleam) on the gleamviz platform (www.gleamviz.com), for example, contains extensive data on populations and human mobility, and allows stochastic simulation of the global spread of infectious diseases using user-defined transmission models. our understanding of the epidemiology of viral infections is being revolutionized by the integration of traditional epidemiological information with novel sources of data. l data streams from the internet are promising sources to enhance traditional surveillance but have yet to be fully validated. l molecular data on viral genomic sequences provide unprecedented opportunities to characterize viral transmission pathways. l phylodynamic and phylogeographic models have been used to estimate r , and characterize the spatial spread of viruses. l network analysis reveals hidden patterns of transmission between population subgroups that are not easy to capture with traditional epidemiological methods. l novel analytical and computational resources are playing a key role in integrating information from multiple large data banks. these more comprehensive methods improve our ability to estimate the impact of infection control measures. the combination of traditional and evolving methodologies is closing the gap between epidemiological studies and viral pathogenesis. these developments have laid the foundation for exciting future research that will complement other approaches to the pathogenesis of viral diseases. with these evolving technologies in mind, it is timely to ask: is the world able to control viral diseases more effectively? it is a mixed score card. on the one hand, smallpox has been eradicated and we are on the verge of elimination of wild polioviruses. furthermore, deaths of children under the age of years (which are mainly due to viral and other infectious diseases) have decreased by almost % in the last few decades. on the other hand, the aids pandemic continues to rage in low-income countries, with only a slight reduction in the annual incidence of new infections. the united states has not done any better in reducing hiv incidence which has been unchanged for at least years. the - ebola pandemic in west africa reflects the limited capacity for dealing with new and emerging viral diseases on a global basis. in conclusion, epidemiological science continues to advance with evolving new technologies, but their application to public health remains a future challenge and opportunity. new technologies for reporting real-time emergent infections google trends: a web-based tool for real-time surveillance of disease outbreaks unifying the epidemiological and evolutionary dynamics of pathogens studying the global distribution of infectious diseases using gis and rs spatial analysis of west nile virus: rapid risk assessment of an introduced vector-borne zoonosis pandemics in the age of twitter: content analysis of tweets during the h n outbreak travelling waves in the occurrence of dengue haemorrhagic fever in thailand detecting influenza epidemics using search engine query data rakai health sciences program. the role of viral introductions in sustaining community-based hiv epidemics in rural uganda: evidence from spatial clustering, phylogenetics, and egocentric transmission models middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation the parable of google flu: traps in big data analysis wikipedia usage estimates prevalence of influenza-like illness in the united states in near real-time a molecular basis for the interplay between t cells, viral mutants, and human leukocyte antigen micropolymorphism assessing and maximizing the acceptability of global positioning system device use for studying the role of human movement in dengue virus transmission in iquitos predictive mapping of human risk for west nile virus (wnv) based on environmental and socioeconomic factors a high-resolution human contact network for infectious disease transmission revealing the microscale spatial signature of dengue transmission and immunity in an urban population the evolution of hiv- and the origin of where diseases and networks collide: lessons to be learnt from a study of the foot-and-mouth disease epidemic the use of twitter to track levels of disease activity and public concern in the u.s. during the influenza a h n pandemic air travel and vectorborne disease movement usefulness of commercially available gps data-loggers for tracking human movement and exposure to dengue virus using gps technology to quantify human mobility, dynamic contacts and infectious disease dynamics in a resource-poor urban environment contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission viral phylodynamics measles metapopulation dynamics: a gravity model for epidemiological coupling and dynamics key: cord- -dkwcheu authors: abernathy, emma; glaunsinger, britt title: emerging roles for rna degradation in viral replication and antiviral defense date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: dkwcheu abstract viral replication significantly alters the gene expression landscape of infected cells. many of these changes are driven by viral manipulation of host transcription or translation machinery. several mammalian viruses encode factors that broadly dampen gene expression by directly targeting messenger rna (mrna). here, we highlight how these factors promote mrna degradation to globally regulate both host and viral gene expression. although these viral factors are not homologous and use distinct mechanisms to target mrna, many of them display striking parallels in their strategies for executing rna degradation and invoke key features of cellular rna quality control pathways. in some cases, there is a lack of selectivity for degradation of host versus viral mrna, indicating that the purposes of virus-induced mrna degradation extend beyond redirecting cellular resources towards viral gene expression. in addition, several antiviral pathways use rna degradation as a viral restriction mechanism, and we will summarize new findings related to how these host-encoded ribonucleases target and destroy viral rna. a recurring theme in many virus-host interactions is the attempt to restrict gene expression. for the cell, such restriction is used as an antiviral mechanism. for the virus, dampening gene expression can be used to liberate cellular resources, escape immune detection, and regulate viral transcript abundance. this review will focus on how this virus-host battle plays out at a terminal stage of the gene expression cascadethat of messenger rna (mrna) degradationas research over the last several years has revealed how regulating mrna demise plays important and unexpected roles in the lifecycles of diverse viruses. we will highlight how unrelated viruses have evolved remarkably similar strategies to promote mrna degradation, even though the degree and nature of selectivity often differ. also notable is the apparent viral mimicry of some cellular rna degradation pathways, which themselves have emerging antiviral roles. rates of individual mrna degradation in a cell vary widely, and are regulated by a large cohort of rna binding proteins that control translation, localization, and access to the decay machinery. however, nearly all mrnas are protected by a -methyl-guanosine ( mg) cap and a poly(a) tail, features that physically protect the mrna ends from exonucleolytic decay, and also serve to recruit translation initiation machinery. circularization of mrna during translation through interactions between cap-binding and poly(a) tail binding proteins adds additional protection from cellular decay enzymes. degradation of mrnas at the end of their translational life, termed basal decay, occurs in several stages but initiates with gradual shortening of the poly(a) tail, termed deadenylation, by cellular decay factors including the ccr -not complex and poly(a)-specific ribonuclease (parn). poly(a) tail length is a determinate of mrna stability and translational competence, and thus is tightly controlled (eckmann et al., ) . deadenylation triggers removal of the mg cap by the decapping complex dcp / and its activators. these events expose the mrna to rapid exonucleolytic degradation, primarily from the end by xrn , but also from the end by the exosome and dis l ( fig. ) (gallouzi and wilusz, ) . the fact that basal decay proceeds from the mrna ends allows for tight control of mrna degradation, as removal of the poly(a) tail and cap is regulated, rate-limiting, and in some cases may even be reversible (schoenberg and maquat, ; weill et al., ) . however, the subsequent exonucleolytic decay of the message body is rapid and irreversible. to maintain transcriptome fidelity cells also need to immediately destroy cytoplasmic mrnas recognized as aberrant. in such cases, the strategy for degradation differs fundamentally from that of basal decay, in that mrnas are usually cleaved internally by an endonuclease rather than gradually trimmed from either end. the best-characterized cellular mrna quality control (qc) pathway is nonsense-mediated decay (nmd), which identifies mrnas with premature termination codons (ptc) (fig. ) (popp and maquat, ) . numerous cellular factors comprise the nmd machinery, but the central nmd regulator is upf , whose activation leads to translational repression and accelerated degradation of the ptc-containing mrna. during nmd in mammals, this rapid mrna degradation is triggered by endonucleolytic cleavage of the mrna by the smg endonuclease at the site of the ptc, followed by degradation of the cleaved fragments by components of the basal mrna decay machinery such as xrn (lykke-andersen et al., ; schweingruber et al., ) . other rna qc pathways similarly recognize aberrant translation events such as stalled or non-terminating ribosomes indicative of rna errors and lead to inactivation of the mrna in question through endonucleolytic cleavage (inada, ) . all viruses known to drive widespread mrna degradation do so by causing internal endonucleolytic cleavages or by directly removing the mrna cap structure (fig. ) . regardless of the precise mechanisms used, these strategies have in common one salient feature: they bypass the rate-limiting and regulated steps of deadenylation and cellular decapping, much like the cellular rna qc pathways. this ensures both immediate translational inactivation and exposure of the mrna ends to the processive cellular exonucleases. however, unlike the tightly regulated cellular qc endonucleases, during infection a large proportion of the cytoplasmic mrna population is targeted for cleavage. this allows the viruses to broadly restrict gene expression, as mrnas they target are degraded much more rapidly than they would be if they entered the basal decay pathway. furthermore, akin to cellular pathways like nmd, viruses that cleave mrnas often usurp xrn to complete the degradation process (gaglia et al., ) . four classes of viruses have been shown to cause endonucleolytic cleavage of mrnas for the purpose of restricting gene expression ( table ). the alpha-herpesviruses, gamma-herpesviruses, and influenza a viruses encode non-homologous endonucleases that cleave mrnas directly. sars coronavirus (sars cov) does not encode an rna cleaving enzyme, but nonetheless activates an as yet unknown cellular endonuclease to cleave mrnas. in each examined case, viral specificity for mrnas (as opposed to other types of rna) is conferred by the act of translation or recognition of mrna features associated with translational competence, similar to cellular rna qc pathways kamitani et al., ; read, ) . alpha-herpesviruses such as herpes simplex- (hsv- ) express a fen -like nuclease termed virion host shutoff protein (vhs) that is directed to mrnas through interactions with the translation fig. . overview of cellular and viral decay pathways. basal decay begins with the rate-limiting step of deadenylation, followed by decapping and exonucleolytic degradation of the mrna body. quality control decay pathways such as nmd recognize aberrant mrnas during translation, including the presence of premature termination codons (ptc), and induce endonucleolytic cleavage, whereupon the fragments are degraded by exonucleases. virus-induced decay also bypass early steps of the basal decay pathway and involves internal cleavage of mrnas, usually in a translation-linked manner, which is followed by degradation by host exonucleases. initiation factors eif h and eif ai/ii (feng et al., ; page and read, ) . if this interaction is disrupted but the catalytic endonuclease activity remains intact, no host shutoff occurs, indicating that recruitment of vhs to the pool of translating mrnas is crucial to its ability to dampen gene expression (feng et al., ; sarma et al., ; shiflett and read, ) . in vitro, vhs lacks specificity, cleaving mrnas and non-mrnas indiscriminately, as well as anywhere along the rna (read, ) . however, in cells or in the presence of cell extracts, vhs preferentially cuts mrnas at unstructured sites within the utr or near the start codon of capped mrnas (karr and read, ; shiflett and read, ) . cut sites also cluster downstream of the encephalomyocarditis virus (emcv) internal ribosome entry site (ires), which recruits the vhs-targeting translation factors eif ai/ii, but not near the more minimal cricket paralysis virus (crpv) ires that recruits the ribosome in the absence of eif f (shiflett and read, ) . further support for the hypothesis that vhs accesses its cleavage sites during translation initiation comes from experiments showing that specific cleavage sites can be repressed or enhanced by mutating the target mrna start codon or enhancing its kozak consensus context, respectively (read, ; shiflett and read, ) . however, the observation that an mrna with a capproximal hairpin structure that prevents s recruitment remains fully susceptible to vhs cleavage argues against an absolute requirement for ribosomal scanning (gaglia et al., ) . one possibility is that assembly of the eif f complex on the mrna cap induces local rna structure remodeling that creates vhs accessible sites, but that more directed cleavages occur near the start codon during the process of s scanning. after vhs-induced cleavage, the resulting mrna fragments are degraded by the cellular xrn exonuclease (gaglia et al., ) . gamma-herpesviruses encode a viral endonuclease that, although not homologous to alpha-herpesvirus vhs, also broadly targets cytoplasmic mrnas for cleavage and subsequent degradation. this protein, termed sox in kaposi's sarcoma-associated herpesvirus (kshv), musox in murine gamma-herpesvirus (mhv ), and bglf in epstein-barr virus (ebv), is a member of the pd(d/e)xk restriction endonuclease superfamily. the sox ortholog in hsv- was originally shown to have dnase activity involved in viral dna genome replication (wilkinson and weller, ) , a function it presumably retains in all herpesviruses in addition to the gamma-herpesvirus-specific mrna degradation activity. both the dna and rna cleavage activities of the protein require the same catalytic core region (bagneris et al., ; glaunsinger et al., ) . although sox specifically targets translationally competent mrnas, active translation is not a requirement for target recognition and the molecular features that direct sox to mrnas remain unknown. sox-induced mrna cleavage occurs at one or more specific, but as-yet poorly sequence defined rna elements (z nt) that can be present anywhere along the length of a target gaglia et al., ) . although a sox targeting element can confer a new cleavage event if moved to a different location on an mrna, it is incapable of directing cleavage by sox if introduced into noncoding rnas transcribed by rna polymerase i or iii (gaglia et al., ) . similar to vhs, recombinant sox and bglf display relaxed rna targeting specificity in vitro (bagneris et al., ; buisson et al., ) , indicating that additional mrna-specific features must be required for sox recruitment in cells. single function mutants of sox and musox that are defective for mrna cleavage but retain their dnase activity have mutations that map to regions outside the catalytic core on the protein surface (bagneris et al., ; covarrubias et al., ; glaunsinger et al., ) . furthermore, the crystal structure of bglf revealed the presence of a flexible "bridge" domain that crosses the active site and contains residues involved in host shutoff (buisson et al., ; horst et al., ) . these noncatalytic regions may therefore function in targeting the gammaherpesvirus sox orthologs to translationally competent mrnas, perhaps through interactions with specific mrna binding proteins. similar to vhs, sox-cleaved mrnas subsequently enter the cellular mrna decay pathway and are degraded by xrn gaglia et al., ) . the pa-x protein of influenza a virus (iav) is a recently discovered mrna endonuclease involved in restricting host gene expression (jagger et al., ) . it is expressed by a ribosome frameshifting event during translation of the pa subunit of the viral rna-dependent rna polymerase (rdrp), itself an endonuclease that is responsible for cap snatching in the nucleus. like the gamma-herpesvirus sox orthologs (and many endonucleases involved in cap snatching), pa-x is a member of the pd(d/e)xk nuclease family. pa-x retains the n-terminal pa endonuclease domain but contains a distinct c-terminus that augments the cellular mrna degradation activity of the protein via unknown mechanisms (desmet et al., ; jagger et al., ) . one possibility is that c-terminal sequences are involved in directing pa-x to its mrna targets. in this regard, it would be interesting to determine whether pa-x targeting is linked to translation and feeds into the cellular xrn decay pathway, as has been shown for other viral mrna restriction factors. sars cov expresses a host shutoff factor, nsp , that binds the s ribosome, simultaneously inducing cleavage of mrnas and inactivating the ribosome (huang et al., ; kamitani et al., ) . by binding the s ribosome, nsp is recruited to all translationally competent mrnas, allowing for broad targeting of cellular transcripts. nsp itself does not possess detectable intrinsic nuclease activity, suggesting that nsp -induced mrna cleavage may instead occur through activation of a cellular rna surveillance pathway (almeida et al., ; huang et al., ) . candidate pathways include those involved in monitoring translational efficiency, given that the nsp - s interaction leads to ribosome inactivation in addition to mrna cleavage (narayanan and makino, ) . for example, the no-go decay pathway degrades mrnas with stalled ribosomes, albeit using a currently unknown endonuclease (harigaya and parker, ) . as has been observed for cellular qc pathways like nmd as well as the herpesviral endonucleases, degradation of the cleaved mrnas in nsp -expressing cells is executed by xrn (gaglia et al., ) . poxviruses and african swine fever virus (asfv) are the only viruses known to encode decapping enzymes. similar to cellular shors et al., ; souliere et al., ) . the asfv decapping enzyme g r also contains a nudix domain essential for decapping. both the vacv and asfv decapping enzymes are inhibited in the presence of excess uncapped rnas, but only the vacv d enzyme is also inhibited by cap analogs (parrish et al., ). this suggests that g r recognizes its substrates by binding the rna body rather than the cap, whereas vacv d binds both the methylated cap and the rna body (parrish et al., ) . extensive site-directed mutagenesis of vacv d identified eight amino acids in the catalytic core of d important for decapping activity and showed that d recognizes the cap in a manner distinct from other characterized cap binding proteins (souliere et al., ) . it is unknown why poxviruses expresses two functional decapping enzymes, although the reason may be linked to the fact that d is expressed early during infection while d is expressed later, after dna replication. additionally, there are some differences between the two enzymes, including the observations that d requires longer rna substrates than d , and d mutants have less pronounced phenotypes than d mutants moss, , ) . therefore, the kinetic and functional requirements for decapping may vary as vacv infection progresses. as decapping renders the end of an mrna unprotected, it is likely that d and d cleaved mrnas are digested by xrn , similar to the cleavage products induced by vhs, sox, and nsp . interestingly, a recent rnai screen suggested a positive role for xrn in vacv replication (sivan et al., ) , perhaps indicating that xrn -mediated rna degradation plays an important role in the viral lifecycle. it is often presumed that restriction of cellular gene expression during infection serves in part to divert resources for the selective enhancement of viral gene expression. however, in each of the above documented examples there is not a clear escape mechanism to broadly protect viral mrnas from inactivation. instead, these viruses may benefit from reduced transcript levels during infection, either because mrna inactivation helps them regulate their gene expression kinetics or other aspects of the viral lifecycle. during vacv infection, the decapping enzymes d and d fail to discriminate between viral and cellular mrna. targeting viral transcripts is proposed to help facilitate transitions between the classes of gene expression, as d mutants exhibit delayed onset of early and late viral gene expression (liu et al., ; parrish and moss, ) . similarly, alpha-and gamma-herpesviral mrnas are inherently susceptible to endonucleolytic cleavage. during hsv- infection, vhs plays an important role in mediating the effective transition between the expression of immediate-early (α), early (β), and late (γ) genes (read, ) . there are some discrepancies in the field as to exactly which viral mrnas are susceptible to vhs-mediated degradation during infection. some data suggest that only α mrnas are targeted (shu et al., ; taddeo et al., ) , while data from other groups indicate that α, β, and even some γ mrnas are susceptible to degradation by vhs (kwong and frenkel, ; read, , ) . regardless of the extent of viral mrna degradation, targeting of viral mrnas by vhs helps facilitate the transition between viral gene classes as infection progresses. furthermore, during infection with a vhs null virus, γ mrnas are excluded from polysomes due to 'translational overload', whereby the capacity of the translation machinery becomes overwhelmed due to an excess of mrnas produced earlier in infection . this confirms the long-held hypothesis that host shutoff is a means of liberating translational machinery for viral use-with the twist that both host and viral transcripts must be degraded to ensure efficient translation of γ mrnas. further contributing to the robust accumulation of γ proteins is the inactivation of vhs later during infection by the virion proteins vp , vp , and ul (read, ; shu et al., ) . all three are packaged into the viral particle along with vhs, and it has been suggested that sequestering vhs in this complex represents an early stage in virion assembly and protects mrnas from cleavage late in infection. thus, despite widespread viral mrna susceptibility, vhs targeting of mrnas appears temporally controlled. the sox homologs in mhv (musox) and ebv (bglf ) have also been shown to target viral mrnas for cleavage (abernathy et al., ; horst et al., ) . unlike vhs, however, there is no indication that sox or its orthologs are inactivated as infection progresses. during mhv infection, musox broadly targets viral mrnas from all three kinetic classes, which generally leads to corresponding decreases in viral protein levels in each class (abernathy et al., ) . thus, unlike hsv- infection, the targeting of viral mrnas during gamma-herpesvirus infection is not a mechanism to redirect the translation machinery towards viral genes. this also suggests that translation factors do not become limiting during mhv infection. selective inactivation of the mrna degradation activity of musox results in altered protein composition of progeny virions, which ultimately impacts subsequent rounds of infection by favoring lytic cycle entry over latency. the mutant also exhibits replication defects in multiple cell types (abernathy et al., ; richner et al., ) . deletion of bglf during ebv infection also results in accumulation of several viral proteins, as well as causes nuclear egress defects (feederle et al., ). however, because bglf has dual roles in viral genome maturation and mrna degradation and the bglf mutant virus lacks both functions, it is not possible to ascribe the above phenotypes solely to a defect in host shutoff. nonetheless, these data support the hypothesis that degradation of viral mrna during gamma-herpesvirus infection plays important roles in regulating gene expression and subsequent viral particle composition. unlike herpesviral and poxviral mrnas, sars cov transcripts are categorically resistant to nsp -induced cleavage and degradation. this protection is due to the presence of a protective leader sequence present on all viral mrnas, although the mechanism of protection remains unclear (huang et al., ) . however, while cov mrnas escape endonucleolytic cleavage, they do not escape nsp -induced ribosome inactivation, raising the issue of what advantage is conferred by the protective sequence (huang et al., ; lokugamage et al., ) . one likely possibility is that ribosome inactivation is not complete, and consequently viral gene expression is not as severely impacted as cellular gene expression. whether this represents a mechanism to fine tune viral protein synthesis in a manner important for the viral lifecycle in vivo remains an interesting question for future investigation. degradation of mrna has recently been shown to be highly interconnected with many other cellular processes including transcription, mrna export, and translation (braun and young, ; huch and nissan, ) . it is thus likely that the broad virus-induced mrna decay described above will result in changes to other rna processes as well. one example of this is altered mrna end processing in the nucleus that occurs as a consequence of enhanced mrna decay in the cytoplasm (fig. ) . poly(a) binding protein (pabpc) normally binds to poly(a) tails of mrnas in the cytoplasm, where it contributes to the regulation of mrna stability and enhances translation. however, pabpc becomes strongly relocalized to the nucleus in cells expressing sox, musox, bglf , vhs, pa-x or nsp (arias et al., ; khaperskyy et al., ; kumar and glaunsinger, ; lee and glaunsinger, ; park et al., ) . nuclear import occurs because within its rna binding domains, pabpc harbors noncanonical nuclear localization signals (nls) that are masked when it is bound to poly(a) tails in the cytoplasm. however, during accelerated mrna degradation by these viral proteins, pabpc is released from poly(a) tails, exposing its nls for interaction with the nuclear import machinery . such aberrant accumulation of pabpc in the nucleus causes hyperadenylation of nascent transcripts by cellular poly(a) polymerase ii (kumar and glaunsinger, ; lee and glaunsinger, ). these hyperadenylated mrnas are retained in the nucleus, presumably because they are recognized as aberrant by the nuclear rna qc machinery. this process thus contributes to the overall magnitude of host shutoff, as the cytoplasm cannot be efficiently repopulated with newly transcribed mrnas. accelerated cytoplasmic decay may also lead to inhibition of stress granule (sg) formation. sgs are storage sites for translationally stalled mrnas, and form in response to translational arrest that often occurs during viral infection (valiente-echeverria et al., ) . many viruses have evolved mechanisms to block their formation, presumably to ensure continued translation of viral proteins. viral nucleases can contribute to sg dispersal, presumably through the bulk reduction of mrnas needed to nucleate sg formation. for example, along with several other iav-encoded proteins, the endonuclease pa-x was recently identified as a potent inhibitor of sgs (khaperskyy et al., ) . pa-x-mediated sg inhibition coincides with pabpc relocalization, hinting at a link between host shutoff and sg dynamics. similarly, the vhs nuclease of hsv- is required for the sg disruption that occurs during hsv- infection (finnen et al., ) . however, vhs has also been implicated in translational enhancement of viral late genes (a role separable from its rnase activity) (dauber et al., , making it difficult to ascribe the sg dispersal phenotype solely to mrna depletion. nonetheless, viral mrna-targeting nucleases provide a unique system to dissect the link between mrna decay and sg assembly. widespread dampening of gene expression during infection is presumed to contribute to viral immune evasion, both by inhibiting expression of cellular immune regulatory genes and by reducing the abundance of viral antigens available for detection. indeed, viruses containing mutations in hsv- vhs, mhv musox, coronavirus nsp , and vacv d exhibit more severe phenotypes in a mouse model of infection than in cultured cells (liu et al., ; richner et al., ; smiley, ; zust et al., ) , suggesting mrna degradation contributes to virulence. activation of the innate immune response leads to expression of hundreds of genes involved in establishing an antiviral state. vhs suppresses the expression of several of these genes including tetherin and viperin, which would normally act to restrict hsv- infection (shen et al., ; zenner et al., ) , as well as many pro-inflammatory cytokines (suzutani et al., ) . some of the differences in the in vivo infectivity of wt versus the vhs mutant hsv- are alleviated in interferon receptor defective (ifnar ko) mice, suggesting that vhs-induced suppression of the innate immune response contributes to viral fitness (leib et al., ; smiley, ) . selective inactivation of the musox mrna degradation activity leads to a severe attenuation of mhv in b cells during the phase of peak latency establishment (richner et al., ) . this could be due to improper immune evasion and/or cell-type specific replication defects, as the musox mutant virus replicates to wt titers in the mouse lung but traffics inefficiently to b cells and displays cell type specific replication defects in cultured cells (abernathy et al., ; richner et al., ) . similar to the ability of vhs to degrade immune modulatory mrnas, ebv bglf also reduces expression of immune molecules, in particular hla i and ii (rowe et al., ; zuo et al., ) . however, this activity is redundant with other ebv proteins that specifically combat hla processing and transport and thus appears to have only a small effect on cd þ t cell recognition (quinn et al., ) . whether cd þ t cell recognition or innate immune signaling are influenced by mrna degradation during in vivo infection with other gamma-herpesviruses remains to be determined. both vacv decapping mutants and cov nsp mutants also display altered virulence phenotypes, although further research is needed to determine the extent to which these are directly linked to mrna degradation. mice infected with vacv d stop and catalytic mutants show less weight loss and mortality compared to a wt infection, and these mutant viruses replicate to lower titers in all organs (liu et al., ) . although there is not in vivo data for nsp of sars cov, the nsp protein of the coronavirus mouse hepatitis virus (mhv) retains the mrna degradation function, as well as several additional roles in inhibiting immune signaling pathways. these activities align well with the observation that an mhv nsp deletion virus is severely attenuated in wt mice, but is completely rescued in ifnar ko mice (zust et al., ) . determining the extent to which nsp -induced virulence links to its host shutoff activity will require the use of single function nsp mutants selectively defective for mrna cleavage or immune pathway impairment. in this regard, the recent characterization of a panel of sars cov nsp mutants that exhibit selective functional defects should help determine the contribution of mrna degradation to the nsp virulence phenotypes (jauregui et al., ) . the use of rna-targeting nucleases is also a component of many of the cellular antiviral defense pathways, some of which play dual roles in regulating normal cellular metabolism and viral restriction (fig. ) . for example, in addition to its well-established role in eliminating ptc-containing mrna, the nmd pathway has recently been shown to function in the restriction of positive strand (þ) rna viruses in plants and in mammalian cells (balistreri et al., ; garcia et al., ) . a genetic screen uncovered the central nmd effector upf as a cellular restriction factor of the plant (þ) rna viruses potato virus x (pvx) and turnip crinkle virus (tcv) (garcia et al., ) . nmd-based restriction is hypothesized to act upon the input genomic viral rna undergoing initial rounds of translation, and thus might function before the onset of rnai, the major antiviral pathway in plants. one of the known activators of nmd is an unusually long utr on the target mrna, as this can be associated with less efficient translation termination (inada, ; kervestin and jacobson, ) . in this regard, many (þ ) rna plant viruses encode subgenomic (sg) rnas, which creates the appearance of a long utr on the genomic mrna and select sgrnas. indeed, these long utrs are required for degradation of pvx and tcv rnas via nmd, confirming that intrinsic features of viral rnas render them susceptible to cellular qc pathways (garcia et al., ) . in mammalian cells, depleting nmd factors upf , smg , or smg leads to increased replication of the alphaviruses semliki forest virus (sfv) and sindbis virus (sinv), suggesting that nmd may target viral genomic rna for degradation (balistreri et al., ) . unexpectedly, although like pvx and tcv these viruses have long utrs, deletion of the long utr of sfv does not alter the restriction by upf (balistreri et al., ) . furthermore, viral restriction does not require the nmd endonuclease smg . thus, the mechanism of decay and the viral rna feature(s) that trigger virus-induced activation of nmd in mammalian cells remain to be elucidated. however, a broader role for nmd in controlling mammalian viruses is supported by the fact that multiple retroviruses have evolved mechanisms to restrict nmd. this can occur through inhibitory interactions with nmd components or through viral rna sequences that protect against nmd (mocquet et al., ; withers and beemon, ) . in plants and insects, rna interference (rnai) is the primary antiviral defense mechanism. the rnai pathway restricts gene expression by processing the long double stranded rnas frequently generated during viral replication into short interfering rnas (sir-nas), which guide endonucleolytic cleavage of complementary target mrnas. although mammalian cells possess the rnai machinery, in most cases rnai does not appear to play a significant antiviral role, and has instead been supplanted by the protein-based interferon response (cullen, ) . however, recent data reveal that in select cell types such as es cells, rnai indeed functions in an antiviral capacity maillard et al., ) . one hypothesis is that an antiviral role for rnai is retained in these cells because they lack a fully functional interferon response. furthermore, mice express an oocyte-specific n-terminally truncated isoform of the nuclease responsible for generating mature forms of the effector small rnas (mirnas or sirnas), termed dcr (flemr et al., ) . dcr has increased sirna-processing activity relative to the full-length dcr nuclease, perhaps explaining why undifferentiated cells contain rnaibased antiviral activity (cullen, ; flemr et al., ) . because dcr is not expressed in primates, whether similar rnai-based antiviral activity is active in human es cells remains an open question. the interferon (ifn) pathway is the primary effector of the mammalian innate immune response, and its activation can induce the expression of proteins that drive either selective destruction of viral rna or more indiscriminate destruction of viral and cellular rna. an example of the former is the zinc-finger antiviral protein (zap), which binds specifically to viral rnas that contain a zap response element (zre). upon binding to viral rna, zap recruits cellular rna decay machinery, including the deadenylase parn, the rna - directed exonuclease complex called the exosome, and the dcp / decapping enzymes via their p helicase co-factor (zhu et al., . cellular nucleases with antiviral roles. (a) viral rna of ( þ ) rna viruses can be recognized by cellular qc pathways like nmd, in some cases due to long utrs which are inherent to subgenomic rnas (sgrna). this leads to their degradation by smg and perhaps other nucleases. (b) rnai cleaves viral dsrna, which is loaded into a rna induced silencing complex (risc) that targets viral rna for endonucleolytic cleavage by ago. (c) ifn-activated mrna degradation pathways include zap and rnase l. zap binds viral rna at specific response elements (zre) and recruits cellular decay factors, including deadenylase parn, de-capping enzyme dcp , and the - exosome. ifn also induces - a synthase (oas) to synthesize the rnase l activator - a, leading to rnase l dimerization and cleavage of viral and cellular rnas. including hiv, filoviruses, sindbis virus, and mhv (bick et al., ; muller et al., ; xuan et al., ; zhu et al., ) . zap contains four ccch-type zinc fingers in its n-terminal domain that specifically bind rna and recruit decay factors (zhu et al., ; zhu and gao, ) . as with many other mrna decay pathways, zap-induced degradation is preceded by inhibition of mrna translation. zap restricts translation of its target mrnas by interacting with the eif a helicase in a manner that disrupts the ability of eif a to associate with eif g (zhu et al., ) . translational repression appears selective for zre-containing transcripts, suggesting that zap only binds eif a associated with its target mrnas (zhu et al., ) . unlike the zre-specific targeting of zap, the ifn-activated cellular endonuclease rnase l cleaves a much broader spectrum of rnas. rnase l is inactive as a monomer, but becomes active upon binding - oligoadenylates ( - a) that are produced by another ifninduced protein, - a synthase (oas). binding of - a allosterically activates rnase l by inducing its dimerization, whereupon it cleaves both viral and cellular rnas, usually at a -unn- consensus (bhattacharyya, ; han et al., ) . that said, two features of rnase l might cause it to favor viral over cellular mrnas. first, the uu/ua dinucleotides that often make up the rnasel cleavage site are relatively rare in the coding regions of cellular mrnas, possibly as an evolutionary trend to avoid rnase l cleavage (al-saif and khabar, ) . second, cellular rnas contain a variety of nucleoside modifications, some of which confer increased resistance to rnase l (anderson et al., ) . not surprisingly, many viruses have evolved mechanisms to counteract the activation of rnase l, including blocking ifn induction and directly disrupting - a production (bhattacharyya, ; silverman and weiss, ) . the expanding number of viruses shown to exert control over the cytoplasmic mrna population through the activity of virally encoded endonucleases or by activating cellular nucleases highlights the importance of this process in diverse viral lifecycles. although we have highlighted select examples of viral endonucleases that promote mrna decay, many other viruses impact rna fate by inactivating mrna degradation enzymes, hijacking or competing with the cellular decay machinery, and relocalizing cellular proteins that control mrna stability (moon and wilusz, ) . furthermore, as is frequently the case in virology, the study of this virus-host interplay is sure to offer new insights into the regulation of cellular rna decay pathways. the field is now beginning to uncover how cellular rna degradation enzymes with central roles in basal and qc-associated rna decay are also key contributors to the antiviral response. yet, in some cases the precise players or their regulation may differ from their previously characterized roles in the context of uninfected cells. in this regard, revealing how viral rnas are recognized and marked for degradation by pathways such as nmd remains an important endeavor. this should simultaneously provide insight into cellular rna features that impact qc surveillance, especially given the numerous parallels between mrna degradation by viruses and cellular qc pathways. furthermore, additional research is required to define the importance of rnai in the mammalian antiviral response, including the cell context in which it operates as well as whether it plays antiviral roles in primates. much remains to be discovered about the mechanisms underlying mrna targeting by viral endonucleases as well. questions surrounding the precise roles of translation factors in recruiting or activating nucleases, what sequence elements and context confer cleavage, as well as how active translation impacts targeting all remain active areas of research. finally, although the data all point to important roles for virus-induced mrna degradation in replication and immune evasion in vivo, very little is known about the relative importance of regulating host versus viral mrna abundance in these processes. ongoing and future research should provide answers to these questions, as well as reveal the impact of virus-induced mrna degradation on a diversity of other cellular processes. gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mrna degradation uu/ua dinucleotide frequency reduction in coding regions results in increased mrna stability and protein expression novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein from the severe acute respiratory syndrome coronavirus nucleoside modifications in rna limit activation of - -oligoadenylate synthetase and increase resistance to cleavage by rnase l activation of host translational control pathways by a viral developmental switch crystal structure of a kshv-sox-dna complex: insights into the molecular mechanisms underlying dnase activity and host shutoff the host nonsense-mediated mrna decay pathway restricts mammalian rna virus replication expression of the zinc-finger antiviral protein inhibits alphavirus replication coupling mrna synthesis and decay a bridge crosses the active-site canyon of the epstein-barr virus nuclease with dnase and rnase activities coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease xrn host shutoff is a conserved phenotype of gammaherpesvirus infection and is orchestrated exclusively from the cytoplasm viruses and rna interference: issues and controversies the herpes simplex virus vhs protein enhances translation of viral true late mrnas and virus production in a cell type-dependent manner the herpes simplex virus virion host shutoff protein enhances translation of viral late mrnas by preventing mrna overload identification of the n-terminal domain of the influenza virus pa responsible for the suppression of host protein synthesis control of poly(a) tail length the epstein-barr virus alkaline exonuclease bglf serves pleiotropic functions in virus replication mrna decay during herpes simplex virus (hsv) infections: protein-protein interactions involving the hsv virion host shutoff protein and translation factors eif h and eif a the herpes simplex virus virion-associated ribonuclease vhs interferes with stress granule formation a retrotransposon-driven dicer isoform directs endogenous small interfering rna production in mouse oocytes a common strategy for host rna degradation by divergent viruses a distinctively novel exoribonuclease that really likes u nonsense-mediated decay serves as a general viral restriction mechanism in plants the exonuclease and host shutoff functions of the sox protein of kaposi's sarcoma-associated herpesvirus are genetically separable structure of human rnase l reveals the basis for regulated rna decay in the ifn response no-go decay: a quality control mechanism for rna in translation the "bridge" in the epstein-barr virus alkaline exonuclease protein bglf contributes to shutoff activity during productive infection sars coronavirus nsp protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp -induced rna cleavage interrelations between translation and general mrna degradation in yeast quality control systems for aberrant mrnas induced by aberrant translation elongation and termination an overlapping protein-coding region in influenza a virus segment modulates the host response identification of residues of sars-cov nsp that differentially affect inhibition of gene expression and antiviral signaling a twopronged strategy to suppress host protein synthesis by sars coronavirus nsp protein the virion host shutoff function of herpes simplex virus degrades the end of a target mrna before the end nmd: a multifaceted response to premature translational termination influenza a virus host shutoff disables antiviral stress-induced translation arrest nuclear import of cytoplasmic poly(a) binding protein restricts gene expression via hyperadenylation and nuclear retention of mrna importin alpha-mediated nuclear import of cytoplasmic poly(a) binding protein occurs as a direct consequence of cytoplasmic mrna depletion herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mrnas aberrant herpesvirus-induced polyadenylation correlates with cellular messenger rna destruction interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo rna interference functions as an antiviral immunity mechanism in mammals the d decapping enzyme of vaccinia virus contributes to decay of cellular and viral mrnas and to virulence in mice severe acute respiratory syndrome coronavirus protein nsp is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation human nonsense-mediated rna decay initiates widely by endonucleolysis and targets snorna host genes antiviral rna interference in mammalian cells the human tlymphotropic virus type tax protein inhibits nonsense-mediated mrna decay by interacting with int /eif e and upf cytoplasmic viruses: rage against the (cellular rna decay) machine inhibition of filovirus replication by the zinc finger antiviral protein interplay between viruses and host mrna degradation a mutant of herpes simplex virus type exhibits increased stability of immediate-early (alpha) mrnas control of mrna stability by the virion host shutoff function of herpes simplex virus the virion host shutoff endonuclease (ul ) of herpes simplex virus interacts with the cellular cap-binding complex eif f nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(a)-binding protein are distinct processes mediated by two epstein barr virus proteins the african swine fever virus g r protein possesses mrna decapping activity characterization of a vaccinia virus mutant with a deletion of the d r gene encoding a putative negative regulator of gene expression characterization of a second vaccinia virus mrnadecapping enzyme conserved in poxviruses vaccinia virus d protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression organizing principles of mammalian nonsensemediated mrna decay cooperation between epstein-barr virus immune evasion proteins spreads protection from cd þ t cell recognition across all three phases of the lytic cycle virus-encoded endonucleases: expected and novel functions global mrna degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency host shutoff during productive epstein-barr virus infection is mediated by bglf and may contribute to immune evasion small interfering rnas that deplete the cellular translation factor eif h impede mrna degradation by the virion host shutoff protein of herpes simplex virus re-capping the message nonsense-mediated mrna decay-mechanisms of substrate mrna recognition and degradation in mammalian cells herpes simplex virus counteracts viperin via its virion host shutoff protein ul mrna decay during herpes simplex virus (hsv) infections: mutations that affect translation of an mrna influence the sites at which it is cleaved by the hsv virion host shutoff (vhs) protein down regulation of gene expression by the vaccinia virus d protein the nuclear-cytoplasmic shuttling of virion host shutoff rnase is enabled by pul and an embedded nuclear export signal and defines the sites of degradation of au-rich and stable cellular mrnas viral phosphodiesterases that antagonize doublestranded rna signaling to rnase l by degrading - a human genome-wide rnai screen reveals a role for nuclear pore proteins in poxvirus morphogenesis herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral rnase? characterization of the vaccinia virus d decapping enzyme provides evidence for a two-metal-ion mechanism insights into the molecular determinants involved in cap recognition by the vaccinia virus d decapping enzyme the role of the ul gene of herpes simplex virus type in evasion of non-specific host defence mechanisms during primary infection the herpes simplex virus host shutoff rnase degrades cellular and viral mrnas made before infection but not viral mrna made after infection viral modulation of stress granules translational control by changes in poly(a) tail length: recycling mrnas the role of dna recombination in herpes simplex virus dna replication structural features in the rous sarcoma virus rna stability element are necessary for sensing the correct termination codon zap inhibits murine gammaherpesvirus orf expression and is antagonized by rta herpes simplex virus counteracts tetherin restriction via its virion host shutoff activity zinc-finger antiviral protein inhibits hiv- infection by selectively targeting multiply spliced viral mrnas for degradation zap-mediated mrna degradation translational repression precedes and is required for zap-mediated mrna decay the dnase of gammaherpesviruses impairs recognition by virus-specific cd þ t cells through an additional host shutoff function coronavirus non-structural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines key: cord- -itqwhi a authors: haddad, christina; davila-calderon, jesse; tolbert, blanton s. title: integrated approaches to reveal mechanisms by which rna viruses reprogram the cellular environment date: - - journal: methods doi: . /j.ymeth. . . sha: doc_id: cord_uid: itqwhi a rna viruses are major threats to global society and mass outbreaks can cause long-lasting damage to international economies. rna and related retro viruses represent a large and diverse family that contribute to the onset of human diseases such as aids; certain cancers like t cell lymphoma; severe acute respiratory illnesses as seen with covid- ; and others. the hallmark of this viral family is the storage of genetic material in the form of rna, and upon infecting host cells, their rna genomes reprogram the cellular environment to favor productive viral replication. rna is a multifunctional biomolecule that not only stores and transmits heritable information, but it also has the capacity to catalyze complex biochemical reactions. it is therefore no surprise that rna viruses use this functional diversity to their advantage to sustain chronic or lifelong infections. efforts to subvert rna viruses therefore requires a deep understanding of the mechanisms by which these pathogens usurp cellular machinery. here, we briefly summarize several experimental techniques that individually inform on key physicochemical features of viral rna genomes and their interactions with proteins. each of these techniques provide important vantage points to understand the complexities of virus-host interactions, but we attempt to make the case that by integrating these and similar methods, more vivid descriptions of how viruses reprogram the cellular environment emerges. these vivid descriptions should expedite the identification of novel therapeutic targets. mammalian ribonucleic acid (rna) viruses persist to pose serious threats to human health and global economies. as this article is being prepared, the world is living through a viral pandemic rna is a diverse, multifunctional biomolecule that is involved in both the transfer and storage of genetic information as well as the modulation of a myriad of biological processes by virtue of its capacity to fold into complex structures and to catalyze biochemical reactions [ ] . it is therefore no surprise that rna viruses take advantage of the unique physicochemical properties of their viral genomes to assemble functional complexes, which in turn drives almost every aspect of their replication cycles within host cells (figure ). many of these complexes are formed through the recruitment of cognate rna-binding proteins (rbps) to specific genomic (or sub-genomic) loci, and the nature of these interactions manifest as signals that regulate each step of viral gene expression [ , ] . thus, studying viral rna structures and their interactions with cognate rbps are essential to understanding the pathogenesis of rna viruses and to further assist the design of novel antivirals. in this article, we attempt to describe how integrating methods that probe rna structures and its interactions can inform on mechanisms that regulate viral gene expression for two representative positive-sense rna viral families, namely enteroviruses and coronaviruses. members from both of these families have caused wide-spread outbreaks in recent history. non-polio human enteroviruses (ev) are persistent pathogens that cause millions of infections in the united states and globally each year [ , ] . infections typically manifest with mild illness; however, protracted infections in the immunocompromised (mostly infants, children and teenagers) can lead to severe neurological disorders, morbidity, paralysis, respiratory failure and death [ , ] . the national institutes for allergies and infectious diseases identified ev-a and evd as emerging infectious pathogens [ ] , and the world health organization discussed including both viruses in its blueprint list of priority diseases [ ] ; emphasizing the serious threat that these viruses represent to public health. in a ev-a outbreak in vietnam, , children were hospitalized and six died [ ] . similar cases with significant mortality rates have been reported in taiwan and other parts of asia-pacific, thus, reiterating the urgency to develop antivirals or vaccines, and the necessity to better understand the molecular mechanisms involved in host-virus interactions [ ] . ev-a is a non-enveloped single-stranded rna virus that contains a , nucleotide (nt) positive sense genome; a dual-purpose rna element that must serve as template for both viral translation and genome replication [ , ] . cellular entry is initiated through interactions between the viral capsid and host membrane receptors such as the scavenger receptor b (scarb ), p-selectin glycoprotein ligand- (psgl- ), heparan sulfate and annexin ii (anx ) and sialic acid-linked glycan. [ ] upon cellular entry, the single strand positive-sense viral rna genome is released into the host cytoplasm. given its limited coding capacity, ev-a uses multiple strategies to usurp host factors and modulate viral protein synthesis and replication. particularly, the virus takes advantage of its highly structured ' untranslated region ( ' utr) to initiate translation in a capindependent pathway [ ] . the 'utr is predicted to fold into six stem loops. stem loop (sl) i adopts a 'cloverleaf' structure known to interact with the viral c protease to promote genome replication, whereas, stem loops ii-vi form the active type i internal ribosome entry site (ires) involved in the recruitment of the ribosome [ ] . the viral genome is translated in a cap-independent pathway, such that a single polyprotein is synthesized, which is further processed by the viral-encoded proteases a and c into structural and non-structural proteins [ ] . additionally, the viral-encoded proteases facilitate the shutdown of the host translation and transcription machinery, which produces the ideal environment for viral translation and replication, and ultimately apoptosis [ ] . the ev-a genome cannot undergo translation and replication concurrently on the same genomic rna, as the ribosome blocks the '- ' progression of the elongating viral rna polymerase [ ] . thus, the virus coordinates complex processes to transition between these two particular stages of its replication cycle. specifically, genomes undergoing replication are translocated to virus-induced vesicles, allowing for spatial separation from those undergoing translation in the cytoplasm [ ] . the ev-a genome is subsequently replicated through a negative strand intermediate and packaged into the viral capsid [ ] ( signaling changes to normal cellular functions [ , ] . competition between the positive and negative regulators suggests a mechanism by which the virus fine-tunes its protein synthesis while simultaneously coordinating the reduction of the host cells translation levels and overall physiological homeostasis [ , ] . interestingly, almost all of these itafs are known to interact with the 'utr of other picornaviruses to regulate ires activity and replication, suggesting an evolutionary preference to conserve rna structural features that drive specific rbp recognition [ , ] . the current dogma supports a model in which itafs cycle the ires through different conformational states to modulate ribosome assembly; however, the molecular mechanisms by which itafs interact with the ires to regulate this process remain poorly understood. therefore, knowledge of the ires structures and how itafs remodel it to assemble functional complexes is essential to understand how ev-a promotes viral protein synthesis to produce progeny virions. december of , the city of wuhan in china witnessed an outbreak of the novel coronavirus disease (covid- ), spreading to countries and regions in months [ ] . according to the johns hopkins coronavirus research center, globally more than million cases and more than , deaths were recorded as of june , . the rapid spread of the infection is attributed to its ability to target the respiratory system [ ] . this virus demonstrates similar symptoms to previously known coronaviruses, such as dry cough, dyspnea, and fever; however, it has the ability to infect lower respiratory airways leading to multiple organ failure in severe cases [ ] . no treatment or vaccine are available to date. covid- is an infection caused by the severe acute respiratory syndrome-related coronavirus, namely sars-cov- and formally known as -ncov [ , ] . the sars-cov- virus belongs to the coronaviridae family, subfamily orthocoronavirinae, and genera betacoronaviruses, similar to mers-cov and sars-cov [ , , ] . the sars-cov- genome displays . % sequence identity to sars-cov, and % similarity to the bat-related coronavirus [ ] . the enveloped coronavirus (cov) genome is a positivesense, single-strand rna, which varies in size from to kb, specifically . kb in sars-cov- [ , ] . the enveloped virion carries a surface spike protein which binds to the host cell surface receptor, angiotensin converting enzyme (ace ). [ ] this interaction promotes fusion of viral and cellular membranes, subsequently releasing the viral contents into the host cytoplasm. upon cellular entry, the viral rna genome is uncoated and released into the cytoplasm where it serves as a template for capdependent viral protein synthesis [ , ] (figure ). the open reading frames, orf a and orf b, covers two-thirds of the viral genome to encode two large polyproteins (pp a and pp ab), which are post-translationally cleaved into nonstructural proteins (nsps) [ , ] . polyprotein translation utilizes a ribosomal frameshifting mechanism that requires ' cap formation and ' polyadenylation of the viral genome [ , ] . nsp or papain-like protease and mainly nsp or c-like protease ( cl pro ) are responsible for processing the polyprotein into mature nsps [ , , ] . structural and accessory proteins are encoded by the remaining one-third of the viral genome [ ] . in covs, ' and '-untranslated regions (utrs) consisting of phylogenetically conserved stem loops are required for replication and transcription [ , ] . multiple nsps and a number of host factors assemble to form the replicase-transcriptase complex (rtc) at the '-utr in order to synthesize genomic and subgenomic rna (sgrna) through negative strand template intermediates [ , ] (figure ). rna-dependent rna polymerase (rdrp) or nsp replicates genomic rna and transcribes sgrna. transcriptional regulatory sequences (trss) guide nsp and are present at the 'leader sequence (trs-leader or trs-l) and at the genomes encoding for accessory and structural proteins (trs-body or trs-b) [ ] . rdrp continues transcription after encountering trs-b sequences and switches to the trs-l to transcribe the ' leader sequence; however, this mechanism is poorly understood [ ] . having common '-ends, sgrnas translate only the ' segments of their orfs into accessory and structural proteins and recognize the rest of the sequence as an untranslated region [ , ] . comparatively to other coronaviruses, nsp promotes degradation of host mrna and inhibits host cell translation [ ] ; a common strategy by which positive-sense rna viruses reprogram the cellular environment [ ] . in murine hepatitis virus (mhv), a betacoronavirus, hnrnp a and polypyrimidine tract-binding protein (ptb) bind to the '-utr, specifically at the trs, and play a role in rna synthesis [ , ] . in addition, eukaryotic initiation factors, eifs, i, f, and e along with other host proteins assemble in the microenvironment of the replicase-transcriptase complex (rtc) [ ] . the sirnamediated knockdown of these initiating factors showed a reduction in rna replication, indicating their involvement in virus-dependent reprogramming of the cellular environment [ ] . these aforementioned virus-host interactions and their mechanisms of action are yet to be understood. the novelty of sars-cov- raises many questions concerning the mechanisms by which the virus regulates its gene expression. obtaining structural details of the utrs and identifying functional binding sites of rbps will be deeply insightful in elucidating how this virus replicates within host cells. focusing on essential rna-rna and rna-protein interactions, such as rdrp, cl pro [ , ] , or cellular rbps will inform on novel targets to therapeutically inhibit sars-cov- , while simultaneously shedding light on the cellular pathways hijacked by the virus. despite differences in the life cycles of entero-and coronaviruses, sufficient similarities allow for the parallel discussion of shared features of their biology (figure ). implemented to identify functional rna structural motifs unlike traditional methods, such as align-and-fold or fold-and-align, which rely on previous sequence alignments [ ] . scanfold decouples these steps, which minimizes computational time and aids researchers studying systems with poor sequence alignments [ ] . potential functional regions on the rna are identified by analyzing the thermodynamic parameter z-score from which a single base pair arrangement is assigned to each nucleotide in the input sequence and a structural model is built [ ] . the scanfold pipeline was previously benchmarked against experimentally supported models of the well-studied hiv- genome [ ] , and it has been used recently to identify thermodynamically stable rna structures throughout the sars-cov- genome (https://www.biorxiv.org/content/ . / . . . v ). a similar approach can be employed to identify probable functional regions along the genome of other rna viruses, which are thought to coordinate multiple aspects of their replication cycles through co-opting rbps (figure ) . furthermore, scanfold results can be complemented with sequence alignment data to identify functional regions that have been evolutionarily conserved in rna viruses. although scanfold can identify rna sequences with potential to form stable structures, the obvious "limitation" is that the structures are predicted and therefore need to be validated experimentally by other structural methods described in this article (figure ) . particularly, these structures predicted in silico can be further confirmed experimentally [ ] . dms modifies non-base paired adenosine and cytosine nucleotides present in bulges, loops, and other regions where the watson-crick edges of these bases are exposed. using thermostable group ii intron reverse transcriptase (tgir-ii), the modified rna is reverse transcribed by creating a mutation when it comes across a methylated nucleotide. high throughput sequencing of the products will generate dms driven rna secondary identify the accessibility profile of rna regions that are exposed and this information can aid in the determination of the structural changes that accompany rna-rna and/or rnaprotein interactions [ ] . in the aim of studying structural rna accessibility, antisense rna probing utilizes a structural sensing system (irs ), a previously designed in vivo sensor, that utilizes an antisense rna probe that hybridizes to the rna of interest. this probe constitutes of a to nucleotide rna complementary to its target, a stem loop structure that blocks the ribosome binding site (rbs) by binding to the cis-acting region, and a gfp encoding region to generate a fluorescent output signal [ ] . the clip-seq framework was used in a recent study to identify binding sites for the splicing regulators hnrnp a and hnrnp h along the hiv- genome [ ] . that study revealed rbp binding sites proximal to splice acceptor and donor signals that control hiv splicing. mutations of select binding sites resulted in changes in hiv splicing patterns that had impacts on viral replication. nmr spectroscopy experiments carried out on protein-rna interaction identified from clip-seq offered additional mechanistic insights into sequence specific recognition of the hnrnp h protein for its hiv targets. given the large number of rbps known to interact with genomic and subgenomic viral rnas to modulate translation, replication and the shift between these two stages, clip-seq can be employed to understand virology at the molecular level. altogether, this methodology provides the experimental scaffold to study host-viruses interactions more comprehensively and design novel strategies to design antivirals. as demonstrated for the hiv clip-seq study, it is useful to couple clip-seq with other structural approaches to provide more mechanistic details on the rna physicochemical features that contribute to form functional rnp complexes (figure complementary information on all nucleotide types exposed within unpaired regions and those likely involved in long-range tertiary interactions. once secondary structural models are known, high-resolution d nmr structures can be obtained in association with computational methods (https://doi.org/ . / . . . ), producing models based on integrated data sets. by adapting this integrated approach, viral rna structural models, which are cross-validated, can be determined for the 'utr of sar-cov- as well as others. in addition to structure, the rna-protein interactions and their effect on rna folding are yet to be studied in the sars-cov- rtc. clip-seq can identify the rbp rna-targets at the nucleotide level in vivo. with prior knowledge of the binding sites, antisense rna probing can confirm these interactions and identify their effects on rna folding. coupling with nmr, binding sites for sub-domains of the rtc can be mapped onto the rna structure. since nmr assignments will be available, it should be straightforward to assess the extent of binding induced conformational changes. although the rtc is used as one example, these integrated technologies and others like them should provide mechanistic insights into a wide-range of host-virus pathways (figure ) . for instance, rdrp plays a primary role in viral transcription and replication [ ] . uncovering its binding site on the '-utr can aid in designing a site-specific drug to prevent this interaction. also, monitoring the '-pseudo-knot's structural interactions under the effect of rdrp can give insight on the molecular switching mechanism for rna synthesis. this can be tested under the influence of different viral or cellular factors at different environmental conditions. in addition, rdrp transcribes sgrna through the guidance of trss and host proteins [ ] . identifying the binding sites of specific host proteins to the trs will help investigate the mechanism of protein recruitment for transcriptional purposes. several other rna involved processes can be studied under the effect of viral or host factors. similar to sars-cov- , ev-a and other viruses can be effectively studied by integrating structural techniques. this blueprint can be adopted for any rna virus. studying rna structural interactions and the effects of viral-host rbps on rna structure and function are essential for understanding translation, replication, and transcription processes in order to better understand how viruses reprogram the cellular environment. the effectiveness of integrating these approaches can help design and test more effective and site-specific small molecules. as we deal with the covid- pandemic and prepare for the next viral outbreak, we hope that this article will encourage adopting integrative (collaborative) approaches that will enhance our understanding of viral rna structures, their interactions with cognate proteins, and in turn the mechanisms by which viruses reprogram the cellular environment. with this comprehensive level of knowledge, we expect that the discovery of novel therapeutic agents will be accelerated. insights into rna structure and function from genome-wide studies diverse roles of host rna binding proteins in rna virus replication virus and human disease an apparently new enterovirus isolated from patients with disease of the central nervous system neurotropic enterovirus infections in the central nervous system understanding enterovirus 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interacts with the enterovirus ′ untranslated region and participates in virus replication hnrnp a alters the structure of a conserved enterovirus ires domain to stimulate viral translation regulation mechanisms of viral ires-driven translation hur and ago bind the internal ribosome entry site of enterovirus and promote virus translation and replication far upstream element binding protein binds the internal ribosomal entry site of enterovirus and enhances viral translation and viral growth research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases covid- and the cardiovascular system the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- bat coronaviruses in china. viruses a pneumonia outbreak associated with a new coronavirus of probable bat origin the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak-an update on the status cell entry mechanisms of sars-cov- coronaviruses: methods and protocols sars and mers: recent insights into emerging coronaviruses direct rna sequencing and early evolution of sars-cov- . biorxiv inhibition of the main protease cl-pro of the coronavirus disease via structure-based ligand design and molecular modeling recombinant sars-cov nsp and the use of thereof and the method for producing it potential therapeutic agents for covid- based on the analysis of protease and rna polymerase docking coronaviruses: an overview of their replication and pathogenesis continuous and discontinuous rna synthesis in coronaviruses. annual review of virology viral and cellular mrna translation in coronavirus-infected cells a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp protein. nature structural & molecular biology polypyrimidine tract-binding protein binds to the leader rna of mouse hepatitis virus and serves as a regulator of viral transcription heterogeneous nuclear ribonucleoprotein a binds to the transcription-regulatory region of mouse hepatitis virus rna determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling. elife structural elucidation of sars-cov- vital proteins: computational methods reveal potential drug candidates against main protease, nsp rna-dependent rna polymerase and nsp helicase coronavirus cl pro proteinase cleavage sites: possible relevance to sars virus pathology ribosolve: rapid determination of three-dimensional rna-only structures. biorxiv progress and challenges for chemical probing of rna structure inside living cells advances in clip technologies for studies of protein-rna interactions high-throughput determination of rna structures integrated structural biology to unravel molecular mechanisms of protein-rna recognition scanfold: an approach for genomewide discovery of local rna structural elements-applications to zika virus and hiv mapping the rna structural landscape of viral genomes viral rna structure analysis using dms-mapseq antisense probing of dynamic rna structures identifying the rna targets of rna binding proteins with clip genome-wide analysis of heterogeneous nuclear ribonucleoprotein (hnrnp) binding to hiv- rna reveals a key role for hnrnp h in alternative viral mrna splicing applications of nmr to structure determination of rnas large and small this work was funded by national institutes of health grants u ai (the center for hiv rna studies) and r gm (bst). key: cord- - x j iqg authors: bösl, korbinian; ianevski, aleksandr; than, thoa t.; andersen, petter i.; kuivanen, suvi; teppor, mona; zusinaite, eva; dumpis, uga; vitkauskiene, astra; cox, rebecca j.; kallio-kokko, hannimari; bergqvist, anders; tenson, tanel; merits, andres; oksenych, valentyn; bjørås, magnar; anthonsen, marit w.; shum, david; kaarbø, mari; vapalahti, olli; windisch, marc p.; superti-furga, giulio; snijder, berend; kainov, denis; kandasamy, richard k. title: common nodes of virus–host interaction revealed through an integrated network analysis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x j iqg viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses continue to be a major contributor to the global burden of disease through acute and chronic infections that cause substantial economic impact in addition to increased mortality and morbidity ( ) . despite the tremendous improvement in the understanding of the antiviral immune response and the availability of therapeutics, existing and emerging viral diseases are an ever-growing problem, particularly in developing countries. development of antiviral resistance of hepatitis c virus (hcv), influenza a virus (iav), herpes simplex virus (hsv), human cytomegalovirus (hcmv), human immunodeficiency virus (hiv), and other viruses is a major concern ( - ). one of the main reasons for increasing resistances is the accumulation of mutations in the viral genome caused by multiple factors including the polymerase infidelity ( , ) . therefore, the world health organization (who) and the united nations have urged for better control of viral diseases. this has led to turning the focus on the host for therapeutic intervention. targeting the host factors has been proven to be useful for restricting viral infections ( , ) . the small molecule ccr inhibitor maraviroc and the anti-cd monoclonal antibody ibalizumab are examples of successful use of host-directed therapies for combating hiv in clinic ( ) ( ) ( ) . viruses have evolved to evade the host antiviral response at various stages starting from viral sensing to antiviral proinflammatory responses ( ) ( ) ( ) . multiple studies attempted to understand global principles of the viral evasion employed by various viruses, including dengue virus (denv), ebola virus (ebov), iav, and hiv ( ) ( ) ( ) ( ) ( ) ( ) . global systems-level approaches including functional rnai screens, interactome mapping technologies such as affinity-purification mass spectrometry (ap-ms), quantitative proteomics, and crispr/cas -based screens have provided unparalleled details and insights into the dynamics of host proteome in immune cells ( ) ( ) ( ) ( ) , host-virus interactome ( - , , ) , and also identified important host dependency factors of various viruses ( , , ) . meta-analyses of such high-dimensional datasets have been crucial for identifying novel host factors as drug targets such as ubr in iav infection ( ) . moreover, some of these factors represent drug targets for multiple viruses ( ) . we hypothesized that combining a meta-analysis of host-virus protein-protein interactions of multiple viruses and functional rnai screens would provide novel insights for developing broadspectrum antiviral strategies. for this, we assembled a host-virus protein-protein interactome of , host-virus interactions (hereafter referred to as "hvppi") covering viral proteins from different viruses and , host proteins. we performed extensive bioinformatics and network analysis and integrated this dataset with genome-wide or druggable-genome rnai screen data from published studies. this resulted in the assembly of critical nodes of viral evasion and identification of core cellular processes and druggable nodes that were verified by a drug re-purposing screen using broad-spectrum antivirals. host-virus protein-protein interactions were downloaded from published studies ( - , , - ) we performed gene-set enrichment analysis using david bioinformatics resources [version . , ( ) ]. for all enrichment analysis, a p-value cutoff of ≤ . was used as significant. protein disorder analysis was performed using iupred a software. we used the offline version with protein sequences downloaded from uniprot. statistical analysis of disordered region distribution was performed by kolmogorov-smirnov test in r statistical environment. annotation of human proteins was mapped from uniprot id to ensembl using ensdb.hsapiens.v . the index of subcellular localization of interaction partners of single viral proteins was calculated for all viral proteins with ≥ five host targets. localization of host targets was mapped using compartments ( ) , filtered for a minimum evidence score of in the knowledge channel, excluding non-experimental based localization predictions. evidence for all protein was subsequently divided by the absolute number of host-targets per viral protein. multiple sequence alignment was performed using clustal x [version . , ( )]. genome-wide rnai screen data for hcv ( ) and hpv ( ) , through genomernai database [( )-gr ], as well as druggable rnai screen data for hpv ( ) , vacv ( ) , and sv ( ) were integrated in the existing network as z-scores. drug-gene interaction data was downloaded from dgidb and drugbank. the identifiers were mapped to uniprot ids and then compared with hvppi. for the hmpv nl/ / -gfp screen, approximately × human retinal pigment epithelial (rpe) cells were seeded per well in -well plates. human non-malignant rpe cell line represents excellent model system for studying replication of many viruses including respiratory ( , , ) . the cells were grown for h in dmem-f medium supplemented with % fbs, . % nahco , and µg/ml streptomicine and iu/ml penicillin. the medium was replaced with virus growth medium (vgm) containing . % bovine serum albumin (bsa), mm l-glutamine, . % nahco , and µg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin in dmem-f . hcv screen-associated cell culture conditions are described in kim et al. ( ) . the compounds were added to the cells in fold dilutions at seven different concentrations starting from µm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. after h of infection, the medium was removed from the cells. to monitor cell viability, celltiter-glo reagent was added ( µl per well). this assay quantifies atp, an indicator of metabolically active living cells. the luminescence was measured with a plate reader. to determine compound efficacy, hmpv nl/ / -mediated gfp expression was measured. the half-maximal cytotoxic concentration (cc ) and the half-maximal effective concentration (ec ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version . a. the relative effectiveness of the drug was quantified as the selectivity index (si = cc ec ). cytotoxicity and antiviral activity of the compounds against gfp-expressing hcv in huh- . cells was determined as previously described ( ). to provide new and critical insights into viral evasion mechanisms we performed a comprehensive meta-analysis of the host-virus interaction landscape. we assembled the hostvirus protein-protein interaction data ("hvppi") from published studies ( figure a ) ( - , , - ) . this dataset comprised of protein-protein interactions from two different types of experimental methods-affinity purification mass spectrometry (ap-ms) and yeast two-hybrid screens (y h). altogether, this combined dataset includes viral proteins, , host proteins, and , protein-protein interactions ( figure b and figure s ). many interactome networks including yeast and human are scale-free networks, where a large portion of the nodes (e.g., a protein in the network) have few interactions and only a few nodes have large number of interactions. the latter are often referred to as "hubs" which are crucial in keeping the network intact ( ) . we performed network topology analysis to infer the properties of the host proteins targeted by the viral proteins in the context of the human protein interactome. we considered two important parameters-relative betweenness centrality (which reflects the amount of information that passes through this protein in the human interactome) and degree (number of binding partners in the human interactome) of the host proteins targeted by each virus. the targets of all the viruses showed higher betweenness centrality and degree as compared to an average protein in the human interactome (figures c,d) . this shows that viruses, by targeting "hubs" and proteins that serves as key communication nodes, have evolved the best way to disrupt the scale-free human interactome. this topological property thereby shows how viruses having small genomes achieve the maximal effect in rewiring the human interactome to benefit viral survival and replication. our analysis is in agreement with several previous studies, which have highlighted this property ( , , , , ) . we propose that this could be a general principle for all viruses. proteins typically fold into stable three-dimensional structures that mediate specific functions. in addition, there are substructures in proteins termed "intrinsically disordered regions (idrs)" which lack stable structures under normal physiological conditions. idrs are required for multiple cellular functions even though they lack these defined structures ( ) . many studies have highlighted the presence of such idrs in viral proteins ( ) ( ) ( ) , such as e from human papilloma virus, that are crucial for hijacking the cellular machinery. we analyzed the host proteins from the hvppi for presence of idrs using the prediction software iupred ( ) . it is estimated that the human proteome more than , short linear binding motifs in idrs ( , ) . proteins with idrs are often signaling hubs and might form dynamic complexes with other proteins through specific motif based interactions ( ) . we found a statistically significant enrichment (p-value < . × − ) of idrs in the host proteins targeted by viruses (figure a and figure s ). we then identified the subnetwork in the hvppi which contained the top host targets with high disorderness score ( figure b) . the top five proteins with large idrs include cd antigen (cd ), serine/arginine repetitive matrix protein (srrm ), myristoylated alaninerich c-kinase substrate (marcks), bag family molecular chaperone regulator (bag ), and mitochondrial antiviralsignaling protein (mavs; figure c ). cd is a marker of exhausted cd + t cells ( ) and replication of hcv in t cells was shown to decrease cell proliferation by inhibiting cd expression and signaling ( ) . srrm is a serine/arginine-rich protein involved in rna splicing ( ) . srrm is differentially phosphorylated in hiv- infected cells and absence of srrm lead to increased hiv- gene expression, since it regulates the splicing of hiv- ( ) . in the hvppi, srrm is targeted by multiple viral proteins including the tat protein from hiv- . tat protein has an important role in the stimulation of the transcription of the long terminal repeat (ltr) ( ) . in addition, ns protein from influenza b virus has also been reported to interact with srrm ( ) . proteins of the marcks family are involved in a range of cellular processes including cell adhesion and migration ( ) . marcks is a negative regulator of lipopolysaccharide (lps)-induced toll-like receptor (tlr ) signaling in mouse macrophages ( ) . mavs is an adaptor protein in the rig-i signaling pathway involved in the sensing of rna. ablasser et al. ( ) reported that double-stranded dna serves as a template for rna polymerase ii and is transcribed into a ′ triphosphate containing double-stranded rna, which activates the rig-i signaling pathway. in the hvppi, mavs is targeted by several proteins from dsdna viruses such as ebv and hpv. altogether, our analysis shows that the idr-high part of the human proteome is an essential part of the viral evasion strategy and some of the selected targets highlighted here could show novel insights into the viral evasion mechanisms. however, the very flexible protein structure of disordered proteins also makes them also difficult to target with drugs. to assess the signaling pathways and cellular processes within the hvppi, we identified highly connected subnetworks within hvppi network. we constructed a host-host interaction network based on the host targets in the hvppi and identified a number of highly connected subnetworks/clusters (figure ) . we then performed a gene-set enrichment analysis of significantly enriched biological processes. we found one or more enriched processes for each of this subnetwork including core cellular processes such as proteasome, spliceosome, protein translation, protein/rna transport, and cell cycle. next we listed the viruses that target one or more of these processes, and found that almost all the core pathways and processes are targeted by all the viruses that are part of the hvppi (figure s ). this analysis highlights the core components of the cellular process subnetworks which are targeted as part of the viral evasion strategies and thus could be broad-spectrum antiviral hot-spots from a therapeutic point of view. given that the viral proteins were interacting with a large number of host proteins, we analyzed the sub-cellular location of the host proteins. we performed gene-set enrichment analysis of sub-cellular localization information provided by uniprot database. we binned the localization into compartments and estimated the percent of host proteins in a given compartment as compared to the total number of host proteins targeted by a given figure | clusters of hvppi involved in core cellular processes. network view of the "clusters" or highly-connected sub-networks and their associated cellular processes. each cluster is marked in a unique color. virus. we found that the viral targets were distributed across multiple subcellular compartments with cytoplasm being the most common ( figure s a ). the hvppi includes two different strains of iav-pr (h n ) and udorn (h n ). the subcellular localization analysis showed that both strains were enriched for nuclear proteins. non-structural protein (ns ) from both the strains had the highest number of nuclear targets but their targets were very different ( figure a) . ns of udorn was enriched for a large number of histones as compared to ns of pr that had large number of heterogeneous nuclear ribonucleoproteins (hnrnps), such as hnrnpu−a known restriction factor for many viruses. this corroborates with the observation that ns protein has short linear histone mimicry motifs that can suppress the host antiviral response ( ) . in our analysis, we found that it is ns of udorn that has a histone mimicry motif "arsk" (figure s b) . similarly, hpv and hpv e proteins interact more often with host proteins located in the endoplasmic reticulum (er). we found both common and specific subsets of er proteins targeted by the e protein ( figure b ). hpv e protein er targets were enriched for phospholipid biosynthesis as well as gpi anchor related proteins, such as phosphatidylinositol glycan anchor biosynthesis class s/t/u (pigs, pigt, and pigu), glycosylphosphatidylinositol anchor attachment (gpaa ) and phosphatidylserine synthase (ptdss ). hpv e protein er targets were enriched for er-associated ubiquitindependent protein catabolism involving host proteins such as er degradation enhancing alpha-mannosidase-like protein (edem ) and er lipid raft associated (erlin ). er targets common to hpv and hpv e protein were enriched for unfolded protein response, n-linked glycosylation and protein folding involving host proteins such as srp receptor alpha/beta subunit (srpra/srprb) and catalytic subunits of the oligosaccharyltransferase complex (stt a and stt b). two independent crispr/cas screening studies identified multiple er associated components including stt a and stt b as host factors for denv, zika virus (zikv) and japanese encephalitis virus (jev) ( , ) . the non-canonical function of stt a and stt b is required for denv replication and that ns protein of denv interacts with these proteins ( ) . our orthogonal approach can lead to the identification of critical host factors, and similar functions of er components, such as stt a and stt b, are used by hpv and hpv as well. thus, targeting the non-canonical function of stt a and stt b could be a broad antiviral strategy. overall, the enrichment analysis clearly shows that there is commonality and specificity in the subcellular targets of the viral proteins and that detailed interrogation of these targets can give vital clues into the viral evasion mechanisms. rnai screens have been a powerful high-throughput method to identify various cellular functions, including for identification of host restriction factors of viruses ( ) . in order to explore the functional relevance of the host targets in the hvppi, we integrated it with five published rnai screens that performed genome-wide or druggable-genome-wide rnai screens for identifying host factors of hcv ( ), hpv ( ), hpv ( ), sv ( ) , and vacv ( ) . we found that host targets from the hvppi were spread across the spectrum of genes with proviral as well as antiviral phenotype (figure s ) , thus showing that targeting of the host protein by the virus could lead into any direction that favors the virus. we then investigated the top proviral genes that are also targeted by the viral proteins as seen in the hvppi. we identified host proteins ( figure a ) that were significantly enriched for coatomer protein complex (copi), protein translation/transport and proteasome ( figure b) . this further substantiates the findings from the earlier section on the core cellular processes targeted by the viruses. network analysis of these top hits showed high level on connectivity and crosstalk-for example between the translation and proteasome machinery (figure c ). vesicle carriers are involved in the transport of membranes and proteins. copi system is one of the three vesicular carrier systems that is involved in the early secretory pathway ( ) ( ) ( ) . moreover, it has been pointed out that there is a strong similarity between vesicular transport and viral transport [viral entry to budding process, ( ) ]; thus making copi system important for the viral life cycle. in addition to the findings of the present study, sirna-based silencing of copi lead to a decrease in entry and subsequent gene expression of iav, vsv, lcmv, and hpiv and disruption of the copi complex inhibited the production of infectious progeny virus ( , ) . copi coatomer inactivation results in a direct decrease of vsv attachment and uptake, but not for membrane fusion or rnp release; however the direct mechanism remains unclear ( ) . altogether, these top hits including the copi system could serve as targets for developing therapeutic antiviral intervention strategies for a broad group of viruses. our analyses pointed out that viral evasion mechanism observed in one virus could also be relevant for other viruses. to test this, we obtained known drug-gene interactions from dgidb ( ). we selected investigational/experimental/approved antivirals compounds ( ) which had dgidb annotated targets that are part of the hvppi. we added agents as controls (table s and figure s ) . we tested broad-spectrumantivirals against gfp-expressing human metapneumovirus (hmpv) nl/ / strain ( ) . seven different concentrations of the compounds were added to hmpv or mock-infected cells. hmpv-induced gfp expression and cell viability was measured and after h to determine compound efficiency and toxicity. after the initial screening, we identified five compounds, which lowered gfp-expression without detectable cytotoxicity (with si > ). we repeated the experiment with these compounds. the experiments revealed novel activity of azacytidine, lopinavir, nitazoxanide, itraconazole, and oritavancin against hmpv ( figure s a and table , table s ). similarly, we examined toxicity and antiviral activity of broad-spectrum-antivirals against gfp-expressing hcv in huh- . cells using previously described procedures ( ) . our test identified azithromycin, cidofovir, oritavancin, dibucaine, gefitinib, minocycline, and pirlindole mesylate as novel anti-hcv agents with si > ( figure s b and table , table s ). in summary, our metaanalysis approach of the hvppi could provide novel and faster approaches for the re-purposing of existing drugs as antiviral agents. using integrative analysis of orthogonal datasets our study provides a comprehensive view of viral evasion mechanisms. in particular, our analysis of the hvppi network revealed that all the viruses have evolved to target proteins that are central and have strong control over the human interactome. host proteins targeted by viruses contain a high proportion of intrinsically disordered regions. we identified the core cellular processes and associated proteins that are targeted by all viruses. detailed comparative analysis of the subcellular localization of the host proteins showed commonality and specificity both between viral proteins from different strains of the same virus; and between viruses. integrating hvppi with functional rnai screens showed that % of the hvppi are host factors of one or more virus. hvppi data-based drug re-purposing screen identified novel activities for various broad-spectrum antivirals against hmpv and hcv. this unique dataset can be used for further detailed interrogation of the mechanisms behind viral evasion. this could serve as a starting point for identifying novel host targets and generating hypothesis in the context of viral evasion and development of pan-viral therapeutic intervention strategies. the methods described here also provide unique ways of dissecting the orthogonal datasets. various analyses from this study have highlighted the existence on pan-viral evasion points that could be utilized for the development of host-directed antiviral therapies. it is also intriguing to see that there is commonality and specificity at the level of sub cellular localization of the viral targets. our analyses have underlined some salient features in the context of iav, hpv, denv, and hcv. further detailed analysis in this context along with protein sequence features, such as short linear motifs [slims; ( ) ] would provide novel insights as well as deeper understanding of how small sequence features are involved in the hijacking of the host machinery. integration of such data with known drug-gene interactions provides a clear estimate of the druggable proportion in the hvppi. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. to prove the concept, we tested bsas against hmpv, hcv, sindbis virus (sinv), cytomegalovirus (cmv), and hepatitis b virus (hbv). importantly, bsas have dgidb annotated targets that are part of the hvppi, whereas were used as controls. these safein-man drugs have already been used as investigational agents or experimental drugs in different virus infections (table s ) . we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv. azithromycin, is an fda-approved antibiotic of the macrolide family. it is also an investigational agent against rsv and experimental agent against ebov, hrv-a, zikv, and rsv. cidofovir is an fda-approved anti-cmv drug. it is also investigational agent against adv, bkv, hpv, hsv- , hsv- , and experimental drug against b v. dibucaine is an fdaapproved amide local anesthetic. in addition, it is experimental anti-hev-a, hev-b, hev-d, and ebov agent. gefitinib is an fda approved anticancer drug. it has also antiviral activity against bkv, cmv, and vacv. minocycline is a broad-spectrum antibiotic and experimental anti-denv, hiv- , and wnv agent. oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections. pirlindole is an antidepressant, which is also experimental anti-hev-a, hev-b, and hev-d agent. azacitidine is a chemical analog of cytidine, which is used in the treatment of myelodysplastic syndrome. it is also an experimental anti-adv, the measurements were repeated three times (p < . ). fluav, rvfv, hiv- , and hiv- agent. itraconazole is an antifungal medication. it is also used as experimental anti-hev-b, hrv-b, hrv-a, par-a , and safv agent. nitazoxanide is a broad-spectrum antiparasitic drug, which is also investigational agent against fluav and hcv and experimental anti-chikv, rsv, hbv, hiv- , vacv, rv, jev, mers-cov, nov, ruv, and zikv agent. lopinavir is an fda-approved antiretroviral of the protease inhibitor class. it is also investigational anti-mers-cov and experimental anti-zikv agent (table s ). in addition to inhibition of viral proteases (table s ) , lopinavir was reported to induce host rnasel production in infected and non-infected cells ( ) . rnasel is endoribonuclease that is a part of interferon (ifn) antiviral response, which is the most critical node of virus-host interactions. although, the antiviral mechanisms of action of other compounds are still unknown, these agents could inhibit steps of viral infections, which precede reporter protein expression from viral rna. in summary, our results indicate that existing bsas could be re-purposed to other viral infections. to further expand a spectrum of their activities, these bsas could be tested against other viruses. re-purposing these and other safe-inman antiviral therapeutics could save resources and time needed for development of novel drugs to quickly address unmet medical needs, because safety profiles of these agents in humans are available. effective treatment with broad-spectrum-antivirals may shortly become available, pending the results of further pre-clinical studies and clinical trials. this, in turn, means that some broad-spectrum-antivirals could be used for rapid management of new or emerging drug-resistant strains, as well as for first-line treatment or for prophylaxis of acute virus infections or for viral co-infections. the most effective and tolerable compounds could expand the available therapeutics for the treatment of viral diseases, improving preparedness and the protection of the general population from viral epidemics and pandemics. all datasets used for this study are accessible as stated in the materials and methods section . . we thank christian sinzger and group for the egfp-expressing tb e cmv strain. we thank vironovative and erasmus mc for the gfp-expressing hmpv nl/ / strain. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material redefining chronic viral infection antiviral drug resistance as an adaptive process emerging virus diseases: can we ever expect the unexpected? emerg microbes infect herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy mechanisms of viral mutation complexities of viral mutation rates host-directed therapies 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title: pathogenesis of viral infections date: - - journal: veterinary clinics of north america: small animal practice doi: . /s - ( ) - sha: doc_id: cord_uid: wwaa ls the article considers factors that influence pathogenesis, initiation of infection, dissemination of virus within a host, lytic viral infections, viral immunosuppression, viral immunopathology, and viral oncogenesis. genetic characteristics of the host · · or environmental influences. genetic host factors include animal species, breed, and organ or tissue susceptibility. · · · such restrictions function at the cellular level either as the presence or absence of appropriate cell surface receptors (in some instances, they have been shown to be inherited as dominant alleles in a mendelian manner) · · · · · ·u · or the intracellular hospitality of the cell (several genetic host restrictions on virus replication have been identified). · · · · · · · · restricted growth of several dna viruses in some cells results in transformation without production of progeny viruses. to a degree, the animal's ability to respond immunologically or by interferon production is an innate property of the host. , , . , , , , . nongenetic factors include passive immunity, acquired immunity, age, stress, trauma, hormonal levels, nutritional status, environmental temperature, pregnancy, and concurrent infections (which may either enhance or interfere with a virus infection). , , , , o, , , , , virus factors certain virus properties materially affect the manner in which disease is elicited. cytocidal viruses such as alphaherpesviruses effectively inhibit cellular metabolism, · · · · ·ii · · · and others result in pathophysiologic permeability of cell membranes. certain viruses contain cytotoxins, which may be structural proteins (such as the penton adenovirus protein) or induce production of cytotoxic substances. , ,u,us, , , , noncytocidal viruses may result in steady-state infections, · · · · • but these in turn can result in perturbations of the immune response. , , , , , s-so,oo, · · · , ·m· cell-mediated destruction of tissues, immune complex disease, autoimmunity, and disseminated intravascular coagulation are possible sequelae. the presence of the enzyme reverse transcriptase in virions of members of the family retroviridae imparts two important characteristics to these viruses-the capacity for latency as "proviruses" and for transformation of the cell. • · · strain variation in virulence and in tissue tropism is well recognized. · · ·uo.u · these phenomena are undoubtedly related to variations in the nucleotide sequences of the viruses. ·u · some viruses, as a result of their genomic structure or their particular replication strategy, induce interferon production in the host more efficiently, which in turn may result in greater resistance to the virus. · several instances have been documented in which viruses modify cell function without detectable cell injury. · examples include disease as a result of virus modulation of immunocompetent cells and cells with endocrine functions. · · - b,lll of particular concern in viral pathogenesis is the tissue tropism of the virus, which is determined by complex interactions of virus structure, cell receptors, cell metabolism, intracellular hospitality, and virus replication strategy. , , , , , , ,oo, , , o, , the host is more vulnerable if cell destruction occurs in vital tissues or organs such as the heart, central nervous system, immunocompetent cells, liver, endothelium, and certain endocrine tissues. , , , , , , , , , whether a virus initiates a successful infection in a particular host is often dependent on the amount of inoculum that the host is exposed to and the route by which virus gains entrance into the body. · · · for such viruses, a certain threshold amount of virus is required for successful infection, and, in some instances, the severity of the ensuing disease may be determined by dose of the virus. as alluded to earlier, the first barriers that viruses must penetrate are various epithelia of the skin and its mucosal extensions, alimentary canal, respiratory tract, and the urogenital tract. · · · these epithelia may have various products that aid in resistance to virus, such as film, mucus, keratin (skin), acidity (stomach), and so forth. these barriers must be overcome by viruses to initiate infections. some viruses (orthomyxoviruses, for example) penetrate mucus by enzymatic action (neuraminidase); others are more phresistant and may more readily infect the intestinal tract, and some viruses initiate infection after mechanical injury to epithelia. · · ·u · cell surface receptors characteristic for a particular species and for a particular tissue may determine species (or breed) and organ susceptibility. · · · ·u · these receptors are genetically specified. in most instances, the chemistry of cell receptors is not well characterized. it is best known for the ortho-and paramyxoviruses for which it is a neuraminic acid-containing mucoprotein. ·u others include lipoglycoproteins (picornaviruses), histocompatibility complex antigens (some alphaviruses), and fe and complement receptors on macrophages. · · a · · · the latter receptors on some flavi-, alpha-, bunya-, rhabdo-, and reoviruses result in enhanced infections in the presence of subneutralizing concentrations of antibody or heterologous strain antibody that promotes attachment to fe receptors. receptors that are ubiquitous on the surface of cells of various species (such as neuraminic acid-containing glycoproteins) are not important determinants of host range or tissue tropism. · receptors for picornaviruses often do determine these phenomena. · a receptor map has been made for four groups of nonenveloped viruses, and it is clear that even unrelated viruses may share the same receptor. viruses that share the same receptor can interfere with one another since they compete for the receptor (homologous interference). · host range and receptor specificity are particularly important in the retroviridae family. · · for instance, the susceptibility of certain genetic strains of chickens to various oncovirinae depends on the nature of cell surface receptors for the largest viral envelope glycoprotein. these receptors are specified genetically and are inherited as dominant alleles in a mendelian manner. the viral protein and cell receptor have clinical significance because occupation of a receptor by an avirulent oncovirus will block infection with a highly oncogenic virus. another factor in the initiation of infection should be considered. attachment of ortho-and paramyxoviruses to receptors is not important in determining host range and tissue tropism, but it appears that the step of penetration does determine these phenomena. · · · · certain peplomeres of paramyxoviruses have a fusion (f) protein that, after attachment, results in the fusion of the viral envelope with the plasma membrane, al-lowing the viral rna to enter the cell to initiate virus replication. the fusion protein exists as a precursor and is not activated until cleaved by specific cellular proteases probably on the surface of the plasma membrane. cell susceptibility is determined by the presence of the appropriate proteases. a similar event occurs with the orthomyxoviruses, but in this virus group, cleavage of the hemagglutinin facilitates penetration (but does not affect attachment to cells). • • · however, the cleavage of the hemagglutinin occurs during assembly of the virions late in the replication cycle. thus, virion infectivity is dependent on the virus undergoing a full replication cycle in susceptible cells that contain the appropriate protease. a novel concept with regard to receptors on cell surfaces is that cells infected with viruses may permit adherence of certain pathogenic bacteria, leading to bacterial colonization. the phenomenon appears to be mediated by virus-induced receptors on the surface membrane of cells and may be one mechanism of the often-encountered secondary bacterial infections associated with viral diseases. some viruses initiate infections as a result of mechanical injuries of epithelial barriers. • • this can occur as a result of insect bites, which is particularly important for the arthropod-borne viruses (many of the togaviridae, reoviridae, bunyaviridae, and so on). iatrogenic introduction of viruses with needles and so forth is not uncommon. rabies virus constitutes a classical example of a disease initiated by a wound and influenced by dose of virus. • · the latter virus is deposited in a wound usually as a result of a bite from an animal with salivary secretion of virus. if sufficient virus is present, nerve endings are immediately penetrated. however, often the virus first replicates in local muscle cells, which allows for amplification of the infectious dose and penetration of the nerve endings followed by centripetal spread along the axoplasm of nerves to the central nervous system. variations in pathogenesis occur when this general progression of virus is delayed or stopped at some point. replication of virus in muscle cells at the inoculation site may be responsible for variations in incubation period. survival may occur when infection is localized for some reason at the inoculation site or peripheral nervous system. for a variable period of time (before the virus enters the nerves), it is susceptible to antibody. the respiratory tract is a very common site at which virus infections are initiated, usually as the result of airborne infections. • • • • droplets often originate from the respiratory tract and mouth of infected animals, but aerosols of urine, fecal material, and so on can infect the respiratory tract. small droplets of less than j.lm in diameter dry to droplet nuclei of i to j.lm in diameter that remain suspended for long periods and easily gain entrance to bronchioli and alveoli. viruses that resist desiccation remain viable in droplet nuclei for extended periods of time. film and mucus afford some protection, but myxoviruses that are entrapped by receptor-like mucoproteins in mucus may be released by the peplomere enzyme neuraminidase, thereby allowing the virus to move on and finally become attached to a cell surface receptor. , ,ns, , a number of viruses remain localized in the respiratory tract during the course of infection-some in the upper respiratory tract (rhinoviruses, some herpesviruses) and others in the lower respiratory tract (parainfluenza virus). · · although restricted to the respiratory tract, some viruses (influenze, for example) still cause generalized clinical signs such as fever, malaise, and muscle pains as a result of the products and inflammation associated with cell injury and destruction. · · many viruses become rapidly disseminated throughout the body after the primary infection of the respiratory tract (canine distemper virus is a good example). • · · usually there is no evidence of respiratory tract infection in the initial phase of these diseases and the animal usually is not infectious during this phase. the virus spreads via macrophages or lymphatics to regional lymph nodes before spreading further. generally, viruses initiate infection in epithelial cells of the respiratory tract, but some viruses achieve this by successfully infecting alveolar macrophages. · · infection of alveolar macrophages has important sequelae in the pathogenesis of viral respiratory tract disease. · · viral respiratory tract disease is a consequence of mechanical and biochemical injury to epithelial cells and alveolar macrophages, which can, in the most severe instances, result in secondary bacterial infection, pneumonia, and death. · · · · · · denuded respiratory tract, impaired mucociliary escalator, and the growth medium provided by exudate have been thought to enhance bacterial growth and colonization in the respiratory tract. however, these mechanisms may not be as important as originally thought. injury to various biocidal mechanisms may be of greater consequence. · · · the alveolar macrophage, which is of critical importance in pulmonary resistance to bacterial colonization, may be affected in several ways either as direct consequence of virus replication in this cell or as a result of immunemediated cytotoxicity directed at the virus-infected macrophage. the latter is of particular consequence. it occurs late in the disease process when the immune response is initiated and when macrophages are ingesting virusladen cell debris. not only is the number of alveolar macrophages reduced by viral infection, the function of the remaining cells may be impaired. · · - · · · · evidence suggests that such macrophages have suppressed immunologic (fe) and nonimmunologic membrane receptor binding activity, fe and nonspecific receptor-mediated phagocytic ingestion, phagosome-lysosome fusion, intracellular killing, and bacterial degradation. some of these observations are the result of low levels of lysosomal enzymes and impaired biochemically mediated killing mechanisms. the alveolar macrophage may be impaired also because of hypoxia (it is an aerobic cell) and reduced surfactant levels. · the latter facilitates phagocytosis and is produced by type-ii alveolar pneumocytes. viral injury to the latter causes reduced production of surfactant in the lung. as alluded to earlier, certain viruses (such as influenza virus) promote bacterial colonization by altering the plasma membrane of infected cells, which facilitates bacterial adherence to the surface of such cells. the phenomenon appears to be the result of virus-induced receptors on the cell surface. some viruses appear to produce disease primarily by suppressing immunity, which is relatively sequestered from the systemic immune func-tions. • • • • • apart from the pulmonary macrophages, pulmonary cellmediated immunity is derived from bronchus-associated lymphoid tissue and augmented by the influx of blood monocytes and neutrophils. locally synthesized lgg, primarily synthesized in the lung, or transudated serum immunoglobulins function as opsonins, whereas secretory iga (primarily from the upper respiratory tract) prevents bacterial adherence and colonization and probably aggregates bacterial particles to facilitate mucociliary clearance. various viruses attach and replicate in epithelia of the mouth, pharynx, tonsils, and (or) the gastrointestinal tract. · • however, the stomach, abomasum, and intestine are not very hospitable to viruses because of an unfavorable ph and the presence of bile. enteric viruses usually are tolerant of a low ph. certain viruses (rotaviruses, for example) remain restricted to the gastrointestinal tract. · • • secretory immunity (and not humoral immunity) is protective against such viruses. • • many other viruses that invade intestinal epithelia subsequently become disseminated with varying frequency (enteroviruses). • • certain parvoviruses (canine parvovirus ii and feline panleukopenia virus) initiate infection in the pharynx-tonsil area, spread by the blood stream to various organs, and finally infect and destroy the crypt cells of the intestinal tract. the intestines thus become infected by a very circuitous route. villous atrophy results because of impaired replacement of enterocytes, which are derived from the crypt cells. central nervous system infection by the coronavirus, hemagglutinating encephalomyelitis virus of swine, occurs as a result of nerve tract migration from the gastrointestinal tract to peripheral ganglia. infection of neurons that regulate peristaltic functions of the intestinal tract results in gastrointestinal disease and subsequent starvation. certain viruses such as rinderpest and african swine fever initiate submucosal infection without infecting epithelial cells and appear to have the capacity to pass through the epithelial barrier. ·m some enteric viruses destroy the absorbtive columnar enterocytes on the tips of intestinal villi. • • the upper portion of the intestine is first affected, but infection often spreads throughout the entire length of the intestine. the affected cells are desquamated and are replaced by cuboidal or even squamous cells, resulting in a dramatic atrophy of the villi. these target cells are important for the digestion of disaccharides and the absorption of macrosaccharides, and contribute to osmoregulation. replacement of these cells occurs by m'igration of undifferentiated cells from the crypts, which are resistant to infection. however, the new villous tip cells are immature (contain thymidine kinase instead of sucrase, which is an enzyme profile similar to crypt cells) and result in a disturbed sodium transport system with a net extracellular fluid-to-lumen flux of sodium ions. pathophysiologic changes include loss of water, sodium, chloride, bicarbonate, and potassium. metabolism of glucose and lactate becomes severely disturbed, and the hypoglycemia, lactic acidosis, and an elevated efflux of potassium to hypovolumic plasma may lead to acute shock, heart failure, and death. the disease is more severe in neonates because of their milk diet and their dependence on readily available nutrients, and because replacement of sloughed epithelial cells is slower. the inert superficial protective layers of the intact skin usually are impervious to virus infection. • • • injury to the integument can allow several viruses to penetrate into susceptible tissues. trauma, insect bites, needles, and so on may be responsible for the introduction of virus (see earlier discussion). viruses that commonly initiate infection in this manner include most of the arboviruses, rabies, papular stomatitis, and some herpesviruses. some viruses penetrate directly through mucosal epithelia. • infection initiated in the urogenital tract few viral venereal diseases, affecting primarily the genital tissues, have been described. • the principal diseases in this category include those caused by herpesviruses and perhaps genital papillomaviruses of various species. • • however, the potential of infection occurring in the genital tract is high because many viruses may be present in semen. several viruses are capable of establishing infection in the placenta of pregnant animals and subsequently result in congenital infections. vertical infection occurs when a virus is transmitted to progeny with the germ cell. the latter occurs primarily with retroviruses in which viral genetic material becomes incorporated in host dna. it is difficult to consider the establishment of viral disease in a fetus without considering the infection of the dam, because the latter usually is infected for a variable time before the fetus. transplacental infection is perhaps the principal route of fetal infection by a virus. • it may occur by a variety of mechanisms depending on the nature of placentation of the dam and the nature of the virus. • in some instances, the virus appears to have selective pathogenicity for the fetus, because disease may not occur in the dam (for example, some strains of bluetongue virus). • • perhaps a primary infection of the fetus is one that is transmitted with the germ cells. this apparently occurs only with retroviruses, in which viral genetic material (provirus) is passed along as a cellular gene insert. . , fetal infection is a prominent feature of several viruses. the outcome of the disease in the fetus depends on both virus and host factors. some cytolytic viruses (for example, some herpesviruses and parvoviruses) uniformly result in fetal death, whereas other viruses (bluetongue virus, bovine virae diarrhea virus) may not. • • • the nature of the disease in the fetus with some of the latter viruses may depend on the age of the fetus. • this has been quite well documented with porcine parvovirus, bluetongue virus, and bovine viral diarrhea virus. the fetus is very vulnerable in the first third of pregnancy, and infection at this gestational age often results in death, but the fetus becomes progressively resistant to the effect of virus infection. infection in the last third of pregnancy may not have serious consequences. however, frequently the fetus is partially resistant only during the middle third of pregnancy, and infection during this stage may result in various lesions. the latter may include malformations of the central nervous system such as cerebellar hypoplasia (feline panleukopenia, bovine viral diarrhea virus), hypomyelinization (bovine viral diarrhea virus, border disease), hydranencephaly (bluetongue virus, bovine viral diarrhea virus), porencephaly (bluetongue virus, bovine viral diarrhea virus), and hydrocephalus (parainfluenza virus, japanese encephalitis virus). porencephaly is a later manifestation than hydranencephaly in lamb fetuses infected with bluetongue virus. stillbirths, weak neonates, and skeletal malformations may also occur. the progressive development of resistance in a fetus is correlated with the ontogeny of the immune response. some viruses (such as rubella virus) reduce the mitotic rate of infected fetal tissues and thereby affect organ development. • • offspring may be stillborn or runted. infection of the fetus at certain stages of gestation by a number of viruses (pestiviruses, arenaviruses) may result in "immunotolerant" offspring that are persistently viremic and without a detectable immune response. • • • in some instances, superinfection of such animals with the same virus or certain strains of the same virus results in serious, often fatal, systemic disease that may be immune-mediated. fetal disease can occur as a result of viral infections of the dam, even if the fetus itself is not infected. • • • changes in the placental circulation (vasoconstriction, congestion, and hemorrhage) as a result of viral placentitis can rapidly and severely affect the fetus. coxsackie b virus-induced pancreatic acinar atrophy in pregnant mice results in malnutrition owing to the mices' inability to digest and metabolize protein. fetal wastage and growth retardation are two sequelae of this condition. the hyperthermia associated with some viral infections in pregnant animals (for example, influenza) can cause abortions, stillbirths, and malformations (anencephaly, microencephaly, hydroencephaly) of fetuses. local extension of viral lesions occurs in susceptible hosts. · • • it may occur by virus release and dispersal from infected cells to neighboring susceptible cells. in some instances, lysis of infected cells is necessary before virus is liberated. herpesviruses, paramyxoviruses, and others may spread from cell to cell by a fus·ion mechanism. virus-induced cell proliferation, as occurs with poxviruses, papovaviruses, and retroviruses, is another mechanism of lesion extension. dissemination of the virus from the initial focus of infection occurs by several mechanisms. · • • • • the first step in generalized dissemination of a virus is the spread from the local lesion to the regional lymph node, either free in lymph or, more frequently, within carrier cells such as lymphocytes or phagocytic cells. virus concentration may be amplified by replication in the regional lymph node; secondary spread of the virus in blood vessels, either free or, more likely, within carrier cells, to other tissues and organs in the body may occur. this secondary spread of the virus may be detected clinically as the second part of a biphasic fever. in many instances, the critical event in viral dissemination is the successful infection of phagocytes. • the macrophage provides an important resistance mechanism of viral infections, and virulent viruses overcome their antiviral action. impaired macrophage function markedly enhances the susceptibility of animals to viruses. neonatal and corticosteroid-treated animals may be more susceptible for this reason. it is likely that several mechanisms of antiviral action exist in macrophages. • often virus penetration occurs in both susceptible and resistant macrophages, but uncoating of the virus is blocked in resistant macrophages, thereby terminating virus replication. various relatively sequestered organs may become infected with certain viruses. the central nervous system (cns) has nonfenestrated vasculature, tightly packed cellular components, and lacks a conventional lymphatic system. • the endothelial cells are joined by tight junctions and are surrounded by dense basement membranes, which in turn are tightly packed against astrocytic footplates. these structural barriers impede virus invasion of the cns from the bloodstream, but transendothelial migration of an infected lymphocyte and endothelial injury (either as a result of the virus infection or some other cause) may result in cns infection. • • the traffic of leukocytes into the cns is very limited under normal circumstances. some viruses are transported across the cells in pinocytotic vesicles and are deposited in the cytoplasm of the adjacent astrocytes. the cns may become infected by virus migration along nerve trunks (herpesviruses, rabies, hemagglutinating encephalomyelitis virus). • • • several mechanisms have been described • : movement within the central axoplasm, along endoneural spaces, and cell-to-cell infection of schwann cells. both centripetal and centrifugal spread along nerve trunks to and from the cns are possible. rabies virus infects salivary glands by the latter route. viral disease of the cns requires that the virus either has the capacity to invade these tissues or that its entry is facilitated by some unrelated event. • virus infection that leads to injury by direct or indirect mechanisms of oligodendrocytes results in demyelinization. • • • neurologic dysfunction may develop in the absence of obvious cell injury. persistent canine distemper virus infections of rat glioma cells cause a reduction of betaadrenergic receptors. some viruses specifically infect neurons of the cns. virus specificity may be so restricted that only certain subpopulations of neurons become infected. in humans, poliomyelitis viral infection is restricted mainly to motor neurons. rabies virus, during the early stages of infection, is confined primarily to neurons of the limbic system. this facilitates transmission by biting because the cortical-neurons are not involved early, and instead of seizures and motor deficits, alertness and aberrant behavior are the predominant clinical signs. virus selectivity determines also the development of hydranencephaly and porencephaly in newborn lambs infected in utero with bluetongue virus. the virus selectively destroys the germinal cells of the subventricular zone, the precursors of the neurons and glial cells of the forebrain. before the development of the cortical mantle in early gestation, necrosis and hydranencephaly occur owing to the destruction of these cells. after the formation of the cortical mantle before midgestation, porencephaly develops because the glial cell precursors are destroyed, resulting in focal white matter necrosis. several parvoviruses, which replicate only in cells in s-phase mitosis, selectively destroy the germinal cells of the cerebellum, resulting in hypoplasia of the cerebellum with abnormal foliation, depleted granular cells, and aberrant synaptic organization. this condition, which occurs in cats after infection as neonates or in late gestation, is known as spontaneous ataxia of kittens and is the most common neurologic disease in cats. for many years, it was thought to be an inherited condition. it has become evident that viruses can injure endocrine tissues specifically with the concomitant deficit in endocrine secretion. • · • • • in some instances, functional impairment develops without obvious cell injury. certain picornaviruses cause selective destruction of beta-pancreatic cells, resulting in diabetes. • evidence suggests that this may occur in humans also. impaired growth hormone production, growth rate, and glucose metabolism result from selective noncytopathic infection of anterior pituitary gland cells with lymphocytic choriomeningitis virus in mice. • immunosuppression vastly increases the potential of a virus to advance from a localized to a generalized infection. • ° complex interactions occur between various microorganisms in natural infections. • • • • a virus that induces immunosuppression in an animal can significantly enhance the severity and change the nature of a concurrent viral disease. one report suggested that canine parvovirus, which is lymphocytolytic, may enhance the neuropathogenicity of modified live canine distemper vaccine virus. bovine viral diarrhea virus, which also affects lymphocyte function, greatly enhances the dissemination of a herpesvirus, infectious bovine rhinotracheitis virus. various host-and virus-related restrictions determine tissue or organ invasion by a virus. cell surface receptors and the virus penetration step have already been discussed. • • • • intracellular hospitality for the particular virus may determine whether virus replication (either partial or complete-with progeny) occurs in a particular cell. • • · defective interfering particles are viruses of subgenomic size that contain most or all of the normal viral proteins. • they lack complete genomes and are unable to replicate in the absence of the parental virus. paradoxically, they interfere with the replication of the parental virus, apparently because the defective particles with their smaller genomes have a replication advantage and are able to sequester the replicase systems. defective interfering particles have been demonstrated in most groups of viruses. they may mediate the cessation of a viral disease or may convert an acute, lytic disease to a persistent or chronic infection. evidence indicates that the generation of defective interfering particles is controlled by host cells. blockage of host dna synthesis blocks generation of these particles when vesicular stomatitis virus free of defective interfering particles is used as the infecting virus. defective interfering particles are produced in such cells if they are introduced with the parenteral virus. this evidence, together with evidence that the defective particles can alter the pathogenesis of viral infections, suggest that cells may have evolved mechanisms to protect themselves against viruses that successfully enter and penetrate. the most rapidly produced defense against viruses is a family of proteins secreted by tissue cells in response to various stimuli such as viruses, bacteria, foreign cells, foreign macromolecules, and several other compounds. • • interferons act indirectly by stimulating surrounding cells to produce protein(s) that, in turn, may regulate virus replication, the immune response, cell growth, and other functions. interferons are unexpressed genetic functions of mature cells and may have a normal role in cell regulation. certain viruses are much more efficient in eliciting interferon production than others, which, of course, affects the course of the virus infection. • generally, the most potent interferon stimulators are also the most susceptible to interferon. · there are many examples of exquisitely sensitive genetically controlled expression of viral disease functional within the host cell. an example of \iral genetic control of pathogenesis is that of reoviruses in newborn mice. reovirus type produces nonfatal infection of ependymal cells after intracerebral inoculation, whereas type results in fatal encephalitis with neuronal destruction. the tropism for ependymal cells or neurons seems to be regulated by a single gene (the s genome segment) that codes for the sigma- capsid protein. another genome segment (m ) determines the ability of these viruses to initiate local or systemic infection in newborn mice after oral inoculation. an example of host genetic control of pathogenesis at the intracellular level is the fv- system in mice, which determines susceptibility to murine leukemia viruses. this mechanism operates after penetration but before integration of viral genetic material in host dna. the cellular gene, known as fv- , is present on chromosome , and the virus determinant with which it interacts to determine this tropism is the p viral protein. this mechanism is illustrated by murine leukemia viruses that preferentially infect cells from ~ih swiss mice (n-tropic) or balb/c mice (b-tropic). cells may be destroyed when they become infected with certain viruses, particularly dna viruses. several mechanisms are responsible for this effect on cells. , , , , , , , , , , , , , , , in some instances, cell metabolism is drastically altered. macromolecular synthesis may be inhibited by a variety of mechanisms and may occur at the level of replication, transcription, or translation. certain viruses 'interfere with host dna replication. vaccinia virus mrna competes with cellular mrna. this virus may also result in disruption of polysomes and impaired rna processing. poliovirus inhibits the initiation factor in mrna translation. increased permeability of virusinfected cells causes increased intracellular sodium ions, which favor viral mrna translation. the rate of formation of viral products may be greater than their release, which has an adverse effect on cellular metabolism. the virus may deplete substrates essential for vital cellular functions, and the physical presence of viral products in a cell has an adverse effect on cell metabolism. complete or partial replication of a virus is necessary for cell injury in many instances, but virus replication does not necessarily result in cell injury, particularly with many rna viruses. several viruses produce a toxin-like substance that is either a direct effect of a virus-induced product or a secondary effect caused by the activation of lysosomal enzymes by a virus. viral cytotoxins have been described for poxviruses and adenoviruses (the penton capsid protein which reversibly modifies cell membranes from the outside). these cytotoxins may be either structural virion components (preformed) or induced during virus replication. vaccinia cytotoxin has been identified as a surface tubular virion protein, whereas adenovirus cytotoxin is contained in the penton capsomere (the hexon capsomeres and fiber antigen inhibit macromolecular synthesis). the cytotoxic effect is markedly amplified in certain virus infections. t lymphocytes infected with dengue virus produce a cytotoxin that induces macrophages to produce a cytotoxic factor. the exact mechanism by which various viruses affect cell metabolism and produce cytopathogenesis is not well known. however, it is known that poxviruses rapidly cut off host macromolecular synthesis, disaggregate host polysomes, interfere with processing of rna, and redistribute lysosomal enzymes. one hypothesis is that the cytopathic and pathophysiologic changes may be due to a common cause-an alteration of the permeability of cell membranes. leakiness of cell membranes occurs during infection with viruses from several virus families and usually precedes cytopathogenesis. certain physiologic and clinical manifestations of virus infections can be attributed to membrane leakiness. these include excess respiratory tract mucus production with rhinovirus infections, loss of vision in corneal keratitis caused by herpesvirus infections, and excessive loss of water and electrolytes from the gastrointestinal tract during rotavirus infections. one of the most important aspects of infectious diseases that is receiving increasing recognition is the interaction and synergism of pathogenic microorganisms in the manifestation of the pathologic state. in many instances of enhanced disease due to microorganism interaction, it is the result of virus-induced immunosuppression. • • - • - a virus-causing immunosuppression can enhance dramatically diseases caused by viral, bacterial, or protozoan infections that normally are relatively innocuous. viral immunosuppression may facilitate dissemination of microorganisms in a host and promote persistent or chronic infections. the cellular basis and consequences of viral-induced immunodeficiency are discussed in detail elsewhere in this issue. a brief summary will be presented here. the most readily recognized types of viral immunosuppression are those infections that result in destruction of immunocompetent cells such as macrophages, neutrophils, and lymphocytes. however, in the majority of instances of viral immunosuppression, cytocidal infection of immunocompetent cells is not recognized, and the phenomenon appears to be the result of impaired function. · · · · - · · · · · ·m virus-induced immune modulation has been recognized in macrophages, neutrophils, various subsets oft lymphocytes, and b lymphocytes. many of the immune responses inhibited by viruses may be attributed to alteration of macrophage function (see the discussion on the antibacterial activity of alveolar macrophages). influenza virus, lymphocytic choriomeningitis virus, bovine viral diarrhea virus, and several other viruses are capable of affecting phagocytic cells. several studies have indicated suppression of phagocytic, chemiluminescence, and chemotactic responses of macrophages and neutrophils. delayed wound healing in mice has also been attributed to viral alteration of macrophage function. paradoxically, impaired function of some macrophages (such as the alveolar macrophage) may be due to immune-mediated injury as a result of virus-specific cytotoxicity directed against the virus-laden macrophage. virus-induced modulation of macrophages conceivably could alter the outcome of numerous cellular interactions, either due to impaired direct participation in lymphocyte functions or because of interference of secretion of regulatory factors. functional impairment of lymphocytes by viruses may be the result of changes in cell surface receptors, competition between virus and immunogen for protein or dna synthetic machinery, generation of suppressor interferon, interruption of the cellular communication network in the immune response, and stimulation of suppressor t cells. • · · · ·m as has been observed for macrophages, viral infection in lymphoid cells may result in the production of a lymphocytotoxic immune response, which may lead to the premature senescence of t cells. another mechanism that may occur in some viral infections is virus-induced alterations in normal lymphocyte traffic and recirculating patterns in the host. this results in redirecting immunocompetent cells away from lymph nodes and spleen and decreasing the immunologic reserve of lymphatic tissues. an active area of investigation is immunosuppression by retroviruses, which seems to be mediated by the pl e envelope protein of some of these viruses (particularly feline leukemia virus). · · · · · impaired immunologic functions associated with exposure to pl e include monocyte chemotaxis, lymphocyte blastogenesis, erythroid colony formation, macrophage accumulation, and tumor immunity. it has been suggested that pl e causes immunosuppression by blocking the production of interleukin by lymphocytes. virus-induced immunosuppression is often accompanied by multiple deficits of the immune response. during certain viral infections, there is both an activation and increase of suppressor t cells, which, in turn, suppress autologous t-cell proliferation to antigens as well as b-cell antibody production. · · ·m certain paramyxoviruses cause silent infections of lymphocytes, which fail to generate natural killer (nk) cell activity (an important antiviral mechanism) or produce antibodies. · several viruses have been identified that inhibit interferon production in a host, resulting in increased susceptibility to other viruses. • host-damaging immune responses in viral infections have been reviewed adequately in the literature. • • the mechanisms and consequences of viral immunopathology will be summarized in this section. generally, host immune responses are responsible for recovery from virus infections but, occasionally, they can initiate or enhance cell injury. certain viruses, such as herpesviruses and picornaviruses, are cytolytic, and disease results from direct destruction of tissue cells. in such instances of viral infection, the immune response can limit infection by destroying infected cells before progeny virus is assembled and released. immune-mediated cytolysis can be deleterious to the host if it occurs late in the replication cycle of the virus, because then it serves only to release infectious virus. noncytolytic viruses often do not cause direct cell injury, but immune responses may injure cells persistently infected with such viruses. a chronic inflammatory response may occur when such cells are not rapidly destroyed by immune processes. such responses frequently are harmful to the host, especially if they interfere with the function of certain tissues and organs. immunopathologic chronic inflammation usually involves antigen-sensitized t lymphocytes and/or antibody immune complexes that also activate complement with inflammatory consequences. viral immunopathology may be divided into two categories: lesions due to antibodies, either directly or with other nonspecific effector mechanisms (for example, antibody-dependent cellular cytotoxity-adcc) or lesions due to specific cellular immune responses. the classical example of virus antibody immunopathology is immune complex disease. • ,· • • · • • • the development of immune complex disease is associated with circulating antibody-antigen complexes in serum, deposition of the smaller complexes (in instances of antigen excess) in tissues with limiting basement membranes (glomeruli, arterioles, choroid plexus, joints, and so on) to produce injury, and the presence of antigen, antibody, and complement components at the site of injury. the initial step in immune complex disease is the release of vasoactive substances, such as histamine and serotonin, and the subsequent increase in vascular permeability. serotonin is released from platelets when they react with immune complexes in the presence of complement. other mechanisms of immune complex-mediated vascular permeability, such as kinins generated from activated hageman factor, may also occur. such platelets may contribute also to immune complex lesions by causing capillary thrombosis. increased vascular permeability facilitates trapping of immune complexes, usually those of small size that develop in moderate antigen excess and in the walls of arterioles and capillaries. the trapped complexes activate complement locally and chemotactically attract polymorphonuclear cells. the circulating and fixed macrophages can degrade immune complexes, but the phlogogenic effect with anaphylotoxins (and the consequent release of vasoactive amines), neutrophil release of cathepsins and proteolytic basic proteins, and subsequent activation of hageman factor (which leads to kinin and plasmin production) cause direct vessel wall injury. arteritis, glomerulonephritis, arthritis, and choriomeningitis are common sequelae of immune complex disease. immune complex disease is particularly prevalent in infections with noncytopathic viruses such as those that cause equine infectious anemia, aleutian disease of mink, and feline infectious peritonitis (fip), but it may also cause some of the lesions of cytocidal viruses (for example, canine distemper virus). evidence indicates that fip is an immune complex disease. • • • humoral immunity is not protective in this disease, but a functional cell-mediated immunity appears to prevent lesions caused by fip virus. impaired cellular immunity is associated with manifestation of the disease (the effusive form with greatly deficient cell-mediated immunity, and the noneffusive form with a partially impaired cellular immunity. • thus, in this disease, we have paradoxical mechanisms of immunopathogenesis. one facet (the humoral response) is enhanced, whereas another (cell-mediated) immunity, may be impaired. this may be the reason fip is associated frequently with feline leukemia virus, a powerful suppressor of lymphocyte function. virus-infected cells may be destroyed by antibody-dependent cellular cytotoxicity (adcc). in this synergistic action of specific antibody and effector cells, the immunologic specificity is provided by the antibody molecules (only minute quantities are required), whereas the effector cells act nonspecifically. the latter must bear fe receptors and include t cells, b cells, killer cells, macrophages, and neutrophils. complement-assisted cytotoxicity occurs when complement enhances adcc. interferon also has been reported to enhance adcc. complement-mediated cytotoxicity involves virus-infected cell destruction by complement following activation by the classical and alternative pathways with specific immunoglobulins. activation of complement by the alternative pathway by virus-infected cells, in the absence of antibody, may also occur. a special case of antibody-induced host injury is the enhancement, by specific antibody, or infectivity of certain viruses of host cells. this has been described for flaviviruses (for example, dengue virus) and for fip virus, for which target cells are macrophages. although mononuclear cells appear to be the only cells that support replication of dengue virus, the latter is not internalized efficiently in the host cell unless complexed with non-neutralizing antibody, which attaches to fe receptors on the cells. some investigators have speculated on the possible allergic immunopathologic responses to viruses. • • the potential for lge-mediated type i hypersensitivity in virus infections exists, but little evidence has been presented to support this hypothesis. an interesting hypothesis has been proposed to explain certain antibodymediated hypersensitivities in virus infections. • it envisages a viral infection (such as dengue virus) in a host with pre-existing parasitic infection leading to depletion of suppressor t lymphocytes. the resultant augmented production of igg and ige could then result in type iii and type i hypersensitivities, respectively. t-lymphocyte cytotoxicity is an immunologically specific and highly effective lysis of cells with viral antigen expression on the plasma membrane. • • ·m in some species (most notably mice), genetic restriction oftcell cytotoxicity occurs in that there is a requirement, not only for specific antigen binding, but also for recognition of certain antigens of the major histocompatibility region. the cytolytic event requires contact between the effector and the target cell. the former can lyse a number of target cells in sequence, but the number of immune t cells at a particular site may not be high; for this reason, indirect t-cell events such as macrophage recruitment may be more important in immunity and immunopathology. cytotoxic lymphokines (lymphotoxins) are soluble products oft cells following reaction with viral antigen. lymphotoxins cause cytolysis by affecting the permeability of the plasma membrane of cells. natural or normal killer (nk) cells are lymphocytes with cell surface fe receptors that nonspecifically destroy certain virus-infected and neoplastically transformed cells. interferon and complement can facilitate cytolysis by t lymphocytes and thus enhance viral immunopathology. macrophages can be directly cytotoxic, but this appears to be a nonspecific event. they may be localized at sites of specific antigen by t cellmediated and phlogogenic recruitment. evidence is accumulating that certain viruses may elicit tissue-reaction antibodies and t lymphocytes, which results in tissue and organ injury. • • • • • in some autoimmune diseases, the organ and tissue specificity is quite broad, whereas in other instances, it is very restricted to specific cell types. a polyendocrine disease affecting several hormones as a result of virus-induced autoimmunity has been described. • several mechanisms of the pathogenesis of viral pathogenesis have been described. one possibility is the virus-induced access of the immune system to sequestered antigens, such as the cns (protected by the blood-brain barrier), which are normally "immunologically privileged" sites and are not normally monitored by recirculating lymphocytes. this may occur during the acute phase of the disease. subacute or chronic demyelinating encephalomyelitis that occurs with some strains of canine distemper may be the result of such a mechanism. another mechanism that is plausible is virus-induced changes in host cell membrane. the expression of new host antigens such as embryonic antigens or alloantigens is induced. a related mechanism may be the result of virus antigens expressed at the cell surface that share antigens with the host cell. the immune response to either the new or cross-reactive antigens is capable of reacting with normal tissues. the final mechanism is one that has been alluded to already-that of loss of immune regulation, such as impaired suppressor cell function. tolerance to self-antigens may be maintained by suppressor cell activity, at least in part. diminished activity of these cells then results in increased responsiveness to foreign antigens and an immune response to self-antigens. a special case of autoimmune disease may occur in mucosal disease and bluetongue virus-associated hemorrhagic disease in cattle that have "tolerant" infections as a result of in utero infections. superinfection with a heterologous strain or an overwhelming dose of a homologous strain may induce an immune response to the "tolerant" viral antigens that are present in most tissue cells and thereby induce cell injury. much of the discussion of viral pathogenesis has focused on structural and functional injury of cells. another virus-induced response of cells is that of immortalization due to oncogenic transformation. the mechanism by which various viruses transform cells depends on the nature of the virus. oncogenic dna viruses belong to several virus families, but oncogenic rna viruses are members of the retroviridae. viral oncogenesis is a complex phenomenon and is an area receiving considerable attention by the scientific community. transformed cells have altered morphology, changed behavior, and altered biochemistry. often new membrane proteins and glycoproteins appear (for example, tumor-and/or virus-specific surface antigens and fetal antigens). co-carcinogens have been identified that appear to promote transformation by certain viruses. immunosuppression, a prominent feature of infection by several viruses, may be an indirect oncogenic mechanism of these viruses. it is still not clear which of the complex of changes induced in cells by viruses cause transformation and which are secondary events. at best, this review can only summarize the information on viral oncogenesis, and in order to achieve this, the subject material is greatly oversimplified. for a more detailed description of viral oncogenesis, please consult the reviews by dues berg, ° fenoglio and lefkowitch, weiss, generally, dna viruses replicate in cells and destroy the cell after progeny is produced. cells that support virus replication in this manner are known as permissive cells. however, in some cells (nonpermissive cells), virus replication is not completed and progeny virions are not produced. on rare occasions, these nonpermissive cells become transformed. defective virus may also, on rare occasions, transform both permissive and nonpermissive cells. an essential event for dna virus-induced transformation appears to be integration of viral dna into host dna. poxviruses seem to be the exception. indeed, poxvirus-induced tumors consist of hyperplastic rather than trans-formed cells. neither the cellular site of integration nor the location on virus dna is unique or even specific, and integration seems to be the result of random "illegitimate" recombination between nonhomologous regions of the virus and the host. there is insufficient evidence that oncogenes are involved in dna virus transformation. although the entire viral dna genome may be integrated in transformed cells, all transformed clones contain at least part of the early region of the viral genome. various early functions seem to initiate and/or maintain the transformed phenotype, whereas late viral functions do not have a role in transformation. thus, many dna viruses mediate transformation through the action of some viral gene products, which generally are required for the virus to complete its normal cytolytic cycle. it is not known exactly how the transformation genes originate, but, unlike the retroviruses, there is little evidence that they are derived from normal host genes. however, there is some evidence for limited transcription from virus dna sequences into flanking host sequences. in addition, recent transfection experiments with burkitt's lymphoma cells resulted in the discovery of an oncogene of cellular origin. this oncogene is activated in burkitt's lymphoma. furthermore, one locus in the human genome has some homology with the gene-enhancer sequences of a human papovavirus, indicating that some of the oncogenic enhancer dna sequences of dna viruses can be evolutionarily related to host cell sequences. it appears also that dna virus oncogenes can interact with retroviral (or cellular) oncogenes. a synergistic interaction of the "ras" oncogene and an adenoviral oncogene has been reported. viral dna can mediate transformation through several possible mechanisms. viral dna integrated at certain sites in host dna could act as a mutagen, thereby destroying the control of cellular genes. perhaps integrated viral genetic material may contain promoters of viral gene expression and also coincidentally affect host gene expression. finally, viral dna may specify protein(s), which when synthesized, may directly cause transformation of the cell. as mentioned earlier, several viral early proteins have been identified that initiate and/or maintain transformation. the large t and small t antigens of sv virus are proteins that function in this manner. co-carcinogens promote oncogenesis by several dna viruses. the flavinoid (quercetin) from bracken fern is associated with progression of upper alimentary tract papillomas to carcinomas in cattle. burkitt's lymphoma, a malignent b-cell lymphoma of humans, is associated with infection by the herpesvirus epstein-barr virus. the most likely co-factor is holoendemic malaria. aflatoxin appears to be a co-factor in primary liver carcinoma associated with hepatitis b virus. the oncovirinae subfamily of the retroviridae, due to its unique intracellular biology, has an exquisitely well-developed mechanism for cell transformation. • • • • the enzyme, reverse transcriptase, constitutes the fundamental basis of the molecular biology of these viruses. the diploid rna genome usually contains four major genes in a specific sequence. the gag gene, which is closest to the ' end, codes for a poly protein, a precursor of four structural proteins of the nucleoid. the next gene, pol, codes for the reverse transcriptase, whereas the env gene codes for the two glycoproteins on the surface of the envelope. the final major gene codes for a phosphoprotein with protein kinase activity and is responsible for neoplastic transformation. the latter gene, known as the virus oncogene, is inconsistently present on oncoviruses. virus replication is initiated when virus binds to specific receptors and penetrates into a cell. in permissive cells, virus progeny is produced and the host cell may or may not become neoplastic. nonpermissive cells may be transformed occasionally, but viral replication is never completed. defective viruses may efficiently transform both permissive and nonpermissive cells. within a few hours, virion reverse transcriptase synthesizes linear double-stranded dna copies of the haploid genome. linear dna molecules migrate to the nucleus and become circularized. the latter becomes integrated into cellular dna, a step essential for retrovirus replication and gene expression. integrated virus dna is known as the provirus, and the number of copies in the haploid host genome varies from to . the integrated provirus retains the topography of the viral genome and is subjected to expression and control by host mechanisms. proviruses can be transmitted vertically and thus inherited in germ cells as genes by mendelian genetics. viral gene expression relies on the hospitality of the host cell. transcription of proviral dna is catalyzed by cellular rna polymerase ii. expression may be governed by genetic determinants of the cell that are closely linked but separable from the provirus. two general types of oncoviruses exist. the sarcoma-type viruses transform fibroblasts in vitro, have an oncogene, and usually are highly oncogenic. often they fail to produce progeny virus because of a deletion of the env function and are known as replication defective (rd). coinfection with a related virus containing intact env activity may provide the deficient envelope glycoproteins to allow full maturation and progeny sarcoma virus but with surface antigen specificity of the helper (or associated) virus. prior infection or occupation of cell surface receptors by an oncovirus with identical envelope characteristics will result in interference and prevent superinfection by the second virus. the initial virus is known as the resistance-inducing factor. leukemia viruses do not seem to have an oncogene and cannot transform fibroblasts in vitro. such viruses are transformation defective (td). thus, the transforming segment, the oncogene, is not essential for virus replication and is not of intrinsic importance to the virus. however, many of these viruses are leukemogenic in animals after a prolonged incubation period and seem to be responsible for many of the leukemias and lymphomas of various animals. the mechanism of leukemogenesis is not known, but it is likely that an indirect mechanism occurs with these viruses. perhaps these viruses recombine with other viral genomes, but more likely, because of their insertion in the vicinity of cellular oncogenes, activate the latter. cellular oncogenes are very similar to those of sarcoma viruses, and it seems plausible that the latter may have acquired oncogenes by transduction during past interactions with their host cells. various hypotheses on oncovirus oncogenesis have been proposed as the information on the biology of these viruses became available. the virogene-oncogene theory was proposed in by heubner. temin introduced in the protovirus concept for viral oncogenesis and the existence of endogenous oncoviruses. however, considerable evidence now exists to support the cellular oncogene theory, which contains some of the concepts of the two hypotheses oftemin and heubner. an oversimplified explanation of this hypothesis is that most cells contain the so-called oncogenes, a highly conserved host gene with a useful function. this gene product seems to be a protein kinase catalyzing the phosphorylation of certain proteins and has been implicated in regulating growth of normal cells by activity on cell surfaces. this unique protein kinase differs from the normal cell kinases in that it phosphorylates tyrosine instead of serine or threonine. a large number of viral oncogenes and cellular equivalents have now been identified. they are distinguished by the prefix "c" for cell oncogenes and "v" for viral oncogenes. viral oncogenes are probably of cellular origin and may be merely a passenger acquired by sarcoma viruses from the host dna. viruses with oncogenes are directly oncogenic, whereas oncoviruses without oncogenes are indirectly tumorogenic because they interfere with a cellular oncogene at or near the site of provirus integration. two hypotheses have been proposed to explain the mechanism of oncogene transformation; the mutational hypothesis and the dosage hypothesis. the former requires that the viral oncogene differs, as a result of mutation, from the parent cellular oncogene and, upon expression of the gene, neoplastic transformation occurs instead of its normal regulatory function. furthermore, in the instance of nononcogene-bearing leukemia viruses, a similar mutation occurs in cellular oncogenes during integration. proto-oncogene (inactive) activation by one-point mutation has been best documented in the family of "ras" oncogenes. the dosage (or amplification) hypothesis requires that excessive production of the gene product occurs after infection with a sarcoma virus and formation of one or multiple copies of the provirus in the cell. nononcogene-bearing leukemia viruses probably stimulate cellular oncogene activity because some are inserted as proviruses in the immediate vicinity of this gene. recent evidence suggests that the latter theory may be accurate. normal cellular control prevents the oncogene's transforming capacity. however, when an oncovirus includes an oncogene in its genome, the oncogene is removed from its controlled environment, resulting in increased production during viral replication. enhanced activity of the oncogene probably is due to the viral promoter. an unexpressed oncogene (proto-oncogene), when introduced with its normal promoter into host cells, does not cause transformation; however, transformation occurs when the viral promoter accompanies the cellular oncogene in transfection experiments. the activation of normal cell oncogenes by leukemia viruses is probably due to the viral promoter inserted in the vicinity of these genes. the delayed tumor induction of leukemia viruses may be due to random integration of the viral genome. thus, the location of the viral promoter at the appropriate site for affecting the cellular oncogene is an inefficient event. the appropriate point of integration of the promoter into the cellular dna varies, and evidence indicates that the site can be some distance from the target oncogene. another mechanism of retroviral neoplasia is related to immunosuppression induced by some of these viruses. immune surveillance may be severely impaired, allowing transformed cells to accumulate unchecked. immunosuppression often precedes tumor development in animals infected by oncoviruses. recent evidence indicates that human t-celllymphotropic virus iii may cause the acquired immunodeficiency syndrome, which, in turn, may allow the development of kaposi's sarcoma. retrovirus-mediated immunosuppression is caused, at least in part, by the pl e envelope protein. influenza a virus-induced polymorphonuclear leukocyte dysfunction host-damaging immune 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changes and immunofluorescence vaccinia virus cytotoxin viral myocarditis oncogenic viruses oncogenic viruses virus-induced diabetes mellitus. isolation of a virus from the pancreas of a child with diabetic ketoacidosis key: cord- -n l j authors: gonçalves, antonio; bertrand, julie; ke, ruian; comets, emmanuelle; de lamballerie, xavier; malvy, denis; pizzorno, andrés; terrier, olivier; calatrava, manuel rosa; mentré, france; smith, patrick; perelson, alan s; guedj, jérémie title: timing of antiviral treatment initiation is critical to reduce sars-cov- viral load date: - - journal: medrxiv : the preprint server for health sciences doi: . / . . . sha: doc_id: cord_uid: n l j we modeled the viral dynamics of untreated patients infected with sars-cov- to infer viral growth parameters and predict the effects of antiviral treatments. in order to reduce peak viral load by more than logs, drug efficacy needs to be greater than % if treatment is administered after symptom onset; an efficacy of % could be sufficient if treatment is initiated before symptom onset. given their pharmacokinetic/pharmacodynamic properties, current investigated drugs may be in a range of - % efficacy. they may help control virus if administered very early, but may not have a major effect in severe patients. we modeled the viral dynamics of untreated patients infected with sars-cov- to infer viral growth parameters and predict the effects of antiviral treatments. in order to reduce peak viral load by more than logs, drug efficacy needs to be greater than % if treatment is administered after symptom onset; an efficacy of % could be sufficient if treatment is initiated before symptom onset. given their pharmacokinetic/pharmacodynamic properties, current investigated drugs may be in a range of - % efficacy. they may help control virus if administered very early, but may not have a major effect in severe patients. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ), which originated in wuhan, china, has become a global pandemic. by march , , this virus had infected more than , people worldwide and caused more than , deaths. despite the unprecedented mobilization of the clinical and scientific community, the development and large scale implementation of new antiviral drugs or vaccines will take months or more. to readily propose a first line of defense and combat the virus in hospitalized patients, the world health organization relies on already existing drugs ("repurposed") that are immediately available in large quantities and have a good safety profile. in coordination with other european institutions, france is implementing a randomized clinical trial in hospitalized patients ("discovery", nct ) comparing the efficacy of lopinavir/ritonavir ± ifn-β- a, remdesivir and hydroxychloroquine. given the very limited knowledge of the host/pathogen interaction the clinical efficacy of treatment strategies using these drugs is largely unknown and could be limited [ ] . fitting mathematical models of viral dynamics to in vivo data can provide estimates of parameters driving viral replication. such models can then be used to predict the needed efficacy of treatments and to optimize their use [ ]. by combining these predictions with the expected drug concentrations and ec of drug candidates, one can anticipate the effects of various dosing regimens (doses, timing of treatment initiation) on viral load dynamics. data used for fitting we used published data from untreated patients infected with sars-cov- that were followed in singapore hospitals [ ] . patients were hospitalized in median at day after onset of symptoms (range: - ) and had a median symptomatic period of days (range: - all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint ). viral loads in nasopharyngeal swabs were measured by real time reverse transcriptase polymerase chain reaction (rt pcr, lower limit of quantification: cycles, ct) at multiple time points with an observed peak of viral load at day post onset of symptoms (range: - days). data presented in ct were transformed to log copies/ml using a published relationship in zou et al. [ ] and the model was fit to the log viral load. of note, the transformation from ct to log copies/ml does not affect the estimates of parameters of interest, in particular r and the death rate of productively infected cells. time since infection was assumed to be days before the onset of symptoms [ ] . in a sensitivity analysis, we also examined values of and days. model viral dynamics was fitted using a target cell limited model with an eclipse phase the model considers three populations of cells: target cells, t, infected cells in the eclipse phase, i , and productively infected cells, i . given the timescale of the infection, we neglect target cell proliferation and natural death, and we focused on the process of cell depletion by virus infection. we assumed target cells become infected with rate constant β. after an average time of /k, these cells start producing virus and are cleared with per capita rate δ. virions are released from productively infected cells i at rate p per cell and are cleared from the circulation at per capita rate c. based on this model, the basic reproduction number, all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. we assumed that the target cell concentration is . × cells/ml. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint absence of treatment, i.e., days after symptom onset. the model providing the best description of the data was used for the simulations, and sensitivity analyses were conducted to evaluate the results obtained with different assumptions regarding the delay between time of infection and time of symptom onset either or days (supplemental information, fig s and s ). we relied on the literature to find pk population parameters of lopinavir/ritonavir [ ], hydroxychloroquine [ ] , and ifn-β- a [ ] as well as reported ec values in vitro (see table author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint here we used a "target-cell limited" model with an eclipse phase fig s and table s ). in influenza a, another respiratory infectious disease, estimates of the within host r varied greatly, but the half-life of infected cells was shorter than hours (see more details in [ ]), suggesting a faster clearance of influenza infected cells than sars-cov- . these numbers also inform us both on the time to initiate antiviral treatment, and the level of efficacy that needs to be achieved to reduce viral load [ ] . as limited information is available on the mechanisms leading to viral clearance, and how they may be modulated by treatment, we used our model to predict the effects of treatment at day post symptoms, which corresponds to the time the viral load tends to peak in the absence of treatment [ ] . we considered a simple case where the drug effectiveness is assumed to be constant after therapy initiation (see methods) and we calculated the minimal efficacy that would be needed to generate more than logs of viral decline at peak viral load in the studied patients (fig. ) . as predicted by viral kinetic modeling theory [ ], we found that the impact of treatment on peak viral load is inversely correlated with the time of treatment initiation. for a putative treatment initiated at the time of infection, symptom onset, or days post symptom onset, a median efficacy of at least , and % in reducing viral replication would be needed, respectively, to generate more than log of decline in the peak viral load (fig. ) . author/funder, who has granted medrxiv a license to display the preprint in perpetuity. and the mean antiviral effectiveness during the first days of treatment is given by . given their pharmacokinetic and pharmacodynamic properties (table ) , we calculated a mean antiviral efficacy of up to % for lopinavir/ritonavir, % for ifn-β- a, and % for hydroxychloroquine. given these estimates, these compounds are unlikely to have a dramatic effect on peak viral load if administered after the onset of symptoms. in fact, the effective concentrations will presumably be lower in patients, as relevant drug may be further limited by protein binding (in particular for lopinavir, which has a protein binding rate > %) or capability to penetrate respiratory compartments, which is not well characterized. importantly, levels of antiviral efficacy of ~ % could nonetheless be relevant in a prophylactic setting, before symptom onset, to reduce viral replication in the upper respiratory tract and reduce the risk of large infiltration to the lung before an effective immune response is mounted to clear virus [ ] note, above we calculated the effectiveness of drugs administered in monotherapy for their usual dosing regimen. we also did not consider drugs that could directly target infected cells and lead to their elimination, such as some monoclonal antibodies. overall our results emphasize that the pk/pd properties of lopinavir/ritonavir, ifn-β- a and hydroxychloroquine make them unlikely to have a dramatic impact on viral load kinetics in the nasopharynx if they are administered after symptom onset. given this, it is possible that continued viral replication in the presence of drug will select for drug resistant mutations as has been seen with other rna viruses [ ], although coronaviruses are unusual in that they all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint appear to have low mutation rates due to rna proofreading capability. drug combination therapy and more aggressive dosing, including consideration of loading doses to rapidly achieve therapeutic exposures, may be beneficial to maximize efficacy of these repurposed antiviral agents. however, they may be relevant in pre-or post-exposure prophylaxis administration to reduce viral replication and hence the risk of disease progression. a trial of lopinavir-ritonavir in adults hospitalized with severe covid- acute bacterial or viral infection-what's the difference? a perspective from pkpd modellers epidemiologic features and clinical course of patients infected with sars-cov- in singapore sars-cov- viral load in upper respiratory specimens of infected patients the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies combination antiviral therapy for influenza: predictions from modeling of human infections kinetics of influenza a virus infection in humans model averaging in viral dynamic models integrated population pharmacokinetic/viral dynamic modelling of lopinavir/ritonavir in hiv- treatment-naïve patients all rights reserved. no reuse allowed without permission population pharmacokinetics of hydroxychloroquine in japanese patients with cutaneous or systemic lupus erythematosus compare: pharmacokinetic profiles of subcutaneous peginterferon beta- a and subcutaneous interferon beta- a over weeks in healthy subjects: pharmacokinetics of peginterferon beta- a and s.c. interferon beta- a dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov host-pathogen kinetics during influenza infection and coinfection: insights from predictive modeling all rights reserved. no reuse allowed without permission author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the key: cord- - qydcc e authors: kumar, asit; kodidela, sunitha; tadrous, erene; cory, theodore james; walker, crystal martin; smith, amber marie; mukherjee, ahona; kumar, santosh title: extracellular vesicles in viral replication and pathogenesis and their potential role in therapeutic intervention date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qydcc e extracellular vesicles (evs) have shown their potential as a carrier of molecular information, and they have been involved in physiological functions and diseases caused by viral infections. virus-infected cells secrete various lipid-bound vesicles, including endosome pathway-derived exosomes and microvesicles/microparticles that are released from the plasma membrane. they are released via a direct outward budding and fission of plasma membrane blebs into the extracellular space to either facilitate virus propagation or regulate the immune responses. moreover, evs generated by virus-infected cells can incorporate virulence factors including viral protein and viral genetic material, and thus can resemble noninfectious viruses. interactions of evs with recipient cells have been shown to activate signaling pathways that may contribute to a sustained cellular response towards viral infections. evs, by utilizing a complex set of cargos, can play a regulatory role in viral infection, both by facilitating and suppressing the infection. ev-based antiviral and antiretroviral drug delivery approaches provide an opportunity for targeted drug delivery. in this review, we summarize the literature on evs, their associated involvement in transmission in viral infections, and potential therapeutic implications. cells mediate intercellular communication and modulation of immune responses through shedding and release of extracellular vesicles (evs) [ ] . these evs are diverse and originate from plasma membrane and endosomes and include exosomes, micro-vesicles (mvs, also known as microparticles), and apoptotic bodies. they are categorized based on their biogenesis, release pathways, size, content, and function [ ] . evs shed from plasma membranes are generally referred to as mvs [ ] [ ] [ ] , while vesicles that are generated by inward budding of endosomes to form multivesicular bodies (mvbs) that fuse with the plasma membrane, and release into the extracellular environment, are known as exosomes [ , ] ; whereas, cells undergoing apoptosis can release vesicles or cell filaments exclusively from the plasma membrane, called apoptotic bodies [ , ] . depending on their biogenesis pathway and cellular origin, exosomes are vesicles of endocytic origin and their size usually ranges from - nm [ ] . exosomal markers include tetraspanins (tspan and tspan , escrt components, and tsg ). the invasion of the plasma membrane inwards forms the early endosome and the limiting membrane of the later endosome sprouts further to form the mvbs. mvbs are characterized by the invagination of the inner body membrane, which results in the formation of intraluminal vesicles (ilvs) [ ] . during this process, cytoplasmic components and certain peripheral proteins are integrated into them. the ilvs accumulated in the mvb lumen have two routes. one is to diffuse with the lysosomes, which causes the contents of the vesicles to degrade, and the other is fusion with the cytoplasmic membrane and release of the vesicles to the extracellular space by exocytosis, referred as "exosomes" [ ] . loading of biological cargos into ilvs involves the endosomal sorting complexes required for transport (escrt) complexes (escrt- , -i, -ii, -iii and the vps ) and other accessory proteins such as alix/pdcd ip, tsg , hrs, etc. [ , ] . in addition to escrt, other mechanisms can also produce exosomes of certain biochemical components. for instance, in some cells production of exosomes requires lipid ceramide and neutral sphingomyelinase [ ] , an enzyme that converts sphingomyelin to ceramide, and related proteins including phospholipase d that hydrolyzes phosphatidylcholine into phosphatidic acid and dgk alpha [ , ] . another mechanism of exosome release relies on small gtpases such as rab a/b [ ] , rab , , , and in some cells, or soluble n-ethylmaleimide-sensitive factor attachment protein receptor (snare) family proteins like ykt [ , ] , vesicle-associated membrane protein (vamp ) [ , ] , cd , and cd . these proteins are involved in exosome biogenesis and are commonly used as markers unlike exosomes and microvesicles, which are released during normal cellular processes, apoptotic bodies are formed only during programmed cell death [ , ] . apoptotic bodies' size ranges from - nm. during apoptosis, the cell undergoes morphological changes and shrinks to a smaller size with densely packed cytoplasm and other organelles, and eventually their nucleus disintegrates [ ] . further, the cells form blebs on its surface and disintegrate into small fragments called apoptotic bodies. these are characterized by the presence of organelles within the vesicles [ ] and are cleared from the body by phagocytosis by specific mechanisms [ , ] . the most commonly used identifiers of apoptotic bodies are annexin v, thrombospondin, and c b [ ] . limited knowledge exists in the literature regarding the role of apoptotic bodies in cell-cell communication during viral infection and their contribution to viral pathogenesis. to understand their possible role and function in intercellular communication, numerous in-depth studies are warranted in the future. uptake of ev seems to depend on the type of recipient cell, its physiological state, and recognition of ligands or receptors on the recipient cell and evs. cells broadly internalize evs either by fusion with the plasma membrane or via endocytosis. internalization of evs by recipient cells occurs by various mechanisms of endocytosis including clathrin-dependent and clathrin-independent mechanisms such as caveolin-mediated uptake, macro-pinocytosis, phagocytosis, and lipid raft-mediated internalization [ , ] . ev uptake is an energy-dependent process [ ] . neurons internalize oligodendrocyte-derived exosomes by clathrin-mediated endocytosis [ ] , whereas microglia internalize exosomes by micropinocytosis [ ] . epithelial cells internalize exosomes by caveola-dependent endocytosis [ ] , while dendritic cells internalize evs through lipid raft domains [ ] . different methods are employed to detect ev uptake, among which the most used viruses , , of method is the use of fluorescent lipid membrane dyes to stain ev membranes. examples of such dyes include pkh , pkh , rhodamine b, dii, and did [ , , , ] . the internalization of evs by recipient cells can be measured using methods such as flow cytometry and confocal microscopy [ , ] . evs and viruses are highly heterogeneous in size, structure, and biogenesis, and therefore they cause apparent difficulties in distinguishing and separating evs from viruses. even though evs and viruses overlap in size and biophysical properties, evs far outnumber high-titer viruses during infection [ ] . in the past decade, a multitude of isolation and purification methods for evs and virus particles have been developed. differential centrifugation/ultracentrifugation (uc) technique is widely used for the isolation of evs from cell cultures' media and biological fluids that contain viruses [ ] . although this technique is considered as the gold standard of ev isolation, it often coprecipitates with proteins and lipoproteins that can affect sample purity and may interfere with downstream analysis [ , ] , limiting its use in hospital settings. this limitation can be overcome by including multiple isolations and characterization techniques such as antibody-based immunoaffinity purification, tangential flow filtration (tff), and nano-flow cytometry (nfcm) [ ] [ ] [ ] [ ] . however, each of these methods has its limitations, which need to be considered before planning ev isolation and purification. for instance, ev isolation using the antibody-based immunoaffinity purification method provides a refined ev population but is limited by the sample volume and amount of final product [ ] . moreover, the expression level of ev markers such as cd , cd , and cd can vary depending on the ev origin and physiological condition, requiring a combination of markers to be used. compared to uc, the tff method can be effective in obtaining ev-enriched formulations from a large volume of samples. however, tff is likely to cost higher than conventional ev isolation methods. further studies are required to explore the utilization of tff for clinical studies [ ] . due to limitations associated with isolation procedures, and lack of a standardized isolation process, a validated good manufacturing practice (gmp)-compliant procedure is desperately needed. bari et al. employed conditioned media from mesenchymal stem/stromal cells for the secretome/ev isolation. a key aspect of their study is a large-scale secretome or ev isolation process using uc and tff that complies with gmp, which allows standardized and pharmaceutical grade products suitable for clinical applications [ , ] . the use of nfcm is reported as a new benchmark for quality assessment of evs. phenotyping of single particles is possible through nfcm using immunofluorescent labeling of evs [ ] . however, the limitations in resolution and detection varied depending on the criteria used to define the ev populations based on markers [ ] that have excluded many researchers widely utilizing this technique. besides, an nfcm based method can be challenging to develop and to validate ev characterization, given the specific ev population measurement and due to the lack of standard guidelines for handling and analyzing a variety of samples with appropriate normative controls in nfcm. li k et al. have developed an approach termed cushioned-density gradient ultracentrifugation (c-dguc), a variant of ultracentrifugation, for ev refinement [ ] . in this approach, samples were processed through a density gradient cushion such as iodixanol (optiprep™) and centrifugal force to maximizes ev recovery followed by density gradient ultracentrifugation steps that eventually provide high-purity purification of evs by effectively removing protein aggregates. however, evs can lose integrity while isolated from a fixed density range [ ] . polyethylene glycol (peg) precipitation followed by iodixanol density separation has recently become a useful method to pull down evs, viruses, and proteins or protein-rna aggregates within a sample, followed by an additional centrifugation step. this method results in a significantly higher yield of evs in comparison to the conventional uc method [ ] . the contents of evs vary greatly depending upon the condition of the parent cell. thus, apart from characterizing the vesicles, identifying these contents reveals a breadth of information regarding the parent cells. the international society for extracellular vesicles (isev) guidelines should be followed when isolating evs from cells or plasma/biological fluids for drug encapsulation. the most pragmatic approach appears to be viruses , , of the isolation of evs using a commercial kit and size exclusion chromatography (sec; also known as gel filtration) methods followed by microfiltration of samples using filters with pore diameters of . , . , or . µm depending on the size of vesicles required. in sec, evs are separated from other material according to differences in sizes (hydrodynamic radii) [ ] that gives this technique the upper edge over conventional methods and can be effectively used for a variety of complex biological samples such as body fluid, blood/plasma, urine, and breast milk [ ] [ ] [ ] [ ] . isolation of high-purity evs from samples containing virions is challenging since both evs and some viruses, in this case, retroviruses, are similar in size. as of now, no validated protocol is available to specifically separate evs from virions that are similar in size and carry the same markers [ ] . however, a study has demonstrated that defective viruses could be separated from naturally occurring viruses based on differences in buoyant densities [ ] . evs loaded with drugs to treat viral diseases require them to target majorly infected cells or tissues. when considering evs as personalized therapeutic carriers, surface engineering of evs is required that can be performed using covalent and noncovalent modification [ ] [ ] [ ] . it is important to optimize the method of isolation for evs for drug loading on a case-to-case basis. upon loading drugs to these evs, the evs can be further separated using a sucrose gradient that utilizes iodixanol and characters each fraction for loading efficiency and total loading. the ev fractions with optimally loaded drugs can be further characterized by their size, shape, and marker proteins for further use. evs released by virus-infected cells can incorporate protein molecules, derived from viral genes involved in viral assembly. delivery of the ev-associated virulence molecules affects recipient cells by rendering them particularly vulnerable to viral infection (table ) . moreover, incorporating viral proteins can trigger cell death of non-participating immune cells [ ] that would contribute to the heavy loss of immune cells during the early stages of viral infection or low viral load. intercellular transfer of viral proteins and viral cell surface receptors by evs not only facilitates evasion of the host's immune response by suppressing antibody production in lymphocytes but also makes immune cells susceptible to viral infection [ , ] . however, while evidence indicates that evs can, directly and indirectly, mediate the antiviral response, their role in regulating immune response is not yet fully elucidated in vivo. hiv-infected cell-derived exosomes carrying negative regulatory factor (nef) induces apoptosis in t-lymphocytes; nef-transfected microglia-released nef+exosomes reduce the blood-brain barrier (bbb) integrity [ , ] chemokines and receptors ccr , cxcr , mcp- facilitate the entry of hiv [ ] proinflammatory markers il- , tnf-β, il- hiv-infected cells derived exosome containing tar rna plays a role in the increase of il- and tnf-β in macrophages. hiv-infected u macrophages upon cigarette smoke condensate (csc) treatment enhanced the packaging of il- in evs; il- served as a biomarker for hiv patients with altered immune function due to alcohol and tobacco abuse [ , , ] host protein apobec g inhibit replication of viral infectivity factor (vif) -deficient and wild-type hiv- in recipient cells [ ] mirna vmir- and vmir- triggers endosomal toll-like receptor (tlr) and nuclear factor-κb (nf-κb) signaling, stimulating the release of tnfα by delivering ev to bystander macrophages, and may contribute to chronic immune activation [ ] oxidative stress factors cellular markers cyp ( a , b , and a ), sod , cat gfap induce hiv replication. hiv-infected u macrophages upon csc treatment promotes differential packaging of cyps and aoes in evs increased levels of glial fibrillary acidic protein (gfap) in plasma evs from hiv subjects can serve as a potential biomarker [ , , ] contribute to viral immune-evasion and act in concert to promote tumor development through the interaction with multiple cellular proteins [ , ] mirna mir- , - b, and let- b cancer-associated, cellular pathways targeted by these mirnas. induce tumorigenesis through the effect of these micrornas on their targets [ ] mir- plays a role in cervical carcinogenesis, notably through the downregulation of p and phosphatase and tensin homolog deleted on chromosome (pten) [ ] mir- - p favors cell proliferation [ ] mir- a- p possesses anti-apoptotic properties [ hbv viral proteins large s, core and p proteins hepatocytes secreted exosomes participate in virus replication [ ] viral mirnas hbv-mir- represses viral protein production and hbv replication [ ] htlv- viral proteins gp , tax, and hbz increase cell-to-cell contact and promote a potential increase in viral spread [ ] zika viral genetic material and protein rna and zikv-e evs derived from infected c / cells promote infection and activation of monocytes with enhanced tnf-α mrna expression. [ ] in hiv, evs are thought to play an important role in disease progression through multiple mechanisms. viral components may be packaged in evs, which can then be delivered to uninfected cells, modulating the systemic inflammatory status. for instance, hiv-infected cell-derived exosomes carry viral protein nef that induces apoptosis in immune cells and reduces the blood-brain barrier (bbb) integrity to spread viral infection in the brain [ , ] . it has been shown that evs released during hiv infection are heterogeneous including size variability. a study has shown that treatment-naïve people living with hiv/aids (plwha) contain evs larger in size and numbers compared to plwha who were either virally suppressed, elite controllers, or healthy controls [ ] . additionally, cd counts and the abundance of evs in the blood were inversely correlated, with low cd counts associated with more abundant evs. interestingly, there was no relationship between cd counts and ev size. both size and abundance were also inversely correlated with neutrophils and platelet counts, as well as the cd /cd ratio, all of which are markers of disease progression [ ] . this suggests that evs may function as a biomarker for hiv disease progression. other studies have observed similar findings. in cells treated with antiretroviral drugs (arvs), increases in relative ev production has been observed [ ] , along with decreased loading of genomic, but not non-coding, rna into evs from cells, which were treated with arvs, as opposed to untreated cells. additionally, treatment with interferon-alpha increased the packaging of viral rna into evs. the authors suggest that this occurs because arv or interferon prevents the release of viral particles from cells, which then allows for viral rna to be packaged into evs due to the increased presence of viral rna in the cell. in addition to viral rna, a variety of molecules, e.g., viral & host proteins, cellular markers, mirna, inflammatory molecules such as oxidative stress markers, chemokines and cytokines can also be packaged into evs [ , [ ] [ ] [ ] , ] . a study showed that the viral envelop (env) protein can be packaged into evs from infected cells [ ] . the env-containing evs can increase susceptibility to viral infection in cell culture experiments, and depletion of env-containing evs showed decreased susceptibility to viral infection. altered levels of proteins in plasma evs are often described upon viral infection. for example, various examples of significantly altered expression of proteins, and markers associated with cellular stress, have been reported in plasma evs derived from hiv and htlv- infected patients. however, the mechanism of specific packaging of these proteins and markers in evs and their role in intercellular communication was not elucidated [ , ] . blood plasma can be considered as disease biomarkers since it contains glycoproteins and cellular markers carried in evs [ ] . dysregulation of cytokines and chemokines is often associated with hiv infection and subsequently contribute to the viral pathogenesis [ , , ] . moreover, the use of substances such as alcohol, tobacco, and drugs is prevalent among hiv-infected individuals [ ] [ ] [ ] [ ] . circulating inflammatory cytokines have been found to be elevated in hiv-positive substance users [ , , , ] . in prior studies, we demonstrated that exosomes derived from hiv-infected monocytes/macrophage cells exert a protective effect against cytotoxicity and viral replication in hiv-infected macrophages. however, exosomes derived from hiv-infected cells lost their protective capacity that could be due to the selective packaging of cytochrome p (cyps) and antioxidant enzyme (aoe) mrnas in exosomes [ ] . similar to the previous study, exposure to cigarette smoke condensate (csc) increased the packaging of cytokines, especially il- and cyps ( a and b ) in evs isolated from hiv-infected u macrophages [ ] . conversely, ev packaging of aoes (sod- and catalase) decreased in hiv-infected u macrophages more than in uninfected u macrophages [ ] . recently, our group showed that the astrocytic and neuronal-specific markers (gfap and l cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco [ ] . human cytidine deaminase apobec g (a g) can be packaged in evs and inhibit hiv replication with its potential dna-editing activity [ ] . hpv-infected cells release evs that make other cells more susceptible to infection as they deliver proteins that affect viral expression, and subsequently tumor development [ , , ] . to enhance protein delivery and hpv replication, hpv-infected cells hijack ev signaling pathways to control the quantitative and qualitative release of evs from hpv-infected cells [ , [ ] [ ] [ ] . as tumor genes and proteins are persistently expressed from evs, this contributes to hpv cancer cell growth [ ] , thereby making the signaling pathways of evs harmful to the host. the oxidative stress released from hpv-infected cells into evs should also be considered detrimental to the host as this stress has the potential to induce viral replication of other viruses such as hiv- [ ] . to make matters more complex, the signaling pathways of evs are not limited to increased hpv replication as the release of evs can also promote an adaptive immune response that becomes beneficial to the host [ ] . for example, in the setting of hpv replication and tumor progression, evs have prompted immune activation in head and neck cancers and are being considered as biomarkers for improved clinical outcomes [ ] [ ] [ ] [ ] . besides, endogenously engineered evs are being considered as a novel method to deliver anti-hpv immunotherapy [ ] , thus making them yet another way to improve clinical outcomes. unique mirna signatures were found in evs released from cervical cancer affected cells that were associated with hpv status [ ] [ ] [ ] [ ] ] . during influenza virus infection, evs carrying host mirna or viral epitopes are thought to be integral to antigen transfer, reducing virus spread, and immune regulation [ ] . for example, influenza virus hemagglutinin (ha) epitopes enclosed within exosomes on mhcii molecules have been shown to improve the efficiency of antigen delivery to immune cells [ ] . further, exosomal-like vesicles carrying mucin molecules such as muc , muc , and muc can bind sialic acids and neutralize influenza viruses [ ] , which may help reduce virus dissemination. virus replication can also be blocked by some highly upregulated exosomal mirnas, such as the type i interferon-inducing hsa-mir- and mir- - p [ , ] . also, these evs excite other proinflammatory cytokines, such as il- , tnf-α, and ifn-β [ , ] , although their efficacy may be dependent on cell source, maturity, and mhc molecules. macrophages have been shown to produce thousands of proteins within exosomal vesicles in response to influenza infection. these evs included a variety of host factors, including cytokines and proteins involved in copper metabolism and autophagy [ ] . interestingly, proinflammatory cytokines from macrophages and dendritic cells were suppressed by vaccine-induced evs (e.g., mir- a, mir- , or mir- ) [ ] . although much of the current work has focused on single influenza virus strains, important strain specific ev dynamics have begun to be identified. in one study, nearly half of exosomal mirnas were conserved between h n and h n infection in a cells [ ] . of the differentially expressed evs, they were > -fold during infection with the highly pathogenic h n than with uninfected samples. a better understanding of these dynamics and temporal-and strain-specific differences could provide important insight into pathogenicity and pinpoint new therapeutic and universal influenza vaccine targets. hcv belongs to a family of human virus called flaviviridae characterized by positive-sense single-stranded rna that encodes precursor polyprotein that is cleaved into three structural proteins comprising of core protein p with envelope glycoprotein e & e , and seven non-structural proteins that play a role in viral pathogenesis [ , ] . the chronic viral infection leads to hepatic inflammation that is associated with increased production of pro-inflammatory cytokines and chemokines from liver residential immune cells and immune cells recruited to the liver [ ] . evs are observed as major modifiers of cellular crosstalk between hcv-infected hepatocytes & immune cells [ ] . in hcv pathogenesis evs act as a double edge power by: ( ) delivering vireo-independent hcv rna and ( ) obtaining antiviral immune responses [ ] . the cellular vesicular pathway is exploited by hcv to congregate and release viral particles. this happens by releasing vesicles containing envelope glycoprotein e & e , entire hcv genome & viral particles. when the vesicles containing these components enter the target cells, this helps to establish infection [ ] . in systemic alteration of an immune response, major regulators commonly known as specifically enriched micro rnas (mirnas) are delivered by evs. these are loaded into evs and are involved in post-transcriptional regulation of gene expression, which is known to be influential for hcv replication [ , ] . this confirms that evs have peculiar mirna expression isolated from the sera of chronic hcv patients. exosomes derived from hcv infected cells are responsible for developing infection to other uninfected cells. these exosomes carried viral rna in complex with mir- , ago , and hsp that support virus replication [ ] . evs, isolated from sera of patients with acute or chronic hcv or interferon-stimulated macrophage cultures, mediate inhibitory effects on hcv replication [ ] . in co-culture models, the immunoregulatory effects of evs were assessed on the replication of hcv. stimulation with type i & ii interferon n, which is a fast but short-lasting ev-derived antiviral, leads to the production of macrophages by secreting various cytokines resulting in innate immunity. thus, hcv replication in macrophages derives ev-mediated long-lasting inhibitory effects [ ] . evs released by hcv infected cells contain viral rna that might trigger plasmacytoid dendritic cells to produce ifnα [ ] . the emergence of the life-threatening "atypical pneumonia" caused by severe acute respiratory syndrome coronavirus (sars-cov) in the early st century has led to renewed interest in coronaviruses [ ] . coronaviruses belong to the family of rna viruses and possess the largest genome among them. similar to other viruses, their genome contains essential genes encoded for open reading frames a and b (orf ab), and viral structural proteins, which are required for virus replication, transcription, and virus assembly [ ] . a newly emerged coronavirus disease in (covid- ) is caused by a novel severe acute respiratory syndrome coronavirus- (sars-cov- ). sars-cov- infection spread within a few months after the first outbreak reported in december in china, which later became a worldwide crisis. with high morbidity, the disease is often characterized by an atypical severe pulmonary pneumonia [ , ] . the novel sars-cov- is closely related to sars-cov- coronavirus responsible for the sars outbreak that emerged in late in china. its subsequent worldwide spread had caused cases and deaths by july [ ] . sars-cov- infections, which has already infected > million people and caused the death of~ , people world-wide, are presently occurring and represent an ongoing threat to public health. out of cases in china reported having at least one comorbidity [ ] . the risk of serious adverse outcomes of covid- is especially pronounced in patients with comorbidities such as hypertension, diabetes, kidney, and cardiovascular diseases [ , ] . sars-cov encodes four structural proteins; spike glycoprotein (s), nucleocapsid protein (n), membrane protein (m) & small envelope glycoprotein (e) & several nonstructural proteins of unknown functions [ ] . sars-cov- spike (s) glycoprotein interacts with angiotensin-converting enzyme (ace- ), the same receptor used by sars-cov to enter the target cells, in particular lung alveolar epithelial cells [ ] . it has been demonstrated that evs released by sars-cov- infected lung epithelial cells contain viral rna fragments that were subsequently detected in the cardiomyocytes, suggesting viral rna transmission via evs [ ] . sars-cov- is a positive-stranded rna virus in an envelope with a genome of , nucleotides [ ] . the spike protein s of sars-cov- (sars-s) facilitates the viral fusion that can be triggered following the fusion-mediated conformational changes in the target cell receptor that mediates the entry of the virus into the target cells. once inside the cell, a virus may utilize the exosome secretion pathway to enhance its pathogenesis and viral spread [ ] . to find a vaccine against sars-cov- , researchers performed exosome-based research, where they constructed chimeric s protein of the sars by replacing cytoplasmic and transmembrane domains of sars-s with g protein of the vesicular stomatitis virus. this chimeric s-protein was readily expressed on the cell surface, allowed entry of pseudotyped retroviral vectors, and was incorporated into exosomes. subsequently, chimeric s protein-containing exosomes have been tested as a novel protein for vaccine immunogenicity against sars-cov in mouse models [ ] . recently, preclinical studies have uncovered a therapeutic role of msc-derived secretome or evs in lung regeneration [ ] , which could offer a new therapeutic approach in treating severe covid- infection [ , ] . intravenous transplantation of ace -negative mesenchymal stem cells (mscs) promoted recovery of patients from severe covid- [ ] , thus supporting the hypothesis that binding of sars-s protein through ace expressed on msc-derived small evs could limit the viral infection through competitively inhibit the binding of sars-s to ace expressed on alveolar type ii cells [ ] . epstein-barr virus (ebv) is one of the herpes viruses that hijack its host evs. ebv infected cells release evs that contain ebv-coding/non-coding mirnas and transfer it to uninfected cells including b lymphocytes and epithelial cells [ , ] . the transfer of ebv-coding mirnas to b lymphocytes, especially the akata-lymphoblastoid cell lines-derived evs, causes inflammatory responses of monocytes/macrophages and induces severe lymphoproliferative disease (lpd) [ ] . ebv viral reactivation was recently detected in co-cultured latently ebv-infected bl cells in response to the transfer of evs that contain epithelium-specific mirnas from oropharyngeal epithelial cells [ ] . ebv-infected cells can transfer non-coding rnas such as bart and bhrf mirnas via evs to the target cells. upon entry, mirnas can be directed to cellular sites of mirna-mediated gene repression, causing repression of their target genes cxcl and lmp [ ] . ebv-infected nasopharyngeal carcinoma cells release evs containing galectin- protein that interacts with the tim membrane receptor and induces apoptosis in t cells [ ] . similarly, exosomes released by these cells convey the viral protein latent membrane protein (lmp ) that provoke intrinsic t-cell inhibitory activity and thus modulate immune response mechanisms [ ] . herpes simplex virus (hsv- ) is another herpes virus that hijacked its host evs. hsv- -infected cells release evs with different components based on their stage in the infection cycle [ ] . early in the lytic cycle, hsv- proteins cause remodeling to evs' cargos, which in turn cause virion egress from infected cells to uninfected cells [ ] . hsv- evs contain coding and non-coding rnas and more importantly immune components, such as the stimulator of interferon genes (sting) [ ] . a recent study demonstrated that sting-containing evs play an important role in inhibiting viral replication during the lytic cycle, as well as inhibiting viral gene expression during the latent stage [ ] . another recent report illustrated that mir-h and mir-h are being expressed late in the virus infection cycle and transferred to uninfected cells via evs [ ] ; mirna- induces the formation of gamma interferon (ifn-γ) which blocks viral replication in uninfected cells but not in infected cells [ ] . ifn-γ loaded evs maximize viral transmission between individuals by diminishing the spread from infected cells to uninfected cells [ ] . a study reported that hsv- encoded glycoprotein b (gb) modulates the immune response by manipulating the mhc class ii processing pathway by diverting human leukocyte antigen-dr (hla-dr) molecules into the exosome pathway [ ] . an ev vaccine for the hepatitis b virus (hbv) is currently under investigation. as in most of the viruses, evs carry hbv viral proteins such as large s, core and p proteins which participate in viral replication [ ] . they also play many roles in hbv infection; they are responsible for hbv replication, innate immune response during infection, a biomarker for its diagnosis, and development of a possible vaccine [ , ] . a recent study elucidated that unmodified evs can be attractive coadjutants to hepatitis b recombinant antigen (hbsag), because it triggers the healthy mice immune response due to an increased ifn-γ concentration and accelerates the production of igg antibodies [ ] . hepg . . cells with integrated hbv genome release evs containing hbv-mir- which represses viral protein production and hbv replication [ ] . moreover, the study elucidated that engineered evs that are loaded with exosome-anchoring protein nef mutant (nefmut) and hbv core protein can induce cytotoxic t lymphocyte (ctl) immunization in animals for hbv infection [ ] . on the one hand, evs are responsible for infection transfer from one cell to another. on the other hand, evs are also responsible for antiviral response initiation by inducing the uninfected cells' immune response [ , ] . due to their abilities to activate the innate and adaptive immune response, evs can be the future pathway for the treatment of many viral infections. so far, viruses that impair their host immune response such as human t-lymphotropic virus (htlv- ) only use their host's evs to use viral proteins such as gp , tax, and hbz to increase cell-to-cell contact and promote a potential increase in viral infection [ ] . htlv- evs were found to contain a protein called tax that is implicated with the dysregulation of the recipient cells' immune response [ , ] . interestingly, there are viruses that not only hijack host evs, but also boost the production of evs such as in zika virus (zikv). evs released from zikv-infected (c / ) cells carry viral rna and zikv-e protein that can trigger monocyte activation to induce mrna expression of tnf-α [ ] . zikv-infected cells have incrementation in their neutral sphingomyelinase (nsmase)- /smpd gene expression and activity, which provokes the production and excretion of evs in neurons. treatment of zikv requires the hindrance of ev production through the inhibition of smpd s in neurons to prevent further neuronal death and virus spreading [ ] . with the introduction of antiretroviral therapy (art), the morbidity and mortality associated with hiv infection have drastically reduced [ ] . however, due to the presence of latent reservoirs and inadequate drug concentration in the central nervous system (cns), the virus continues to replicate and causes a wide range of cns pathologies, including hiv-associated neurocognitive disorders (hand) [ ] . therefore, new drug delivery systems that facilitate drug passage across the bbb to effectively suppress the virus in cns, with minimal/tolerable neurotoxicity need to be developed. evs can be used as a potential drug delivery system as they can cross the bbb [ , ] with less immunogenicity. further, in preclinical studies, ev-based drug delivery platforms have been shown to carry therapeutic small molecules across the bbb to help alleviate multiple cns diseases, including parkinson's disease and brain cancer [ ] [ ] [ ] . evs that can be used as a drug delivery platform are mainly derived from exosomes that linked to an endolysosomal pathway. exosomes released from dendritic cells are considered vaccine candidates for immunotherapy in diseases such as cancer. these exosomes can be further taken up by dendritic cells leading to a presentation of mhc-i or peptide complexes [ ] [ ] [ ] . arvs can be loaded into evs to deliver them across the bbb to achieve viral suppression in the cns [ ] . since the autoclaved exosomes show intrinsic stability at a physiological temperature [ ] , sterile drug-loaded evs can be formulated. large scale production of ev drug formulation can be achieved using an endogenous drug-loading method that uses cells to release evs with target drugs encapsulated in vitro. evs with encapsulated drugs are capable of targeting the diseased cell or tissue, with targeting characteristics [ ] . this inherent feature could be used to deliver drugs selectively to their intended targets while abrogating off-target side effects. virus-targeting antiviral drugs can include protease inhibitors (pis), integrase inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors, and nonnucleoside reverse-transcriptase inhibitors. an ev-based drug delivery platform with either hiv pis alone or in combination with ritonavir is used as a pharmaco-enhancer or second line of therapy for the treatment of hiv [ ] . evs can also be used as a vehicle for delivery of crispr-associated endonuclease (cas ) and potentially as the guide rna (grna) to target nucleotide sequences within viral genomes [ , ] . another therapeutic use of evs is vaccination against infectious diseases and viral infection. ev-mediated delivery of mrna encoding pathogenic proteins required for viral infection might be a vaccine candidate that can induce t helper (th )-type immune responses and cell-mediated immunity, without the need to attenuate and inactivate pathogenic viruses or bacteria [ , ] . for the ongoing pandemic of covid- , anti-hiv pis, other pis, or other antiviral and antibacterial drugs can either be encapsulated in evs derived from various cell lines using endogenous loading technique or from the plasma of patients using exogenous loading method for personalized therapy [ ] . repurposing fda-approved antiviral drugs using evs could be a fast way to get tested through clinical trials. although the clinical research done on evs seem promising for therapeutic application, several factors must be considered before translating evs into clinics. at present, available ev isolation methods, such as ultracentrifugation, density gradient centrifugation, precipitation, size exclusion chromatography, affinity, and novel microfluidic techniques are not sensitive enough to distinguish evs subpopulation due to lack of specificity, physical and chemical biomarkers [ ] ; therefore, a high level of standardization is required to compare ev protocols and results used across different laboratories before the adoption of ev therapy to various clinical applications. also, evs' pharmacokinetics, half-life, and plasma stability, as well as the interaction of encapsulated drugs with ev components, ev-targeting, and immune clearance of evs, are other limitations that need to be overcome before realizing the clinical applications of evs in drug delivery. a growing body of evidence suggests that virus-infected cells produce evs, encapsulated with viral proteins and parts of viral genetic material, and in some cases they carry the full infectious viral genome that facilitates viral infection and mediates immune responses ( figure ) . notably, evs can enhance viral infection by: ( ) mediating transfer of chemokine co-receptors or cell surface proteins to null-target cells that do not express endogenous viral co-receptors; ( ) helping viruses to evade the host immune system; ( ) transferring of viral components (viral proteins and rnas) to recipient cells, which induce cytotoxic effects on infected cells, leading to progressive loss of immune cells resulting from the apoptosis of uninfected bystander cells. here, we aimed to shed light on how evs potentially impact infection and the pathogenesis of various viruses. we also evaluated the potential utilization of evs in antiviral and antiretroviral therapy, and in drug delivery. characterizing evs from virus-infected cells and their functional analyses could aid not only in the understanding of the mechanisms of viral infection but also in the utilization of evs as a delivery system for therapeutic agents. overcome before realizing the clinical applications of evs in drug delivery. a growing body of evidence suggests that virus-infected cells produce evs, encapsulated with viral proteins and parts of viral genetic material, and in some cases they carry the full infectious viral genome that facilitates viral infection and mediates immune responses ( figure ) . notably, evs can enhance viral infection by: ( ) mediating transfer of chemokine co-receptors or cell surface proteins to null-target cells that do not express endogenous viral co-receptors; ( ) helping viruses to evade the host immune system; ( ) transferring of viral components (viral proteins and rnas) to recipient cells, which induce cytotoxic effects on infected cells, leading to progressive loss of immune cells resulting from the apoptosis of uninfected bystander cells. here, we aimed to shed light on how evs potentially impact infection and the pathogenesis of various viruses. we also evaluated the potential utilization of evs in antiviral and antiretroviral therapy, and in drug delivery. characterizing evs from virusinfected cells and their functional analyses could aid not only in the understanding of the mechanisms of viral infection but also in the utilization of evs as a 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delivery by extracellular vesicles in mammalian cells and its applications the crispr/cas genome editing methodology as a weapon against human viruses therapeutic applications of extracellular vesicles: clinical promise and open questions extracellular vesicles move toward use in clinical laboratories this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to kelli anne gerth (ph.d. student; university of tennessee health science center) for critical reading and editing of the manuscript. the authors declare no conflict of interest.acknowledgments: the authors are grateful to kelli anne gerth (ph.d. student; university of tennessee health science center) for critical reading and editing of the manuscript. the authors declare no conflict of interest. key: cord- -rswa zdn authors: manjunath, n.; kumar, priti; lee, sang kyung; shankar, premlata title: interfering antiviral immunity: application, subversion, hope? date: - - journal: trends immunol doi: . /j.it. . . sha: doc_id: cord_uid: rswa zdn rna interference (rnai), initially recognized as a natural antiviral mechanism in plants, has rapidly emerged as an invaluable tool to suppress gene expression in a sequence-specific manner in all organisms, including mammals. its potential to inhibit the replication of a variety of viruses has been demonstrated in vitro and in vivo in mouse and monkey models. these results have generated profound interest in the use of this technology as a potential treatment strategy for viral infections for which vaccines and drugs are unavailable or inadequate. in this review, we discuss the progress made within the past – years towards harnessing the potential of rnai for clinical application in viral infections and the hurdles that have yet to be overcome. conventionally, only specialized cells of the immune system and their secreted products are thought to be involved in protecting the body from foreign invaders such as viruses. however, in recent years, a new type of genomic immunity mediated by rna interference (rnai) has emerged and has sparked intense interest as a potential treatment strategy for a variety of diseases, including viral infections, cancer and degenerative diseases [ ] [ ] [ ] [ ] . rnai was first recognized as a naturally occurring anti-viral defense mechanism in plants. in rnai, long double-stranded (ds) rna generated during viral infection is cleaved by an enzyme termed dicer into short, - nucleotide (nt) dsrna molecules termed small interfering (si)rnas that mediate sequence-specific gene silencing [ , ] . the sirna associates with a protein complex called the rna-induced silencing complex (risc) (figure ), following which the sense strand is cleaved by the enzyme argonaute (ago ) [ ] . the antisense strand then guides the risc to the corresponding messenger (m)rna by sequence homology, and the ago nuclease cuts the mrna, resulting in specific gene silencing. in the context of rna viruses, sirnas can be designed to degrade not only viral mrna but also the negative sense viral genomic rna and the complimentary rna that serves as a template for new genomic rna synthesis [ ] . although rnai is a natural phenomenon in plants and worms, long dsrna induces an interferon response in mammalian cells resulting in non-specific global suppression of protein synthesis and cell death [ ] . a landmark development in the field occurred with the discovery that the introduction of -nt-long synthetic rna resembling the dicer-processed sirna into mammalian cells induces sequence-specific gene silencing without evoking the interferon response [ ] . since then, rnai has been widely used as a quick reversegenetics approach for gene-function analysis and for ablating specific genes for therapeutic purposes. the exquisite sequence specificity and high potency of rnai makes it an attractive gene-silencing approach [ ] . rnai was found to be -fold more effective on a molar basis than antisense oligonucleotides [ ] . this is probably owing to the autocatalytic effect of rnai whereby a single sirna molecule is reused for cleaving many target mrna molecules [ , ] . rnai can be induced by the introduction of synthetic sirna or by intracellular generation from vector-driven expression of precursor short hairpin (sh) rnas (box ). the optimal design of sirna or shrna is required for the potent induction of rnai (box ). although rnai is an integral component of the innate immune response to viruses in plants, whether the same is true in mammals is unclear. however, the recently described virus-encoded counter-defense strategies, such as suppressors of rnai and micrornas, suggest a longstanding interaction of viruses with the rnai machinery (box ). given the natural antiviral role of rnai in plants and its induction in mammals by introduced sirna, the phenomenon has generated great enthusiasm as a potential antiviral treatment strategy [ , ] . several viruses with widely differing replication cycles have been inhibited in vitro by targeting viral and cellular genes involved in the viral life cycle ( figure ). these include: the positive-stranded rna viruses, including polio, west nile, dengue and foot and mouth disease (fmd) viruses; the negative-stranded rna viruses, including respiratory syncytial virus (rsv) and influenza virus; the doublestranded rna rotavirus; hiv lentivirus; and dna viruses such as the polyoma virus, papilloma virus and herpes simplex virus (hsv). however, the replicative characteristics of certain viruses can protect them from rnai. for example, although sirnas can inhibit progeny virus production, the genomic rnas of rsv, hepatitis delta virus and rotavirus are resistant to rnai owing to tight figure . rna interference. rna interference can be initiated in cells by the introduction of synthetic double stranded sirna or plasmid or viral vectors encoding shrna. the shrna is transcribed in the nucleus and exported to the cytoplasm, where it is processed into sirna by dicer or, possibly, another ribonuclease. in the cytoplasm, the sirna associates with the risc complex consisting of several proteins, which in human cells include dicer, argonaute (ago- ), hiv- transactivating response rna-binding protein (trbp), protein activator of protein kinase r (pact) and, possibly, other proteins unidentified to date [ ] . the sense (passenger) strand of the sirna is then cleaved by ago- within the active risc [ ] . the passenger strand can also be removed, albeit at a slower rate, by a cleavage-independent 'bypass' mechanism used for microrna processing [ ] . because the exact sequence of the molecular interactions involved in risc activation is unknown, the process is shown in a dotted box and the sirna guide strand is shown as curved to indicate its directional loading into the risc. the anti-sense (guide) strand associated with the mature risc guides the complex to the corresponding mrna because of sequence homology, and the same ago- nuclease then cuts the target mrna at a position corresponding to nt - from the end of the anti-sense guide strand. the cleaved mrna is rapidly degraded resulting in gene silencing. rnai can be induced by synthetic sirna or by vector-driven expression of shrna. in the second method, sirna sequence followed by a w nt loop and a reverse complement of the sirna sequence is cloned in dna or viral vectors to express endogenously the shrna, which is processed in the cytoplasm to sirna. whereas synthetic sirna is introduced into cells by transfection, shrna can be introduced by transfection of a dna vector or transduction through viral vectors. non-replicating, recombinant viral vectors are commonly used for shrna expression because of ease of delivery, particularly in difficult-to-transfect primary cells. adenoviruses and adeno-associated viruses have been used as vectors but lentiviral vectors are generally preferred because they infect actively dividing, and resting and differentiated cells such as stem cells, macrophages and neurons. because the viral dna is incorporated in the host genome, the main advantage of this method is the long-term expression of shrnas and gene silencing. in fact, knockdown persisted for at least six months in the mouse brain following transduction with a shrna-expressing vector [ ] . although viral vectors deliver shrna efficiently, they have several disadvantages, including the vector-induced immune response and possible toxic effects of long-term rnai induction. moreover, retroviral integration into the host genome also enhances the risk of insertional mutagenesis, exemplified by the development of leukemia in patients undergoing retroviral-based gene therapy for severe combined immunodeficiency [ ] . by contrast, synthetic sirna, similarly to drug treatment, provides a way to achieve transient gene silencing without the risks of insertional mutagenesis, immune response induction or the toxic effects of longterm rnai induction. the major challenge is its delivery to cells in vivo. also, because sirna becomes diluted by cell division, the silencing effect generally fades after - days in dividing cells. however, in non-dividing cells such as macrophages and neurons, sirna silencing has been observed for at least weeks [ , ] . thus, whereas sirna seems to be ideal for situations such as acute viral infection, shrna could be useful for treating chronic viral infections and cancer. shrna might also be useful to generate cells resistant to infection by transducing stem cells with shrnaencoding vectors. review trends in immunology vol. no. july shielding by proteins or sequestration in membranous compartments [ , ] . despite success in vitro, many hurdles need to be overcome before using rnai to counteract virus infections in vivo. one major bottleneck is the delivery of sirnas and shrnas to appropriate cell types in vivo but significant progress has been made in the past - years. another limitation is the lack of appropriate animal models for many human viruses. nevertheless, studies in mice have proved invaluable for testing the in vivo efficacy for some viruses. data obtained from these initial investigations provide a glimpse of the successes and challenges that can be expected in a clinical setting for specific viruses in terms of rnai design and delivery. in the next subsections, we describe some of the viral diseases for which substantial progress has been made in moving rnai towards therapy. although mice are not susceptible to hepatitis viruses, transfection with plasmids containing the viral genome recapitulates many steps in the viral life cycle, including dna replication and expression of the core and surface antigens. in one of the first demonstrations of rnai effectiveness in vivo, mccaffrey et al. injected the pthbv plasmid encoding the hepatitis b virus (hbv) genome alone or with a plasmid encoding anti-hbv shrnas by hydrodynamic intravenous injection (dna was rapidly injected intravenously in - seconds in a large volume of o ml) [ ] . this resulted in a substantial knockdown of hbv transcription in the liver and also resulted in o % reduction in serum hepatitis b surface antigen (hbsag) levels and viral core antigen expression by liver cells. hydrodynamic injection is not feasible in humans because it entails the injection of almost the whole blood volume. however, morrissey et al. showed that regular low-volume intravenous injection of a chemically modified sirna (containing a phosphorothioate backbone and fluoro and -o-methyl substitutions to render the sirna nuclease-resistant) can also significantly reduce serum dna and hbsag levels in mice [ ] . chemically modified sirnas have also been encapsulated in lipid nanoparticles for intravenous delivery [ ] . this resulted in extending the serum half-life of sirna from w min to . h and reduced the sirna amounts required for a comparable reduction in viral dna and serum hbsag from box . recent advances in sirna and shrna design until recently, the design of sirna was based on selecting a -bp unique sequence with w - % g:c content immediately following the nucleotides aa or na (n represents any nucleotide) within the target gene, and incorporating a phosphate and -nt overhangs [ ] . the chances of making successful hits in this essentially trial-anderror process required testing multiple target sites for each gene. however, based on analyses of the biochemical properties of a large number of highly effective sirnas, more-stringent algorithms have now been developed for predicting functional sirnas [ ] . the thermodynamic stability at the end of the antisense strand has emerged as a crucial criterion for sirna effectiveness [ , ] . a low internal stability at the end enables proper directional loading of sirna to ensure stable incorporation of the antisense instead of the ineffective sense strand into the risc. this increases the potency of sirna almost a -fold, and, by reducing the amounts of sirna needed for silencing, it also minimizes off-target silencing. lower thermostability can also be ensured by incorporating mismatches in the sense strand at nucleotides complementary to positions - of the end in the antisense strand. because the antisense strand is still unaltered in sequence, target specificity is not compromised. alternatively, guanosines can be replaced with inosines in the first positions to give i:c base pairs that are similar in energy to a:u base pairs, to increase the propensity of this end to fray. low thermodynamic stability in the mrna cleavage region is also important to promote the release of the risc-antisense complex for multiple rounds of activity. using a - nt sirna instead of the conventional -nt sirna increases the potency by - -fold without inducing an interferon response [ ] . this could be because the longer sirna is processed by dicer to generate the optimal sirna endogenously. the pol iii promoters, such as u , h and trna, are commonly used to drive shrna expression because they provide an efficient mechanism to generate small rna transcripts. however, it has recently been shown that inserting the shrna sequence into the backbone of a mirna (e.g. mir- ) at the stem increases the shrna potency enormously, even at the level of single-copy integration [ ] . because the pol ii cytomegalovirus (cmv) promoter is used to drive this longer shrna, this system also enables multicistronic expression of multiple shrnas. rnai is used as a natural antiviral defense mechanism in plants, and plant viruses have also developed mechanisms to evade rnai [ ] . although it is unknown whether rnai is induced naturally during viral infection in mammals, recent studies suggest that mammalian viruses can also suppress rnai [ , ] . nodamura virus encodes a protein called b that interferes with dicer function and the incorporation of sirna into risc [ ] . similarly, the e l protein of vaccinia virus and the nonstructural protein ns of influenza virus can suppress rnai by sequestering dsrna [ , ] . the nonstructural protein nss of la crosse bunyavirus and hiv- tat protein also suppress rnai [ , ] . in addition to sirnas, another class of small rnas called mirnas also use the rnai pathway [ ] . in contrast to sirnas, mirnas are cellular gene products and regulate endogenous gene expression in plants, worms and mammals. in fact, the altered development of t and b lymphocytes has been noted in specific mirna-knockout hematopoietic stem cells and in dicer-knockout mice [ , ] . recently, viruses have also been found to encode their own mirna and manipulate host mirnas. epstein-barr virus encodes a mirna bart , which has been predicted to target several cellular genes [ ] . similarly, human cytomegalovirus encodes at least five mirnas, and the kaposi's sarcoma-associated herpesvirus expresses mirnas that can target host genes [ ] . the exact function of these viral mirnas has yet to be elucidated. however, the sv- encoded mirna downregulates sv- t antigens on infected tumor cells, making them less susceptible for cytotoxic t-cell recognition, providing the virus a way to evade host immunity [ ] . viruses can also use different methods for subverting host mirnas for their own purposes. the liver-specific mirna mir- binds to the end of the hepatitis c viral genome to greatly augment viral replication, and mir- inactivation abolishes viral replication [ ] . by contrast, another host mirna, mir- , targets the retrovirus primate foamy virus- genome to repress viral replication, and the viral tas protein suppresses the rnai pathway to overcome mir- action [ ] . a mirna encoded within the hiv nef gene that can regulate viral transcription has also been documented [ ] . review trends in immunology vol. no. july to mg/kg. moreover, the reduction in serum hbv dna could be sustained for weeks simply by weekly sirna treatment, showing the feasibility of using sirna treatment for a chronic disease such as hbv infection. influenza virus (iav) is the major cause of respiratory infections worldwide. two groups have recently used rnai to suppress iav infection in mice. by targeting a conserved sequence in the viral nucleoprotein and acid polymerase genes, tompkins et al. prevented infection with multiple isolates of iav, including the virulent avian h n strain [ ] . for delivery to the lungs, they injected sirna by hydrodynamic intravenous injection combined with intranasal administration of sirna complexed with the transfection reagent oligofectamine. similarly, ge et al. achieved and polio), for which a single mrna is used to transcribe viral proteins, targeting any part of the coding sequence should result in degradation (scissors) of viral genomic rna and/or progeny mrna. by contrast, for (b) viruses containing segmented rna genomes for example, rotavirus or influenza, and for (c) dna viruses, for example, herpes simplex or papilloma viruses, for which many different rna molecules are used to generate viral proteins, it is necessary to target genes essential for the viral life cycle, such as the key viral enzymes or structural proteins involved in cell binding and fusion. (d) for retroviruses (e.g. hiv), although viral proteins are transcribed either from unspliced or multiple spliced viral rnas generated from the integrated proviral dna, the genomic rna is linear, thus, as for (a), targeting any part of the genome should suppress viral replication. in addition to viral genes, cellular genes involved in viral replication, such as the cellular receptors or co-receptors (e.g. cd and ccr- for hiv), can also be used as rnai targets for all viruses. abbreviation: rt, reverse transcriptase. review trends in immunology vol. no. july efficient lung delivery following regular intravenous injection of sirna-or shrna-encoding dna vector by complexing with the cationic polymer polyethyleneimine (pei) [ ] . this treatment resulted in a - log reduction in viral titers even when administered h post-infection. moreover, the intranasal administration of shrna vector with the surfactant infasurf r , or of sirna complexed with pei, were also effective, providing an intranasal approach to treat respiratory illnesses. respiratory syncytial virus rsv is another major respiratory pathogen that causes epidemics of respiratory illness with bronchiolitis and pneumonia. two studies have shown that sirnas can be used effectively for prophylaxis and treatment of rsv infection. the viral nonstructural protein ns suppresses type i interferon production in host cells, and ns deletion mutants are avirulent in vitro and in vivo. transfecting human dendritic cells with a plasmid encoding shrna to suppress rsv ns- resulted in the efficient induction of interferon and interferon-induced genes following rsv infection [ ] . to test its antiviral effects in vivo, the vector was complexed in a chitosan polymeric nanoparticle. intranasal instillation of the nanoparticle was harmless in mice and substantially reduced viral titers and virusinduced pathology when administered days before infection. importantly, it also reduced lung inflammation and viral titers by nearly logs even when administered days after infection. similar results were also obtained by bitko et al. [ ] . in this report, the authors used a synthetic sirna targeting the viral p protein, an essential component of viral rna polymerase. when administered intranasally with the transit r transfection reagent, the sirna substantially suppressed virus replication and lung pathology. this effect was also seen, although to a lesser extent, when the sirna was administered - days after viral infection. interestingly, the intranasal application of naked sirna without a transfection reagent was also effective ( % activity). similarly, a sirna targeting the p protein of parainfluenza virus was also able to suppress viral replication and virus-induced pathology [ ] . an epidemic caused by the recently emerged respiratory viral pathogen sars corona virus (scv) attracted worldwide attention because of the high degree of morbidity and mortality it carried. a rhesus macaque monkey model has been developed for this virus, in which intranasal instillation of the pumc strain of scv results in a disease that is similar to the human disease [ ] . li et al. used this system to test sirna as a potential therapy for scv [ ] . sirnas were used to target the scv genome at the spike protein coding region. because the lipid-based transfection reagents pei and transit tko r used as sirna carriers in previous studies might not be acceptable for human treatment owing to potential toxicity, the authors in this study used infasurf r or % d-glucose in water (d w) for sirna delivery. infasurf r (a naturally occurring lung surfactant protein) and d w are nontoxic and are currently in clinical use. d w was - fold more effective than infasurf r in delivering sirna to the lungs. for testing the efficacy in monkeys, mg/kg of sirna was intranasally instilled in ml of d w solution. this treatment substantially reduced clinical symptoms, lung pathology and viral burden. although the inhibitory effect was maximal when the sirna was administered h before or with viral challenge, disease mitigation was seen even when it was given h after viral challenge. thus, this study demonstrated for the first time the considerable antiviral potential of rnai in a non-human primate model using clinically acceptable carriers for sirna delivery. herpes simplex virus sirna has also been used as a potential topical microbiocide [ ] . intravaginal application of an anti-greenfluorescent-protein (gfp) sirna mixed with oligofectamine in gfp-transgenic mice resulted in the loss of gfp expression throughout the vagina and cervix but not in distant organs, such as the liver. importantly, the topical application of sirnas targeting the essential hsv- genes ul (envelope glycoprotein b) and ul (a dna-binding protein) before viral infection reduced mortality by % and substantially reduced viral shedding from the vagina. combinations of sirnas were also effective when administered and h after infection. this study highlights the feasibility of using a similar approach for other sexually transmitted diseases, including hiv- . japanese encephalitis (je) and west nile (wn) viruses can cause a devastating neurological illness. two studies have used rnai to suppress these viruses in mouse models. bai et al. injected a sirna targeting the viral envelope gene hydrodynamically h before an intraperitoneal wnv challenge and observed a % increase in survival [ ] . kumar et al. targeted conserved regions in the viral envelope genes of jev and wnv and administered sirna through a lentiviral vector or as synthetic sirna complexed with the cationic lipid jetsi/dope to enable sirna delivery to neuronal cells [ ] . a single intracranial treatment with lentiviral vector or synthetic sirna was sufficient to provide almost complete protection from fatality. sirna treatment given h after infection was also effective but the treatment failed at later time points. significantly, by targeting a sequence that is highly conserved in jev and wnv, they achieved near-complete protection against fatal encephalitis induced by either jev or wnv. this offers the possibility of using sirna as a broad-spectrum antiviral agent to suppress related viruses across species. foot and mouth disease fmd is a highly contagious and economically devastating disease of domestic animals. chen et al. used a plasmid vector encoding a shrna targeting the viral vp- gene in a suckling mouse model [ ] . the subcutaneous injection of mg of plasmid dna h before fmd challenge resulted in % survival, compared with % mortality in control-dna-injected mice. however, dna vector injection at the time of viral challenge or increasing the challenge virus dose reduced the protection considerably. hiv interest in rnai as an alternative antiviral approach has been particularly strong for hiv- because of the problems of drug resistance, toxicity and the cost of highly active antiretroviral therapy (haart). several investigators have used rnai to suppress hiv in cell lines and primary cells [ ] [ ] [ ] . however, given the specificity of rnai, the propensity of the virus to mutate can pose a serious challenge for therapeutic use. in vitro studies have documented the generation of rnai-escape mutants in long-term cultures [ , ] . nevertheless, recent studies suggest that although the homology requirement for rnai is stringent for the crucial central residues, there is some tolerance for peripheral nucleotide changes [ ] . moreover, because rnai requires only short stretches of homology, it is possible to target one or more regions which are highly conserved because of their essential structural and functional roles. several of these conserved target sites have been identified [ ] [ ] [ ] [ ] . by targeting a highly conserved vif sequence, lee et al. protected cd t cells from all hiv clades, including multiple isolates of clade b, which is prevalent in the west [ ] . host genes important for hiv replication, including the viral receptor cd and the co-receptors ccr and cxcr , have also been targeted to avoid viral escape. synthetic and lentivirally-expressed sirnas have been used to prevent hiv entry by silencing ccr [ , ] . ccr is the favored target because a base pair (bp) deletion of the gene is known to be harmless and confers resistance to hiv infection [ ] . using a combination of sirnas targeting conserved viral sequences and host genes important in the viral life cycle might be the optimal therapeutic approach, akin to antiretroviral drug cocktails. in another study, rnai has been combined with other gene therapy approaches in a single lentiviral vector. the lentivirus was engineered to encode a shrna targeting hiv- rev/tat mrna, a short rna homologous to the viral transactivation response region (tar) to act as a decoy for tat binding, and a ribozyme targeting the host ccr gene. the approach might be a pragmatic way for achieving the stable long-term suppression of hiv replication because each of these therapeutic rnas targets a different gene product and blocks hiv replication by a distinct mechanism [ ] . a novel approach has also been used for targeted sirna delivery to infected t cells. here, a chimeric recombinant protein consisting of a single-chain antibody to hiv- gp fused to the highly positively charged protamine was able to bind to sirna (by charge interaction) and deliver it specifically to hiv-infected cd c t cells [ ] . moreover, when administered with bound anti-tumor sirnas, it specifically targeted and cleared gp expressing experimental tumors in mice. another approach is to generate hiv-resistant progeny t cells and macrophages by transducing hematopoietic stem cells (hsc). the feasibility of this approach was shown in the severe combined immunodeficient (scid) mouse-human chimeric model by transplanting human cd c hematopoietic stem cells transduced with a lentivirus expressing an anti-hiv shrna [ , ] . rnai has already entered phase i clinical trials for macular degeneration and rsv infection. however, there are several hurdles that must be overcome for routine therapeutic use. a significant limitation for its use in viral infections is the natural sequence differences that exist amongst the various serotypes and strains within a given viral species. sequence divergence can also occur because of the accumulation of mutations during viral replication or the active generation of escape mutants [ ] . selecting conserved sequences where the virus is averse to mutate and using a combination of several viral and feasible cellular targets could overcome this limitation. even so, target sequences based on the data available for the few sequenced viruses might not ensure effectiveness against field strains of virus. thus, testing multiple field isolates in vivo might be necessary. although substantial progress has been made, more studies are required to achieve the effective and nontoxic delivery of sirna and shrna in vivo. toxicity can be a problem for in vivo use, particularly when carriers such as transfection reagents are used for delivery. for example, although pei has been used in one study in mice [ ] , several reports suggest that it induces toxic effects that might result in the death of the animals [ , , ] . thus, greater emphasis should be given to developing clinically acceptable carriers for systemic delivery. the encasement of sirna within nontoxic nanoparticles or liposomes might enhance stability and simultaneously avoid toxicity. combining a targeted delivery approach, using antibodies or receptor ligands, the introduction of chemical modifications in the sirna, and the encasement of sirna within nanoparticles or liposomes could be the ideal way to improve systemic delivery and reduce sirna requirements. delivery to the brain is still a challenge because of the blood-brain barrier. however, a recent study used transferrin receptor antibody-coated immunoliposomes to overcome this barrier [ ] . another issue is the induction of interferon and the associated inflammatory effects. in fact, toll-like receptor (tlr) recognition of rna by plasmacytoid dendritic cells seems to be a major mechanism of interferon induction [ ] , and the administration of naked sirna might induce interferon. however, certain motifs within the rna seem to be crucial for interferon induction; avoiding such motifs or encapsulating sirna within nanoparticles could reduce this risk [ , ] . a better understanding of the immunostimulatory motifs within the rna that are needed for tlr activation might help to avoid non-specific immune activation. however, because most viral infections induce interferon and inflammation, this might not be a major limitation for antiviral therapy. the inadvertent targeting of host genes, observed in some in vitro studies [ ] , is another issue that needs more-thorough investigation. although no grossly observable side effects have been reported in vivo, off-target effects might not necessarily manifest as symptoms in animals but could be unacceptable for human therapy. also, because the rnai machinery is used in mammalian cells to regulate cellular gene expression by micrornas (mirnas), the effect of the exogenously introduced sirna on mirna functioning review should be investigated. another concern is the effect of possible subversion of the rnai machinery by viral proteins or virally encoded micrornas (box ). safety is an also an issue in using shrna delivery through viral vectors because of the possibility of insertional mutagenesis and malignant transformation [ ] . another concern is that the effect of long-term sirna induction is unknown. inducible systems for the controlled expression of sirna from plasmid or viral vectors could offer hope in this regard. rnai has tremendous therapeutic potential in viral infections. the major hurdles of delivery to the appropriate cells necessary for its in vivo use as a therapeutic agent are being rapidly overcome and sirna therapy is already being tested in clinical trials. future studies should undoubtedly focus on further refining in vivo delivery methods, and minimizing off-target effects and the emergence of escape mutants in vivo. optimizing sirna design, delivery methods and delivery schedules to achieve sustained sirna levels in the target cells should be emphasized. with these investigations and further advancement in the understanding of the endogenous rnai mechanism, the next few years should prove to be an exciting time to discover whether rnai will become a viable approach to treat viral infections in humans, particularly after the appearance of clinical symptoms. the prospect of silencing disease using rna interference silencing viruses by rna interference rna interference in cancer silencing neurodegenerative disease: bringing rna interference to the clinic mechanisms of gene silencing by double-stranded rna unlocking the potential of the human genome with rna interference passenger-strand cleavage facilitates assembly of sirna into ago -containing rnai enzyme 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mammalian neurons functional sirnas and mirnas exhibit strand bias asymmetry in the assembly of the rnai enzyme complex synthetic dsrna dicer substrates enhance rnai potency and efficacy a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells induction and suppression of rna silencing: insights from viral infections micrornas and viral infection interaction of viruses with the mammalian rna interference pathway a virus-encoded inhibitor that blocks rna interference in mammalian cells induction and suppression of rna silencing by an animal virus three distinct suppressors of rna silencing encoded by a -kb viral rna genome la crosse virus nonstructural protein nss counteracts the effects of short interfering rna evidence that hiv- encodes an sirna and a suppressor of rna silencing silence from within: endogenous sirnas and mirnas micrornas modulate hematopoietic lineage differentiation aberrant t cell differentiation in the absence of dicer identification of virus-encoded micrornas identification of micrornas of the herpesvirus family sv -encoded micrornas regulate viral gene expression and reduce susceptibility to cytotoxic t cells modulation of hepatitis c virus rna abundance by a liver-specific microrna a cellular microrna mediates antiviral defense in human cells regulation of human immunodeficiency virus transcription by nef microrna this work was supported by nih grant u ai to m.n and p.s. key: cord- - w wf authors: vignuzzi, marco; lópez, carolina b. title: defective viral genomes are key drivers of the virus–host interaction date: - - journal: nat microbiol doi: . /s - - -y sha: doc_id: cord_uid: w wf viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. we are beginning to appreciate the surprising versatility of viral genomes and how replication-competent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. this review summarizes current knowledge of the types of defective viral genomes generated during the replication of rna viruses and the functions that they carry out. we highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. v iruses are remarkably resilient microorganisms able to adapt and survive in the complex host physiological and immune environment. accumulating evidence demonstrates that viruses can generate modified forms of their genome during replication as tools to adapt to environmental challenges. while the standard genome encodes all the viral proteins required for sustaining viral replication, viral variants contain random mutations that can serve to enhance the ability of the virus to adapt to new conditions . in addition, defective viral genomes (dvgs) and an expanding family of sub-viral particles (box ) that result from either small mutations or drastic truncations and modifications of the viral genome render the virus unable to complete a full replication cycle in the absence of a helper full-length virus to complement the functions lost. dvgs were identified by preben von magnus in the late s as incomplete influenza viruses able to interfere with the replication of the wild-type virus . more than two decades after their discovery, alice huang and david baltimore coined the term defective interfering (di) particles, or dips, to define viral particles that contain normal structural proteins but only a part of the viral genome. in addition, they stipulated that dips can only replicate in the presence of helper virus and that they interfere with the intracellular replication of non-defective homologous virus . huang and baltimore theorized that dips play a critical role in determining viral pathogenesis. follow-up studies using viruses grown to contain either a large content of dips, or depleted of them, revealed that dips reduced virulence in vivo , , induced high levels of interferon (ifn) during infections in vitro [ ] [ ] [ ] , and promoted viral persistence in vitro [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and in vivo , . however, despite their ubiquitous presence and important functions, the lack of appropriate technology to identify dips in infections in vivo led to a widespread belief that dips and their dvgs were largely a product of in vitro virus replication and were not relevant in natural viral infections , . by the late s, the study of dvgs had slowed down drastically and was limited to their use as tools for studying viral replication or as potential antivirals. today, dvgs have been described in most rna viruses (table ) and technological advances have contributed to establishing their role as de facto danger signals for triggering of antiviral immunity in many infections. in addition, we are beginning to appreciate their impact on the clinical outcome of natural infections and on the evolution of viruses. moreover, we are witnessing rapid advancements in the understanding of the molecular mechanisms that regulate the generation of dvgs and those explaining their paradoxical roles in promoting antiviral immunity and viral persistence. here, we review evidence accumulated over more than half a century of observations on dvg generation and activity during rna virus infections. we highlight recent advances that illustrate their critical impact on viral dynamics and evolution during both acute and long-term virus-host interactions. see box for a glossary of relevant terms. different types of dvgs are defined by the type of genomic alterations present. next generation sequencing (ngs) has revealed a large variety of dvg species present in some infections, and recent studies have begun to elucidate the distinct functions of these different dvgs. point mutations, hypermutations and frame shifts. while the first dvgs to be identified and distinguished from the wild-type fulllength viral genome lacked large parts of the genome [ ] [ ] [ ] [ ] [ ] , there are a number of dvg types that do not involve drastic genomic alterations. point mutations in rna viruses can result in detrimental alterations because of the highly constrained nature of their genome organization. indeed, a majority of randomly introduced mutations are either lethal or confer a significant fitness cost, as observed for vesicular stomatitis virus (vsv) , poliovirus and influenza virus . while it is inherently understood that detrimental mutations give rise to defective genomes, such genome types have historically not been considered dvgs. a genome harbouring a detrimental mutation in a structural protein could replicate, but not assemble properly; while a detrimental mutation in the replicase would yield a genome that can produce proper structural and assembly proteins, but could not replicate. either of these genomes could potentially hijack the lacking functions from (and thus, interfere with) a fulllength co-infecting virus. indeed, studies where mutation rates are increased to perturb a viral population from viability to non-viability, revealed that the defective rnas that appear prior to population marco vignuzzi and carolina b. lópez * viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. we are beginning to appreciate the surprising versatility of viral genomes and how replicationcompetent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. this review summarizes current knowledge of the types of defective viral genomes generated during the replication of rna viruses and the functions that they carry out. we highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. dvg diversity during infection. historically, it was thought that only certain viruses generated dvgs and that only one or a few distinct dvgs existed for any given virus. most studies originally relied on high multiplicity of infection (moi) passaging conditions that favoured the emergence of larger deletions (and thus, smaller dvgs) that could rapidly outcompete the longer full-length genomes owing to reduced replication times. furthermore, these works relied on methods with low sensitivity of detection (such as visualization and purification on agarose gels) that would only isolate the most abundant dvgs. however, evidence for populations of distinct species of dvgs in a single infection has been reported since the s , . we now know the diversity of dvgs to be much larger than initially appreciated and that certain dvgs are better than others at reaching high abundance, likely by retaining certain properties that confer replication advantage such as packaging signals and replication elements, or by acquiring the ability to interfere with the replication of other variants. ngs has revealed that dvgs are present in virtually any and every virus population, and that the diversity of dvgs is immense; however, the relative abundance of any given deletion or rearrangement varies, likely indicating that a variety of factors drive dvg generation and accumulation. the use of single-cell sequencing technology will allow precise quantification of the frequency and diversity of dvgs in an infected cell and will provide data on the distribution of dvg variants among a cell population. since most ngs alignment tools filter out reads with more than two mismatches, reads corresponding to deletion breakpoints or rearrangement junctions are rejected from a typical virus genome alignment. a re-analysis of rejected reads would identify these dvg hallmarks. with growing interest in this neglected part of the viral population, informatics tools such as virema , di-tector and vodka are emerging. these tools re-examine reads that potentially harbour jumbled or rearranged viral sequences and have enabled a broader appreciation of dvg diversity. a current challenge with ngs data is to determine how best to differentiate between true dvgs and background error of ngs, how to quantify the relative abundance of any given dvg with respect to full-length in addition to dvgs, a growing number of viral particle variants and sub-viral agents have been discovered in viruses of plants, arthropods and mammals. these include viroids, satellite viruses, virophages and viral-like extracellular vesicles (vlvs). the definition of these entities seems, at times, blurry, due to their largely intersecting properties. in general, sub-viral agents, similar to dvgs, depend on complementation with the standard virus to replicate and spread. however, differences in their requirements for complementation, target virus and sequence identity with their helper virus are used to sub-classify them. here, we provide current definitions of these sub-viral entities and highlight those aspects that differentiate them from dips and their dvgs. satellite viruses were originally described in plant viruses as linear or circular rnas ( - , nt long) that require a helper virus to propagate but are unrelated in sequence to the helper virus. satellite viruses are generally dispensable for the replication of the helper virus, with some exceptions , . satellite viruses differ from satellite rnas in that they encode a protein that packages the satellite rna into virions. satellite rnas can interfere with the replication of its helper virus and either attenuate or exacerbate disease. an example of a satellite virus is the satellite tobacco necrosis virus . this positive-sense, singlestranded rna satellite virus suppresses the replication of its helper virus and ameliorates tobacco necrosis virus symptoms . viroids differ from satellite viruses in that they do not encode any protein, do not require helper virus for replication, and are not encapsidated. viroids have a circular rna genome ( - nt long) that is highly complementary and structured, and are adapted to carry out their complete life cycle as a result from interactions with the host cell machinery , . virophages are - kbp long dsdna viruses that normally produce icosahedral particles and parasitize from giant dsdna viruses (mimiviruses and others) for propagation. virophages infect the giant virus factory, thereby harming the giant virus . the first virophage discovered, sputnik, was found together with acanthamoeba castellanii mamavirus and interferes with its propagation . several other virophages, as well as a large number of virophage candidates, have been identified since then , , . recent studies show that virophages could resemble bona fide dna viruses and their reclassification as their own viral family has been proposed . vlvs are produced during viral infection and, in contrast to other extracellular vesicles, contain viral proteins and nucleic acids but lack capsid protein or viral genomes and, therefore, are not infectious. vlvs have been described in both rna and dna viruses, including herpes simplex virus- , hepatitis c virus and kaposi's sarcoma-associated herpes virus. vlvs play functional roles during viral infections by facilitating communication among cells and enhancing viral infection [ ] [ ] [ ] . continued virus, and how best to normalize between samples and between sequencing runs-problems that are similar to transcriptome analysis of genetic isoforms. furthermore, with increasing data sets and samples, it is becoming evident that dvg species are not necessarily the same if generated in different host and cell types, and the factors dictating these differences remain to be uncovered. dvgs have been long considered the result of stochastic mistakes introduced by the viral rna polymerase that lacks proofreading activity. however, new evidence suggests that additional factors control dvg generation, opening the possibility of manipulating their generation for therapeutic purposes. random products or encoded in the viral genome? while dvgs are generated by many viruses, the molecular mechanisms that govern their generation are poorly understood. a predominant theory is that dvgs arise from random errors that occur during viral replication at high viral titers due to the combination of a lack of proofreading activity of the viral polymerase and the presence of lower fidelity variants that favour the generation of deletions. in support, analyses using deep sequencing approaches revealed that multiple species of dvgs are generated during infection. for example, in infections with flock house virus, a positive-sense (ps)rna virus, clickseq and nanopore sequencing identified a large and seemingly random population of deletion dvgs early after infection . in addition, sequencing analysis of nasopharyngeal samples from influenza virus-infected humans or infections in vitro with human metapneumovirus or measles virus (mev) revealed multiple dvg species in these infections [ ] [ ] [ ] . however, different from deletion and point mutation dvgs, copy-back dvgs are frequently found in discrete dominant populations in an infected cell or tissue, and the same copy-back dvg seems to arise in independent infections with the same parental virus , or during infections with different virus strains . the demonstration of hotspots for the generation of copyback dvgs from respiratory syncytial virus (rsv) and the identification of specific nucleotides that determine where copy-back dvgs rejoin further demonstrate that the generation of copy-back dvgs is not completely random, but instead that specific sequences encoded in the viral genome direct or facilitate their formation in some infections, dvg generation is not a completely stochastic process and, instead, virus-encoded sequences favour the production and/or amplification of predominant dvgs. it remains to be determined whether conservation is a property of certain dvg types and which specific sequences and/or rna structures lead to dvg generation in these conditions. a number of viral proteins are implicated in the generation of dvgs, the best studied being the viral rdrp. engineered viral polymerases with a decreased fidelity and an increased mutation rate produce highly attenuated viruses , which, in many instances, correlate with enhanced production of dvgs - (fig. a) . although the mechanism mediating dvg generation by mutant rdrp remains speculative, a few models are emerging. for example, in viruses with low-fidelity rdrp, such as sindbis virus or tombusvirus, an increased production of dvgs correlates with an enhanced rate of viral rna recombination , . in addition, during influenza virus infection, variations in the elongation capacity of the polymerase associate with differential generation of dvgs . a role for the polymerase multimerization activity was also recently proposed as a driver of dvg generation during influenza virus infection . viral proteins implicated in the regulation of viral transcription and replication are also associated with the generation of dvgs. mutations in the influenza virus nuclear export protein (nep, also known as ns ), which regulates the synthesis of complementary rna, result in enhanced dvg production . similarly, deletion or mutations of the paramyxovirus c proteins that regulate template switching from antigenomic to genomic replication result in enhanced copy-back dvg production , (fig. a) . curiously, in infections with influenza virus lacking nep or paramyxovirus lacking c, dvg production is observed in conditions that normally restrict the generation of dvgs, such as infections at low moi. it is possible that the enhanced production of copy-back dvgs in these situations is related to higher template availability or to an indirect effect of viral polymerase activity, but the precise mechanism remains to be established. in addition, a single amino acid mutation in the sendai virus (sev) nucleoprotein that reduced the density of the ribonucleprotein associates with dvg generation (fig. b) . lastly, in lymphocytic choriomeningitis mammarenavirus (lcmv) infection, the ppxy domain encoded within the matrix protein drives the production of viral particles containing defective genomes, but not those containing standard genomes (fig. c) . replication-driven template-switching. rna recombination is a major driver of deletion dvg formation. a predominant model proposes that sequences at the break point or structural signals in the template rna promote the replicase to switch to the acceptor rna and resume synthesis (fig. d) . replicase-driven recombination was proven in biochemical assays using rdrps of a large number of rna viruses [ ] [ ] [ ] . variations of this model include the forced template switch mechanism in which the replicase switches template after encountering the ′ end of the template generating headto-tail rna dimers. if the templates are dvgs, these would lead to head-to-tail dvg dimers. the ′ end could also be modified by endo-or exo-nucleases, leading to new versions of these genomes . rna editing as a driver of dvg diversity. editing of viral rna leads to hypermutations and can result in the generation of dvgs, as reported in persistent measles infections . in addition, a high rate of viral adenine-to-guanine (or uracil-to-cytosine) rna editing by adenosine deaminase acting on rna occurs in dvgs from vsv, human metapneumovirus and mev , , . in some cases, rna editing regulates the immunostimulatory potential of dvgs . however, the impact of dvg editing on viral replication seems to be virus-specific . whether dvgs have a higher rate of editing than the standard viral genome, as well as the impact of editing generated diversity in dvg evolution and selection, remains to be determined. dvgs have three well-described functions that relate to their role in pathogenesis: interference with standard viral replication, immunostimulation and establishment of viral persistence (fig. a) . interference with viral replication and viral production. dvgs were discovered during the search for factors responsible for reduced infectivity of influenza virus after passages at high titers . dvgs with the ability to interfere with the replication of their parental virus both in vitro and in vivo are found in most positive and negative-sense (ns)rna viruses , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . dvgs accumulate at higher rates than full-length viral genomes in co-infected cells due to their shortened length and, in the case of copy-back species, their highly efficient flanking trailer promoters , . a predominant theory for how dvgs interfere with the replication of standard virus is based on the observed competition between defective and full-length viral genomes for viral components needed for replication (fig. b) . as dvgs accumulate to high levels, it is predicted that they can directly interfere with helper virus replication by monopolizing the viral polymerase and/or competing for structural proteins , . it should be noted that while most evidence supports the notion that the shortest dvgs with largest deletions are best able to outcompete full-length virus because their much smaller size can be more rapidly replicated, most of these studies consist of high moi cell culture conditions which favour competition dynamics based on replication kinetics. the question arises as to whether a longer deletion dvg that retains more coding sequence, yet contains mutated proteins (presumably replicating faster than full-length genomes, yet slower than the shortest dvgs), could be a better competitor of wild-type virus in certain conditions because it additionally expresses defective proteins that, in turn, interfere with wild-type proteins (for example, in multi-component structures such as capsids or replicases). furthermore, recent studies have demonstrated that dvgs and full-length viral genomes dominate in different cells during infection, dips. defective interfering particles. viral particles containing a fraction of the viral genome able to replicate only in the presence of helper virus and interfere with the intracellular replication of non-defective homologous virus. dvgs. defective viral genomes. viral genomes with defective ability to replicate in the absence of a co-infecting standard virus. viral genomes can become defective due to mutations, deletions or a variety of gene rearrangements. rdrp. rna-dependent rna polymerase. viral enzyme that copies the viral rna during viral replication. tips. therapeutic interfering particles. synthetic dvgs with strong interfering activity proposed as therapeutics to outcompete standard viruses. conferring distinct functions to different infected cells , . while cells dominated with full-length viral genomes are the predominant producers of viral particles containing either full-length or defective genomes, cells enriched in dvgs do not produce many particles of any species . these data highlight the need to consider the single cell versus population level impacts of dvgs during interference. triggers of antiviral immunity. dvgs, especially those of the copy-back type, strongly induce the expression of type i and iii ifns, tumour necrosis factor (tnf), interleukin (il)- , il- β and other pro-inflammatory cytokines, and are the primary stimuli of antiviral immunity in many infections [ ] [ ] [ ] , , , [ ] [ ] [ ] (fig. c) . in addition, dvg stimulation optimizes the antigen presentation capacity of antigen-presenting cells , . accumulating evidence indicates that the immunostimulatory activity of dvgs is maintained in vivo and during natural infections in humans. increased survival of infected mice in infections containing dvgs have been reported for multiple viruses , , . in mice infected with the respiratory viruses sev, influenza or rsv, ifns and pro-inflammatory cytokines are strongly induced only after dvgs have accumulated to detectable levels , . detection of dvgs in respiratory secretions of children infected with rsv correlates with expression of antiviral genes , and highly pathogenic influenza virus isolates that fail to induce potent antiviral responses in humans have an impaired ability to generate dvgs . immunostimulatory dvgs can be recognized by pattern recognition receptors (prrs), including toll-like receptors (tlrs) and rig-i-like receptors (rlrs). while tlr signalling is not essential for production of type i ifns in vitro in response to dvgs, rlr signalling is required , , [ ] [ ] [ ] [ ] [ ] . sev copy-back dvgs are stronger immunostimulators than deletion dvgs and are among the strongest known inducers of the antiviral response. therefore, much of what we know of dvgs immunostimulatory activity is based on the study of sev copy-back dvgs. copy-back dvg rna binds rig-i , , and dvgs from sev, mev and rsv viruses strongly stimulate rig-i-dependent signalling , , . rig-i is triggered by binding of ′ di-or triphosphates present on uncapped rna or short regions of double-stranded (ds)rna. phosphatase treatment suppresses the ability of in vitro-transcribed copy-back dvgs to trigger ifn production following transfection supporting the role of rig-i in dvg sensing , , . sev dvgs can also associate with melanoma differentiation-associated protein (mda ) , , but the role for mda in sensing other dvgs is less clear , . other cellular proteins can also associate with dvgs. for example, mev dvgs associate with the dsrna binding protein named protein activator of the interferon-induced protein kinase to optimally activate rig-i . dvg-induced rlr signalling is not simply a result of higher viral rna content in the infected cells, as increasing the amount of dvg-deficient virus does not increase the ifn response , , . importantly, efficient sensing of copy-back dvgs occurs even in the presence of virus-encoded antagonists of the cellular sensing pathways , . these observations suggest that unique features of dvgs favour their detection during infection. the predicted long dsrna stretch formed by the reverse complementary ends of copyback dvgs was thought to be a critical factor in their immunostimulatory activity , , . however, recent evidence demonstrates the existence of additional features in dvgs that have a larger impact on their immunostimulatory potential. structural modelling identified a nucleotide (nt)-long stem-loop motif (dvg ) in a sev dvg- , a well-characterized and potent immunostimulatory copy-back dvg, which was absent in the sev genome and spans the unique junction formed between the genomic break and rejoin points that form this copy-back dvg. deletion of dvg reduces the dvg immunostimulatory activity, while introduction of the motif into an immunologically inert rna improves its ability to induce the expression of type i ifns and ifn-stimulated genes . dvg acts in concert with the ′-triphosphate motif to activate rig-i and allows for enhanced rlr polymerization, a marker of activation . dvg lose immunostimulatory potential . failure to detect dsrna through immunostaining during sev infection and normal immunostimulatory activity following disruption of the complementarity between the ′ and ′ ends of copy-back sev dvgs further support the notion that the predicted long complementary ends of copy-back dvgs have a minor role in the onset of anti-viral immunity. it remains to be determined when and how dvg - is exposed during infection, and whether similar motifs are present in other highly immunostimulatory dvgs. dvg immunostimulation is also an important factor in the modulation of infections in insects. similar to the rig-i and mda prrs in mammals, insects sense viral rna through the protein dicer- . this protein processes the viral rna into small interfering rnas that protect insects from re-infection with the same virus . dvgs are the primary targets for dicer- (ref. ). these observations indicate that the immunostimulatory activity of dvgs is widespread and may have a significant impact on the spread of pathogens within and across species. , , as well as one study reporting dvgs in the brain of human patients who had died due to post-mev subacute sclerosing panencephalitis . dips and full-length viruses cycle asynchronously in many persistent infections in vitro , and in vivo , . cycling occurs in a predictable pattern and has been mathematically modelled using variations of the predator-prey model . a theory to explain the asynchronous cycling of full-length viral genomes and dvgs during persistency was put forward in (ref. ). this theory, which is based on the interference effect of dvgs on the replication of the full-length genome, proposed the following: dips accumulate during virus replication until they reach high concentrations and become predominant. in this condition, dvgs interfere with the replication of the full-length viral genome by competing for essential replication machinery, driving a reduction in the full-length virus. during this process, some previously uninfected cells are infected by standard virus and re-initiate the cycle. interestingly, in some persistent infections the amount of dips appears constant . what drives these cyclic patterns in some viruses but not others, and whether host factors such as the infected cell type influences the cycling pattern, remain unknown. recent evidence indicates that the mechanisms involved in the establishment of persistence are more complex than simple intracellular competition for the replication machinery among different types of viral genomes . using rna in situ fluorescent hybridization, xu et al. showed that during infection with sev or rsv containing dips, there is heterogeneity in the content of viral genomes in the infected population . while some cells are enriched in dvgs, others are enriched in standard full-length viral genomes. the mechanisms for this heterogeneity are currently unknown, however, striking functional differences among these cell populations are beginning to emerge , . cells enriched in dvgs engage the rlr sensing pathway and produce ifns and other pro-inflammatory molecules, including tnf. in addition, these cells induce a pro-survival program, also dependent on signalling through the rlr pathway. these programs protect dvg-high cells from tnfmediated death, while cells lacking dvgs die during infection. surviving dvg-high cells can be propagated for months as a persistent infection. this mechanism provides an explanation for the paradoxical stimulation of both antiviral immunity and establishment of persistence by dvgs. it remains unclear how the enhanced survival of dvg-high cells leads to persistence, and how this survival fits into the cycling of dvg and standard virus observed in many infections. cycling may occur at the intracellular level (where each infected cell goes through cycles of standard viral genome or dvg enrichment driven by competition and interference with the viral replication machinery) and/or at the population level, where the strong interfering and immunostimulatory activities of dvgs make them attractive candidates for vaccine adjuvants and antivirals , . the ability of dips to interfere with the replication of standard viruses and diminish virus-associated disease has been extensively demonstrated in mice during infection with virus stocks containing dips , , , , , even when the vaccine contained standard virus inactivated by ultraviolet treatment . a similar protection has been reported in ferrets vaccinated with an influenza vaccine containing only dips . synthetically engineered dips with strong interfering potential, or 'therapeutic interfering particles (tips)' , have been more recently proposed as a possible strategy to control viral infections. tips would theoretically replicate faster than the wild-type virus and therefore outcompete the virus hindering spread and transmission. tips would have the advantage of being active only in organisms already infected with the wild-type virus due to their dependence on a helper virus to replicate , . although still in the exploratory phase, it remains to be determined how tips would impact virus persistence, the generation of adaptive mutations and the generation of new infectious viruses by complementation. whether protection elicited by natural or synthetic dips is due to direct interference with the standard virus replication or through strong immunostimulatory ability of dvgs is unclear. sev copyback dvgs augment the antigen presentation capacity of mouse and human dendritic cells, resulting in enhanced activation of t cells . in addition, experimental vaccines against influenza virus and rsv adjuvanted with in vitro-transcribed sev dvgs delivered subcutaneously, intramuscularly or intranasally show improved antibody production and increased protection from virus challenge , . sev dvg-derived oligonucleotides (ddos) containing the immunostimulatory motif dvg are effective adjuvants able to bias the humoral and cellular responses against inactivated virus and protein vaccines towards type i immune responses including antibodies of the igg a/c isotypes, th cd + t cells and cytotoxic cd + t cells in mice , . ddos also synergize with the emulsion antibody addavax and enhance its type i immunity-driving potential by mechanisms that depends on type i ifns . notably, a dip influenza vaccine conferred protection to an unrelated virus through stimulation of type i ifn production , suggesting that they can be used as prophylactic or therapeutic antivirals. dvgs are present in live attenuated vaccines against polio, measles and influenza viruses , [ ] [ ] [ ] [ ] . however, their impact in the development of protective immunity and vaccine efficacy has not been formally assessed. based on their interfering and immunostimulatory ability, it is speculated that dvgs may enhance the efficiency of the vaccine while enhancing safety of the virus by reducing its replication and spread. if correct, it would be important to carefully regulate the amount of dvgs in vaccine preparations to avoid complete interference and drastic reduction of the virus to a point of ineffectiveness. while recent ngs data reveal that hundreds of dvgs can arise within a single viral infection, the fact that a smaller subset of dominant dvgs are repeatedly detected in different samples indicates that complex dynamics are at play within the viral population. these complexities include competition (and possibly compensation or cooperation) between different dvgs and positive selection of the best competitors that implicates their relative fitness in relation to the wild-type parental virus and other dvgs. parameters such as replication fitness, packaging, immunoregulation and other traits determine the virus dynamics. it is important to stress that most events leading to dvg formation, including mutations, deletions, recombination and translocations, are either non-viable or deleterious to the virus. in addition, although hundreds or even thousands of different dvgs are generated during a virus infection, the vast majority of these will be lost during the population bottlenecks that occur in vivo, for example when crossing anatomical barriers or during transmission from host to host . however, there are instances where these genomes could make it through bottlenecks, such as during infections of hosts that are immunosuppressed or have comorbidities where founding populations are increased. in addition, infections may occur with virions that have co-packaged wild-type genomes and dvgs or virions may aggregate during infection enhancing co-transmission, as seen for vsv mathematical models have generally only considered one dominant dvg , ; further development is required to incorporate the potential cooperation and competition among others. while historically dvgs have been considered replication waste, an artifact of cell culture passaging conditions or simply a nuisance to laboratory experimentation, a renewed interest in this part of the viral population may reveal a more functional or biological relevance to their existence. do dvgs exist for a reason? why keep waste lying around? given the notoriously high recombination and reassortment rates of some viruses, it is possible that for viruses that undergo recombination, dvgs can provide a repertoire of mutations that could feed back into the viable virus population to promote adaptation. it remains to be seen whether dvgs can provide a selective advantage to the virus, and whether the high moi or localized co-infection conditions are biologically relevant outside of cell culture. recent work in insects suggests that dvgs, which are a preferred template for the generation of the viral dna form of rna viruses, provide additional substrate to help boost the rna interference response that is responsible for viral persistence in insects . indeed, it was shown that a change in the amount of viral dna generated during rna virus infection in drosophila, altered the persistence and kinetics of a wild-type rna virus infection. the authors suggested that evolution has perhaps fine-tuned the production of dvgs to balance wild-type infection and promote persistence (and ultimately, transmission of viruses, including arboviruses in mosquitoes). additionally, a closer examination of dvg dynamics within viral populations can help better understand the biology of standard viruses. indeed, in addition to distinguishing between the dispensable and indispensable nucleotide sequences, proteins and rna structures, we can identify what viral components can operate in cis or in trans. a recent study of hcv dvgs, for example, identified novel cis-acting rna structures that were required for replication and packaging . emerging technologies that allow the identification of defective and standard viruses in natural infections, as well as those allowing the establishment of specific associations of dvgs with functional outcomes, have been crucial in reviving the interest in studying dvgs. recent work has provided new appreciation for dvg diversity and the potentially critical role of dvgs in defining the clinical outcome of infections; however, a number of questions remain unanswered: what are the molecular mechanisms driving the generation of dvgs? can dvgs be harnessed for the control of virus pathogenesis and spread? how do alterations in the dvg population impact virus evolution and adaptation to new hosts? how do host factors impact dvg accumulation and activity? further technological developments and interdisciplinary research will be required to 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a sting-dependent manner this work was supported by the us national institutes of health national institute of allergy and infectious diseases (grants nos. nih ai , ai and ai to c.b.l.) and the darpa intercept program (to m.v.) managed by j. gimlett and administered though darpa cooperative agreement (grant no. hr - - - ). in addition, this work was supported by a fulbright us scholar award to c.b.l. the views expressed in this article do not necessarily represent the position or the policy of the us government the authors declare no competing interests. reprints and permissions information is available at www.nature.com/reprints.correspondence should be addressed to c.b.l.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -rr zue l authors: cifuentes-munoz, nicolas; el najjar, farah; dutch, rebecca ellis title: viral cell-to-cell spread: conventional and non-conventional ways date: - - journal: adv virus res doi: . /bs.aivir. . . sha: doc_id: cord_uid: rr zue l a critical step in the life cycle of a virus is spread to a new target cell, which generally involves the release of new viral particles from the infected cell which can then initiate infection in the next target cell. while cell-free viral particles released into the extracellular environment are necessary for long distance spread, there are disadvantages to this mechanism. these include the presence of immune system components, the low success rate of infection by single particles, and the relative fragility of viral particles in the environment. several mechanisms of direct cell-to-cell spread have been reported for animal viruses which would avoid the issues associated with cell-free particles. a number of viruses can utilize several different mechanisms of direct cell-to-cell spread, but our understanding of the differential usage by these pathogens is modest. although the mechanisms of cell-to-cell spread differ among viruses, there is a common exploitation of key pathways and components of the cellular cytoskeleton. remarkably, some of the viral mechanisms of cell-to-cell spread are surprisingly similar to those used by bacteria. here we summarize the current knowledge of the conventional and non-conventional mechanisms of viral spread, the common methods used to detect viral spread, and the impact that these mechanisms can have on viral pathogenesis. viruses are intracellular parasites that have coexisted with cells from ancient times. viruses hijack living cells and redirect many cellular processes to allow for efficient viral protein synthesis and replication. the success of the virus depends on the spread of some of these copies to new target cells to initiate a de novo infection. many animal viruses have evolved more than one mechanism of viral spread, revealing a previously unexpected heterogeneity. remarkably, some of these mechanisms allow the transfer of viral particles or components directly from cell-to-cell, without the need for virus release. cell-to-cell spread has important advantages over the well-studied mechanism of spread by diffusion of cell-free viral particles. to achieve direct cell-to-cell spread, some viruses induce a dramatic remodeling of cell architecture to reach uninfected cells and transfer viral particles or components. it is critical for our understanding of viral propagation to comprehend what circumstances promote the use of one mechanism of spread over others, and whether different mechanisms can operate simultaneously at different stages of the infectious cycle. in this review we have focused on the current knowledge of different mechanisms of cell-to-cell spread in animal viruses. release of viral particles into the extracellular space and the subsequent re-entry of these particles into new target cells is one of the best described and understood mechanisms of virus spread. the release of cell-free particles is critical for viral spread between distant cells, or for spread between hosts. virions released from a cell must interact with molecules at the cell surface of a new target cell as the first step to enter and initiate an infectious cycle. these molecules include receptors, co-receptors, and attachment factors. cell-free viral particles can be released into the extracellular space through different mechanisms, such as: (a) cell lysis induced by viral proteins, as is the case for many non-enveloped viruses such as reoviruses, rotaviruses, adenoviruses and picornaviruses (giorda and hebert, ; hu et al., ; nieva et al., ) ; (b) by budding directly from the plasma membrane, where virions acquire their envelope, as is the case of human immunodeficiency virus (hiv- ), influenza, paramyxoviruses, and pneumoviruses (lorizate and krausslich, ; votteler and sundquist, ; weissenhorn et al., ) ; (c) by exocytosis of intracellularly assembled viral particles, as is the case for bunyaviruses, flaviviruses and coronaviruses (cifuentes-munoz et al., ; lorizate and krausslich, ) . once released, different environmental and host factors affect the stability of viral particles and therefore their ability to initiate a new infection. many of the viruses that can be released as cell-free virions can use additional mechanisms of spread that are summarized below. vaccinia virus (vacv) replication occurs in cytosolic factories, and involves the formation of two forms of infectious virus, the intracellular mature virus (imv) and extracellular enveloped virus (eev) (fig. ) . these two forms differ not only antigenically and structurally, but also in the way they exit the cell (smith and law, ) . the majority of imv particles are released from cells after lysis. late in infection, imv particles can also exit the cell through budding, but the significance of this mechanism is not completely understood. budding from the plasma membrane, together with exocytosis, are the proposed ways that eev exits cells. however, an important role of the actin cytoskeleton in vacv assembly was identified after early observations using high-voltage electron microscopy (stokes, ) . in infected cells, microvilli containing enveloped vacv particles at their tips were observed at the cell periphery (stokes, ) . the formation of the microvilli was shown to be sensitive to cytochalasin b but not to nocodazole, suggesting actin polymerization could play a role in their fig. formation of actin tails. vaccinia virus (vacv) can spread from cell-to-cell through different mechanisms. in vacv infected cells, intracellular enveloped particles are transported to budding sites, where the outer viral membrane fuses with the plasma membrane (a). extracellular enveloped viral particles remain attached to the plasma membrane and several cellular factors are recruited to the site through a cascade of events initiated by phosphorylation of the a r protein cytosolic tail (b). polymerization of f-actin underneath the plasma membrane occurs through activation of the cellular pathways n-wasp/arp / and fhod /rac , leading to elongation of actin tails (c). viral particles can thus reach adjacent cells for rapid direct cell-tocell spread. stabilization. the term actin tails was proposed for vacv-induced microvilli, due to their resemblance to actin tails formed in bacterial infections with listeria, shigella or rickettsia (cudmore et al., ; welch and way, ) . actin tails are initially observed at the interior of the cell, but as the infection progresses they project from the cell surface up to μm. enveloped virions located at the tip of actin tails were shown to project toward uninfected cells for direct cell-to-cell spread ( fig. ) (cudmore et al., ) . soon after the discovery of actin tails, it was demonstrated that phosphorylation of the viral protein a r by src and abl family kinases was essential for the actin-based motility of vaccinia (frischknecht et al., a,b; newsome et al., ) . phosphorylated a r recruits the adaptor proteins nck and grb and the downstream effector n-wasp (wiscott-aldrich syndrome protein) together with the wasp interacting protein (wip) (donnelly et al., ; frischknecht et al., b; scaplehorn et al., ) . n-wasp can stimulate the actin-nucleating activity of the arp / complex (taylor et al., ) . in addition, activation of the formin fhod by the small gtpase rac was shown to stimulate vaccinia virus-induced actin tail initiation and elongation, a mechanism independent of the n-wasp-arp / pathway (alvarez and agaisse, ) (fig. ) . two isoforms of cytoplasmic actin, β and γ-actin, are present in actin tails, but only β-actin was found to be involved in actin nucleation induced by vacv (marzook et al., ) . therefore, the signaling pathway initiated through phosphorylation of vacv a r to induce actin tail formation for direct cell-to-cell spread was found to be an elegant mimic of cellular pathways. more recently, it was shown that two vacv proteins, a and a , are necessary and sufficient to induce actin tail formation. the early expression of both proteins was shown to be crucial for rapid spread and repulsion of virions to neighboring uninfected cells (doceul et al., ) . additional cellular factors such as clathrin and the clathrin adaptor protein ap- enhance actin-based motility of vaccinia. clathrin and ap- are recruited by the extracellular virus during its egress to promote clustering of a , thus potentiating actin-based motility and spread of the infection (humphries et al., ) . casein kinase (ck ) also enhances actin tail formation of vaccinia virus, potentially through direct phosphorylation of a and the recruitment of phosphorylated src (alvarez and agaisse, ) . though actin tail formation has been explored in detail for vaccinia virus, the sequence homology of key viral proteins and additional evidence suggest that actin tail formation is a common strategy for orthopoxviruses (duncan et al., ; newsome and marzook, ; reeves et al., ; welch and way, ) . the fusion of membranes from adjacent cells results in multi-nucleated giant cells, also called syncytia. infection with different viruses including paramyxoviruses (takeuchi et al., ) , pneumoviruses (hamelin et al., ; neilson and yunis, ; vargas et al., ) , herpesviruses (cole and grose, ) and retroviruses (nardacci et al., ) results in syncytia formation in vitro and in vivo. cells infected with these viruses display a high concentration of viral fusion protein at the plasma membrane which can promote membrane fusion with neighboring cells. specific fusion proteins have different requirements for triggering the conformational changes needed to drive fusion, such as exposure to low ph or the presence of receptors which bind the fusion protein or an associated receptor binding protein (hernandez et al., ; kielian, ) . if the needed factors are present, the fusion protein can be activated to promote fusion between the membrane of the infected cell and the membrane of the target cell. the process of membrane fusion driven by viral fusion proteins involves several sequential steps, including: (a) activation of the fusion protein, which exposes a fusion peptide (fp); (b) insertion of the fp into the adjacent membrane; (c) clustering of fusion proteins at the site of fusion; (d) refolding of the fusion protein, which pushes together both adjacent membranes; (e) hemifusion, where only the outer leaflets of the membranes merge; (e) formation of a small fusion pore, and (f ) enlargement of the fusion pore until complete merging of both membranes (fig. ) (hernandez et al., ; kielian, ; lorizate and krausslich, ) . iterations of this process generate massive syncytia containing numerous nuclei with the complete mixing of cytoplasmic material, which allows propagation of infection rapidly without the need for assembly of complete virions. large rearrangements of the actin cytoskeleton are induced during cell fusion and syncytia formation. these rearrangements are regulated by the action of rho gtpases cdc , rac and rhoa (eitzen, ) . for example, it has been demonstrated that hiv- env-coreceptor interactions activate the gtpase rac , which promotes actin rearrangements necessary for membrane fusion (pontow et al., ) . for the paramyxoviruses hendra virus and parainfluenza virus type (piv ), constitutively active rac and cdc increased cell-to-cell fusion by their respective viral glycoproteins, as shown by quantitative reporter gene fusion assays. in contrast, constitutively active rhoa decreased hendra virus fusion protein (f)-mediated fusion and had minor effects on piv cell-to-cell fusion (schowalter et al., ) . cell-to-cell fusion mediated by the respiratory syncytial virus (rsv) fusion protein was shown to require rhoa signaling (gower et al., ) . interestingly, a recent report correlated a rsv fusion protein with a hyperfusogenic phenotype with increased pathogenesis in mice (hotard et al., ) . hypersyncytial viral strains (syn) have been extensively described for α-herpesviruses (ambrosini and enquist, ) . mutations associated with the syn phenotype have been mapped to viral genes encoding the glycoproteins, with a predominance in the gb and gk genes (ambrosini and enquist, ; bond et al., ) . early and recent reports have associated the hyperfusogenic strains of α-herpesviruses with increased electrical activity in infected neurons (mayer et al., ; mccarthy et al., ) . additionally, syncytia formed between neurons and satellite cells during varicella-zoster virus (vzv) infection have been suggested to contribute to neuropathogenesis (reichelt et al., ) . initially, the fusion protein is in an inactive prefusion state; (b) upon the proper stimulus (such as binding a receptor shown in purple on the target cell), the fusion protein is triggered, which induces conformational changes, insertion of a fusion peptide into the adjacent membrane, and conformational changes that bring both membranes, viral and cellular into close proximity, followed by a partial merging known as hemifusion (c). full merger of both membranes results in the opening of a fusion pore (d) that expands allowing the mixing of cytoplasmic material and spread of viral components between cells (e). iterations of the fusion process result in formation of large multinucleated syncytia. in contrast, syncytia formation by vzv mutant hyperfusogenic glycoproteins might be detrimental, rather than beneficial, for viral spread and pathogenesis in skin cells (yang et al., ) . whether syncytia formation contributes to pathogenicity of other viruses such as hiv- is still a matter of controversy (compton and schwartz, ) . remarkably, while viral fusion proteins are well studied in enveloped viruses, another type of viral fusogen that promotes syncytia formation has been described for the non-enveloped orthoreoviruses (duncan, ; shmulevitz and duncan, ) . this family of non-structural viral fusion proteins, named fusionassociated small transmembrane (fast) proteins, are unusual in that they do not mediate viral entry but rather fusion between cells using combinations of membrane effector motifs. cell-to-cell fusion mediated by the reptilian orthoreovirus p fusogen is accomplished by recruitment of the src kinase, the grb adaptor protein and n-wasp, thus initiating branched actin assembly (chan et al., ) . the factors recruited by the reptilian orthoreovirus p for cell-to-cell fusion are thus remarkably similar to those recruited by vaccinia virus to induce actin tail formation, as described above. despite their role in orthoreovirus cell-to-cell spread, syncytia formation is not essential for virus replication or progeny production of orthoreoviruses in vitro (duncan et al., ) . however, the maintenance of these proteins across reovirus species strongly suggests they provide a competitive advantage to the virus (duncan, ). cell junctions are structures composed by several different transmembrane proteins, whose main function is to form a seal between polarized epithelial cells. adhesion between epithelial cells is achieved by three main types of seals: tight junctions (tj), adherens junctions (aj) and desmosomes. these barriers cannot be penetrated by viruses unless there is substantial damage to the epithelia . however, epithelial cells can be infected from the apical or basolateral sides, with viral particles then transmitted to adjacent cells through specialized sites located in the vicinity of cellular junctions that are at least partially inaccessible from the extracellular environment. for example, in addition to cell-free spread, hepatitis c virus (hcv) can spread from cell-to-cell in a neutralizing antibodyindependent manner (brimacombe et al., ; timpe et al., ) . claudin (cldn ) and occludin (ocln), components of tight junctions, are critical for cell-to-cell spread of hcv (brimacombe et al., ; timpe et al., ) . in addition, the cellular receptor cd , scavenger receptor bi (sr-bi), apolipoprotein e, and low density lipoprotein receptor (ldlr) were shown to have a role in the cell-to-cell, but not cell-free, spread mechanism of hcv (brimacombe et al., ; fan et al., ; timpe et al., ) . direct transmission of assembled hcv particles was proposed to occur in cell-cell contacts across partially sealed cell junctions (brimacombe et al., ) . robust evidence shows the use of adherens junctions by herpesviruses to spread from cell-to-cell ( johnson and baines, ; johnson and huber, ) . herpes simplex virus type (hsv- ) particles have been shown to be preferentially sorted towards cell junctions rather than to the apical surface of polarized epithelial cells ( johnson et al., ) . the transport of particles towards these sites is primarily directed by the glycoprotein complex gi/ge, with the cytosolic domain of ge having a critical role (dingwell et al., ; dingwell and johnson, ; farnsworth and johnson, ) . indeed, mutant viruses lacking gi/ge grow poorly in monolayers of human fibroblasts, and form plaques of small size (dingwell et al., ) . this phenotype was corroborated in vivo, as the △gi/ge mutant viruses produced small, punctate lesions in the corneal epithelium of rabbits and mice (dingwell et al., ) . the cell-to-cell spread mediated by gi/ge was found to be independent of neutralizing antibodies, suggesting that it occurs at sites of cellular junctions that are not accessible to these molecules (dingwell et al., ) . immunofluorescent staining showed that gi/ge colocalized with the adherens junction protein β-catenin, but not with zo- , a component of tight junctions (dingwell and johnson, ) . in addition, nectin- , a component of adherens junctions, has been reported as a receptor for the herpes simplex virus glycoprotein d (sakisaka et al., ; yoon and spear, ; zhang et al., ) . in this context, one accepted model is that gi/ge complexes accumulate at trans-golgi network (tgn) subdomains, from where nascent virions are sorted to basolateral cell junctions for cell-to-cell spread (farnsworth and johnson, ; johnson and baines, ) . at these sites, release of particles and their subsequent entry into the adjacent cell occur almost simultaneously. however, the critical function of gi/ge complexes extends beyond the spread between epithelial cells, and their role, together with pus , in the anterograde transport (from cell body to axons) of hsv- particles has been extensively studied (howard et al., ; kratchmarov et al., ; mcgraw and friedman, ; snyder et al., ) . remarkably, the complex gi/ge together with pus promote anterograde transport of enveloped particles, nucleocapsids, and glycoproteins through kinesin-mediated vesicular transport using microtubules (diwaker et al., ; johnson and baines, ; kratchmarov et al., ) . the role of the gi/ge complex and us for cell-to-cell spread has additionally been reported for other herpesviruses including pseudorabies and vzv (cohen and nguyen, ; diwaker et al., ; johnson and huber, ; lyman et al., ; zsak et al., ) . the use of desmosomes for cell-to-cell spread has been studied less than spread at the other cell junctions described above. however, some reports describe an important role of keratin in infection by lymphocytic choriomeningitis virus (lcmv) (labudova et al., ) (labudova et al., ) . keratin filaments are an important component of desmosomes, and the lcmv nucleoprotein was shown to bind and stabilize keratin (labudova et al., ). this interaction resulted in increased desmosome formation and cell-cell adhesion capacity, facilitating cell-to-cell spread of lcmv (labudova et al., ). formation of virological synapses (vs) is a mechanism of cell-to-cell spread that has been shown to be used by retroviruses (alvarez et al., ; dupont and sattentau, ; . in the vs, several cellular factors, including cytoskeletal proteins, receptors, and adhesion molecules, are recruited to sites where virus particles can be transmitted quickly from an infected cell to a target cell. in lymphocytes infected with human t-cell leukemia virus type (htlv- ), env is distributed evenly at the cell surface and clusters of gag are observed close to the plasma membrane (igakura et al., ) . upon contact with uninfected t-cells, a rapid recruitment of a large number of env, gag and viral rna molecules at cell-cell junctions is observed. subsequently, transfer of gag molecules and viral rna is detected from the infected t cell to the uninfected t cell, likely utilizing a mechanism in which viral particles are rapidly released by the infected cell and taken up by the target cell in the junction region (igakura et al., ) . the cellular adhesion molecule talin was frequently found at sites of cell-cell contact, and an important reorientation of the microtubule organizing center (mtoc) to cell-cell contact adjacent sites was shown to be critical for transport of gag molecules. the polarization of the mtoc towards sites of cell-cell contact by htlv- is triggered by the transcriptional transactivator viral protein tax (nejmeddine et al., ) . polarization of the mtoc/tax was shown to depend on intact microtubules, actin filaments, and the function of rac and cdc small gtpases (nejmeddine et al., ) . further evidence indicates that the engagement of the adhesion molecule icam- on the surface of htlv- infected cells is sufficient to trigger the intracellular polarization of the mtoc . analogous observations are seen in cells infected with hiv- . dendritic cells (dc) infected with hiv- rapidly recruit virus to cell junctions formed between dcs and t lymphocytes (mcdonald et al., ) . remarkably, the un-infected t cells recruit receptor and co-receptor molecules including cd , ccr and cxcr at the vs, which facilitates hiv transmission from cell-to-cell (mcdonald et al., ) . similar to htlv- , in hiv- -infected t cells (effector cells) env and gag are quickly recruited to the vs (do et al., ; . polarization of the mtoc and associated organelles towards the vs is induced in hiv-infected lymphocytes and dcs to hijack components of the secretory pathway (bayliss et al., ; jolly et al., ) . in adjacent uninfected t cd + cells, an actin-dependent recruitment of cd , cxcr and lymphocyte function-associated antigen (lfa- ) was found to facilitate spread of gag molecules between cells . quantitative d live microscopy revealed "buttons" of oligomerized hiv- gag at sites of cell-cell contact, which were shown to be the preferential route of infection between cd -t cells (hubner et al., ) . thus, the proposed model of cell-to-cell infection involves budding of viral particles into the synaptic cleft and subsequent fusion of viral particles with the adjacent uninfected cell (hubner et al., ; miyauchi et al., ) . remarkably, it has been shown that hiv cell-to-cell spread can occur simultaneously from one infected lymphocyte to multiple adjacent recipient cells, a process named polysynapse formation (rudnicka et al., ). moreover, cell-to-cell transmission of retroviruses through the vs has been shown to play a critical role in viral dissemination in vivo (murooka et al., ; sewald et al., ) . in this context, studies that examined the accessibility of the vs to entry inhibitors have concluded that synapse-mediated spread is less sensitive than cell-free particle spread to neutralizing antibodies in vitro and vivo (abela et al., ; gombos et al., ; smith and derdeyn, ; zhong et al., ) . cell-to-cell contact sites have additionally been described as the main route of viral dissemination for the murine leukemia virus (mlv) by using live-cell imaging ( jin et al., ) . interestingly, a mechanism that resembles the vs was reported for spread of the nonlymphotropic hsv- (aubert et al., ) . infection of cd + and cd + t lymphocytes by hsv- was shown to occur in vivo. further, infection was more efficient by using cell-to-cell contacts than by cell-free virus, in a manner independent of the gi/ge glycoprotein complex (aubert et al., ). whether or not other non-lymphotropic viruses can establish vss warrants future research. htlv- cell-to-cell transmission does not only rely on the formation of vss. lymphocytes infected with htlv- have also been shown to store viral particles in an unconventional way, as clusters located on the surface of t cd + lymphocytes . these clusters correspond to viral assembly sites and are enriched in carbohydrates and extracellular matrix components including collagen and agrin, a heparan sulfate proteoglycan (pais-correia et al., ; thoulouze and alcover, ) . high-level expression of the host cell actin-bundling protein fascin is induced by htlv- tax in infected t-cells (kress et al., ) . fascin contributes to cell-to-cell htlv transmission, and has been suggested to be involved in transport and fitting gag into viral biofilms (gross et al., ) . remarkably, the clusters are transferred from infected cells to recipient cells during cell contact, and it was estimated that % of the infectious capacity of htlv- comes from these structures (pais-correia et al., ; thoulouze and alcover, ) . the mechanism shares several similarities with bacterial biofilms, and both improved spread and escape from the immune response might be advantages of this mechanism of viral cell-to-cell spread. tunneling nanotubes (tnts) were first described in as a new mechanism of cell-to-cell communication (rustom et al., ) . they are thin, elongated membrane-bound structures, between and nm in diameter, that connect two distant cells and allow the transfer of material including organelles, ions, proteins and mirnas. a very recent complete description of nanotube properties and their role in viral spread has been reported by jansens et al. ( jansens et al., ) . based on their diameter and composition, two classes of tnts are reported in macrophages: "thin tnts" that contain filamentous actin (f-actin) and "thick tnts" (greater than . μm) that contain f-actin and microtubules (onfelt et al., ) . organelles such as mitochondria, lysosomes and peroxisomes were detected in thick, but not in thin, tnts. interestingly, bacteria could "surf" along the surface of thin nanotubes using a constitutive flow (onfelt et al., ) . viruses can also exploit nanotubes for cell-to-cell spread, and this was first demonstrated for hiv (sowinski et al., ) . tunneling nanotubes containing f-actin were shown to connect distant t cells, to have a close-ended nature and not to be tethered to the substratum, in contrast to what is seen for filopodia. rapid spread of hiv particles through tnts was found to be receptor-dependent (sowinski et al., ) . the induction of tnts by hiv was additionally demonstrated in macrophages (eugenin et al., ) . interestingly, the authors identified three different processes in uninfected and hiv-infected macrophages: long tnts, short tnts and filopodia, based on intercellular communication and length. the authors reporter that tnts, but not filopodia, are open-ended and longer than filopodia, with lengths up to μm (eugenin et al., ) . another retrovirus, htlv- , was shown to form conduits in t cells that share characteristics of tnts (van prooyen et al., ) . induction of these conduits was shown to depend on the viral p protein and to be facilitated by the cellular factors icam- and lfa- (van prooyen et al., ) . further, the induction of tnts in infected cells was recently reported for influenza. influenza virus nucleocapsids were observed in long intercellular structures, likely tnts formed between lung epithelial cells (kumar et al., ; roberts et al., ) . direct transfer of influenza virus genomes and proteins was shown to require f-actin and actin dynamics. the process was also shown to be unaffected by the presence of neutralizing antibodies or oseltamivir, suggesting an open-ended nature of these tnts (kumar et al., ) . similar observations were made in cells infected with porcine reproductive and respiratory syndrome virus (prrsv) (guo et al., ) . infection by prrsv induces the formation of tnts that contain f-actin and myosin ii-a, a motor-binding protein. viral proteins and rna were observed through the length of tnts, and it was shown that viral proteins might interact directly or indirectly with myosin ii-a. the presence of neutralizing antibodies does not prevent cell-to-cell transmission of a fluorescently labeled prrsv and spread could occur in absence of cellular receptors, suggesting either a direct cytosolic connection between cells through tnts or transfer through sites of close-contact provided by the tnts that are inaccessible to antibodies (guo et al., ) . exploitation of tnts was additionally reported for pseudorabies virus ( jansens et al., ) . formation of tnts capable of intercellular spread of molecules was achieved by the expression of the viral protein us in the absence of other viral proteins (favoreel et al., ; jansens et al., ) . the tnts were associated with enhanced viral intercellular spread and were very stable. this stability was suggested to be the result of the presence of stabilized microtubules and an enrichment of the adherens junctions components β-catenin and e-cadherin at the site of contact between the tnt and the target cell. in this scenario, enveloped virions were shown to be transported by vesicles through the tnt and released by exocytosis from this structure at sites of contact between the tnt and the acceptor cells (favoreel et al., ; jansens et al., ) . recently, the induction of long intercellular connections has been reported upon infection by dna viruses, including vacv and the bovine herpesvirus (bohv- ) (panasiuk et al., ; xiao et al., ) . the lack of adherence to substratum, their open-ended nature, length of up to μm, and presence of f-actin and tubulin led to the proposal that the bohv- -induced structures correspond to tnts. viral proteins were detected along the length of the tnts, and direct transfer of recombinant bohv- in a neutralizing antibody independent manner was observed, similar to what was reported for rna viruses (panasiuk et al., ) . filopodia are cellular structures involved in a wide array of functions including cell migration, wound healing, adhesion to the extracellular matrix and cell-cell interactions (faix and rottner, ; mattila and lappalainen, ) . their formation is induced through actin polymerization underneath the plasma membrane, which generates finger-like extensions that range between . and . μm in diameter. filopodia are filled with parallel bundles of f-actin (mattila and lappalainen, ) . several signaling pathways drive the protrusion of filopodia, including members of the family of rho gtpases like cdc (faix and rottner, ; mattila and lappalainen, ; nobes and hall, ) . because of the heterogeneous nature of the previously described tnts, it can be challenging to differentiate between tnts and filopodia. indeed, it was proposed that tnts arise from filopodium-like protrusions that connect to neighboring cells (rustom et al., ) . a more recent report suggests that two filopodial connections can interact with each other through n-cadherin, forming a bridge (chang et al., ) . rotation of the actin filaments inside these bridges by myosin proteins separates both filopodia, with one of them reaching a cell body and establishing a stable tnt-like connection via n-cadherin/β-catenin clustering (chang et al., ) . moreover, it has been shown that the cdc /irsp /vasp network that is upregulated during filopodia elongation acts as a negative regulator of tnt formation and vesicle transfer function (delage et al., ) . an interesting case of exploitation of filopodia during viral infection is reported for murine leukemia virus. after binding to receptors, mlv particles move along filopodia to reach the cell body and permit cell entry, a process dependent on actin and myosin that has been termed virus surfing (fig. a) (lehmann et al., ) . later in infection, it was shown that mlv particles move directly from infected to uninfected cells using filopodial bridges, named viral cytonemes, of about . μm in length (sherer et al., ) . movement of fluorescently labeled mlv particles was observed on the outer surface of cytonemes. the formation of cytonemes was shown to be dependent on env-receptor interactions, and antibodies targeting the extracellular domain of env could disrupt viral cytonemes. viral surfing through retrograde transport on the outer surface of filopodial structures has been also reported for dna viruses including hsv- and human papillomavirus types and (dixit et al., ; schelhaas et al., ; smith et al., ) . further evidence of animal virus infection inducing filopodia formation has been reported for asfarviruses ( jouvenet et al., ) . it was shown that african swine fever virus (asfv) induces filopodia formation at the plasma membrane with an average length of . μm. each projection contains the cellular proteins actin and fascin and single viral particles at their tips ( fig. b ) ( jouvenet et al., ) . similar observations were made for alphaviruses, as infection induces a dramatic remodeling of the cell architecture including formation of long intercellular projections (˃ μm) (martinez et al., ; martinez and kielian, ) . as observed for other viruses, projections induced by alphaviruses contain actin and microtubules. however, they are different from those seen with asfv, where single virions are present at the tip of filopodia, as multiple alphavirus virions budding from the projections are observed (fig. c ). an interesting observation made for alphaviruses is that the filopodia-like extensions are not able to transfer cytosolic or plasma membrane components, suggesting they are not openended connections like tnts. instead, viral particles are hypothesized to bud into a protected space at the filopodial tip and then rapidly enter the target cell, preventing access of neutralizing antibodies. recent work has demonstrated that filopodia-like extensions are also a mechanism that respiratory viruses use for direct cell-to-cell spread. infection of human bronchial epithelial cells by human metapneumovirus (hmpv) resulted in a dramatic reorganization of the cell cytoskeleton (el najjar et al., ) . two major forms of projections were observed: long intercellular extensions of up to μm, which resemble filopodia, and shorter abundant branching filaments (up to μm) budding from the cell body and from the intercellular extensions (fig. d) . we speculate that the branching filaments correspond to filamentous forms of budding virus. as was seen for other viruses, f-actin and the rho gtpases cdc and rac were important for the formation of hmpv intercellular extensions. cdc is one of the main small gtpases involved in the formation of filopodia (nobes and hall, ) . up to % of cell-to-cell spread facilitated by hmpv intercellular extensions was shown to occur in the presence of neutralizing antibodies and in the absence of appropriate attachment factors, leading to the suggestion that direct intercellular transfer of viral components could occur. like hmpv, rsv induces formation of filopodial structures in lung epithelial cells which facilitate viral spread (mehedi et al., ) . clusters of filamentous structures were observed at the surface of rsv-induced filopodia, similar to branching filaments induced by hmpv (fig. d) . additionally, formation of filopodia was shown to confer increased motility to infected cells, thus facilitating cell-to-cell spread. filopodia induced by rsv were shown to be dependent on the rsv f protein and the cellular actin-related protein (arp ), an important actin-nucleation factor (mehedi et al., ) . recently, a significant induction of filopodia containing budding particles was reported for the severe acute respiratory syndrome virus (sars-cov- ) (bouhaddou et al., ) . filopodia induction was associated with an increase in signaling by casein kinase . indeed, ck was found in filopodia co-localizing with the sars-cov- nucleoprotein (bouhaddou et al., ) . analysis of measles virus (mev) infection in well-differentiated primary cultures of human airway epithelial (hae) cells revealed a previously unidentified mechanism of direct cell-to-cell spread that involves the opening of intercellular pores (singh et al., ) . a recombinant gfp-mev rapidly spreads in hae cell cultures to form infectious centers, but no syncytia were observed. these infectious centers are larger than those formed by other viruses such as rsv, sendai or piv . cytoplasmic material from the infected cell was shown to suddenly flow into the adjacent cell through lateral pores of an estimated size of nm (singh et al., ) . the formation of infectious centers by mev was dependent on an interaction between the actin filament binding protein afadin and nectin- , the cellular receptor for mev entry (fig. ) . recent work from the same group demonstrated that mev ribonucleoprotein complexes can spread to neighbor human airway epithelial cells by moving along the circumapical f-actin ring (fig. ) (singh et al., ) . the circumapical f-actin ring encircles the uppermost portion of cells in hae cultures, close to and at the level of adherens junctions. further studies demonstrated that mev uses cellto-cell spread promoted by cell-cell contacts to move from infected immune it has been suggested that physical constraints due to the presence of the circumapical f-actin ring thwart expansion of the intercellular pores, in contrast to what is observed during syncytia formation. however, the pore has a size that permits direct spread of ribonucleoprotein complexes that can initiate infection in the adjacent cell. blue dots represent polymerase complexes bound to viral rna. cells to epithelial cells, rather than spread by cell-free virus (singh et al., ) . very recently, another mechanism of cell-to-cell spread exploited by mev has been reported. this mechanism involves the capture of membranes containing nectin- by cells expressing nectin- , a process known as trans-endocytosis (generous et al., ) . cargo cytosolic material, including mev ribonucleocapsids, was shown to be transferred by trans-endocytosis and to remain functional in the recipient cell. this mechanism of spread was shown to occur between epithelial cells but also from infected epithelial cells to primary neurons. this type of spread does not require the assembly of complete virions, and demonstrates that transfer of ribonucleocapsids is sufficient to initiate infection in a target cell (generous et al., ) . a novel mechanism for transfer of multiple virus particles has been recently reported for enteroviruses. this virus family has historically been described as non-enveloped viruses that exit the cell by lysis. however, coxsackie virus b (cvb ) particles were shown to alternatively exit the cell enclosed within vesicles (robinson et al., ) . these extracellular microvesicles (emvs) were shown to contain autophagosomal membranes and to be infectious once added to recipient cells (robinson et al., ) . further evidence supporting extracellular vesicles as a mechanism for viral spread came from the demonstration of non-lytic release of poliovirus (pv) capsids (bird et al., ; chen et al., ) . large extracellular vesicles enriched with phosphatidyl serine (ps) of up to nm in size were shown to contain multiple mature pv particles. the same observations were made for other enteroviruses including cvb and rhinovirus (chen et al., ) . the question of how these large pv-containing vesicles can enter a target cell was addressed, and the process was shown to be dependent on both the cellular receptor cd and the presence of ps on the membranes of vesicles. interestingly, it was shown that the pv particles contained within vesicles infect more efficiently than cell-free viral particles on a per-particle basis, potentially due to the synergistic effect of multiple particles entering the cell at the same time (chen et al., ) . non-lytic release of vesicles containing rotavirus from cultured cells was recently reported (santiana et al., ) . the biological relevance of this mechanism was strengthened by the observation of vesicles containing clusters of rotavirus in animal stool samples. on average, more than rotavirus active particles were shown to be present in each vesicle, and these virus-containing vesicles were shed into stool. similar to what was found for pv-containing vesicles, rotavirus within vesicles was found to be more efficient in infection than cell-free viral particles (santiana et al., ) . norovirus particles were also shown to be released non-lytically within vesicles, but these vesicles were slightly smaller in size and had cellular markers of exosomes (santiana et al., ) . the first demonstrations of viral particles released in exosomal membranes came from studies with hcv and the non-enveloped hepatitis a (hav) virus (feng et al., ; ramakrishnaiah et al., ) . exosomes containing viral particles were shown to transmit hcv infection to recipient cells, a mechanism partly resistant to neutralizing antibodies (ramakrishnaiah et al., ) . similarly, enveloped vesicles with markers of exosomes were found to contain multiple hav particles and to mediate cell-to-cell transmission within the liver (feng et al., ) . these quasi-enveloped hav particles (ehav), are resistant to the action of neutralizing antibodies and can enter target cells through clathrin and dynamin-dependent endocytosis (feng et al., ; rivera-serrano et al., ) . endocytosed ehav are recognized by endolysosomal gangliosides that act as receptors, and the ehav is then trafficked to lysosomes, where the exosomal membrane is degraded and virus uncoated (das et al., ; rivera-serrano et al., ) . these novel mechanisms of spread represent an advantage for the virus, as neutralizing antibodies should not affect infectivity of vesicles the same way they neutralize cell-free particles since the particles are protected by the vesicular membrane. this was demonstrated for the human jc polyomavirus that also associates with extracellular vesicles (morris-love et al., ). furthermore, vesicles containing jc polyomavirus were shown to enter target cells in a manner that is independent of cellular receptors (morris-love et al., ). thus, production of extracellular vesicles containing infectious virus seems to be a mechanism shared by a number of rna and dna viruses, whether they are enveloped or not (altan-bonnet et al., ). while budding and release of cell-free virus particles enables dissemination from host-to-host and spread across long distances to infect distant tissues within a host, direct cell-to-cell spread and spread at specialized cell-cell contact sites have several potential advantages for viral spread and infection, and thus has implications for viral disease and pathogenesis. transmission of virus particles at cell-cell contact sites allows rapid viral dissemination within a tissue. in cell-to-cell spread, virus particles or virus genetic material are delivered right to target cells, making infection of target cells faster than with extracellular release and re-infection. cell-to-cell spread also overcomes the rate limiting fluid-phase virus diffusion step encountered by released particles. fitting with this, several studies have shown that the rate of hiv- spread through cell-to-cell transmission is faster than spread with cell-free virus (boulle et al., ; chen et al., ; dimitrov et al., ; sourisseau et al., ; spouge et al., ) . transfer of virus particles across cell-cell contacts can also facilitate receptor binding and entry. cell-cell contacts can specifically recruit receptors to the appropriate site, making the receptor attachment and entry steps more efficient (chen et al., ; sherer et al., ; vasiliver-shamis et al., ) . in some cases, like with measles virus, complete assembly of virus particles does not occur and direct spread is mediated by transfer of virus genetic material in the form of ribonucleoprotein (rnp) complexes to target cells (lawrence et al., ; singh et al., singh et al., , . intercellular spread is mediated by cytoskeletal forces and protein trafficking pathways, thus allowing faster transmission than that seen with particle diffusion. the actin comet tails induced by vaccinia virus act to propel virus particles quickly across long distances, allowing for faster spread (cudmore et al., ; doceul et al., ) . comparative analysis revealed that cell-to-cell spread can be significantly more efficient than cell-free spread for a number of viruses, as determined by higher infection rates that occur under conditions of cell-to-cell compared to lower infection by cell-free virus infection. hiv cell-to-cell transmission is - , Â more efficient than extracellular spread (chen et al., ; kolodkin-gal et al., ; martin et al., ; sourisseau et al., ) . cell-to-cell spread of hsv and prv through intercellular extensions was shown to be essential for efficient spread in vitro (favoreel et al., ) . cell-to-cell transmission was initially identified for viruses that have low infectivity to particle ratio but show efficient transmission in cell culture (bangham, ; carr et al., ; dimitrov et al., ) . several studies have shown that spread of viruses at cell-cell contacts is associated with transfer of many virus particles or genomes from the donor to the target cell (del portillo et al., ; hubner et al., ; jin et al., ; jung et al., ; rager et al., ; russell et al., ; sherer et al., ; shirogane et al., ; sigal et al., ; zhong et al., ) . enhanced budding at cell-cell contact sites and transfer of multiple genomes provide high local multiplicity of infection (moi) (fig. ) . this high moi makes cell-to-cell spread more efficient than cell-free transmission by increasing fig. advantages of cell-to-cell spread. (a) cell-free viral particles released by budding can be affected by the presence of neutralizing antibodies in the extracellular environment. in contrast, viral particles that are disseminated from cell-to-cell at cell junction-specialized membrane contacts (as one example of cell-to-cell spread) are not accessible to neutralizing antibodies (b). once they enter a cell, individual viral particles can be targeted by intracellular restriction factors during uncoating. in contrast, multiple viral particles entering a target cell can saturate restriction factors available in the cell and proceed with infection. after successful uncoating, individual viral particles will initiate genome replication starting from single genomes (d), a highly inefficient process. in contrast, several viral genomes coming from particles will be more efficient in replication, leading to a rapid infection. alternatively, genomes can be transmitted from cell-to-cell directly through intercellular pores (as an example), which can bypass the uncoating step and inhibition by restriction factors, proceeding more efficiently with replication (c). the probability of successful infection through the introduction of multiple genomes into a single target cell, thus allowing spread even with low viral gene expression and poor particle release. it has been suggested that the transfer of multiple genomes during cell-to-cell spread has implications to virus fitness and evolution (sanjuan, ) . other advantages of high moi include trans-complementation of genetic defects, evasion of innate immune responses in the target cell, avoidance of the loss of viral components necessary for replication, alleviation of transmission bottlenecks and maintainance of genetic diversity (sanjuan, ; sanjuan and thoulouze, ) . although many virus particles or genomes can be transferred, only a limited number of genomes likely initiate infection in the target cell (miyashita et al., ) . it has been estimated that as many as or hiv- particles can be transmitted through a virological synapse (chen et al., ; hubner et al., ); however, hiv- infects with an average of . proviruses after cell-to-cell spread (del portillo et al., ) which is relevant in vivo where - genomes are detected in infected cells ( jung et al., ; law et al., ) . the alpha-herpesviruses hsv and prv infect with an average of . and . genomes, respectively, following cell-to-cell transmission although multiple virions were seen transported on distal axon sites (taylor et al., a) . this suggests that although many virions or genomes can be transmitted at sites of cell-cell contacts, only a limited number establish infection which may be advantageous for the virus. further studies are needed to determine if this is true for other viruses that use cell-to-cell spread for transmission. spread of viruses by cell-to-cell transmission allows evasion of multiple barriers that exist for cell-free infection. one of the hallmarks of cell-to-cell spread is that it allows transmission in the presence of neutralizing antibodies (fig. ) . spread at tight cell-cell contacts provides limited exposure time to neutralizing antibodies in the extracellular environment and can physically restrict access of neutralizing antibodies to the virus. neutralizingantibody resistant spread has been shown for viruses that use different modes of cell-to-cell spread. for example, transmission of viruses such as hcv, hiv- and hsv- across tight cell-cell contacts has been shown to shield the virus from neutralizing antibodies (brimacombe et al., ; favoreel et al., ) . viruses that use nanotubes, filopodia, or intercellular extensions, whether open-or close-ended, show a neutralizing antibody-resistant mode of spread. this includes the alphaherpesviruses prv and hsv- (favoreel et al., ; jansens et al., ; sarfo et al., ) , the alphavirus sindbis virus (martinez and kielian, ) , the respiratory viruses hmpv, influenza a virus, and piv (el najjar et al., ; kumar et al., ; roberts et al., ) , prrsv (guo et al., (guo et al., , , and coxsackievirus b (paloheimo et al., ) . antibody resistant cell-to-cell spread of enveloped vacv particles occurs in an actin-dependent or -independent manner likely mediated by protein a (krupovic et al., ; law et al., ) . the formation of viral biofilms by htlv- may also provide a way of physically protecting particles from circulating antibodies which recognize the env protein (pais-correia et al., ; thoulouze and alcover, ) . for hiv- , several reports show conflicting results on the role of neutralizing antibodies in inhibiting cell-to-cell transmission, with some studies showing decreased neutralization during cellto-cell spread and others showing no inhibition of neutralizing antibodies on direct virus transfer. variations in the effects of the neutralizing antibodies depend on the epitope on env protein targeted by the antibody, the donor and target cell type, and virus strain used (abela et al., ; chen et al., ; ganesh et al., ; gupta et al., ; martin et al., ; massanella et al., ; pantaleo et al., ; van montfort et al., ; zhong et al., ) . in addition to evasion from neutralizing antibodies, cell-to-cell spread can play a role in overcoming intrinsic antiviral restriction factors. for retroviruses, restriction factors such as trim α and tetherin can effectively inhibit cell-free virus spread, but are less effective, or fail completely, in preventing cell-to-cell transmission (celestino et al., ; jolly et al., ; richardson et al., ; zhong et al., ) . this may occur due to saturation of the restriction factors by accumulation of virus particles at sites of cell-cell contacts or because cell-associated virus is inaccessible to a restriction factor. it has been suggested that tetherin can promote accumulation of hiv- virus particles at the vs and even help to stabilize the synapse during cell-to-cell spread . cell-to-cell transmission may also act on overcoming the innate antiviral response. it has been suggested that transfer of influenza a virus ns protein through tunneling nanotubes may overcome the innate immune response in the target cell (kumar et al., ) . cell-to-cell spread has implications for the pathogenesis of viral infection as it contributes to viral persistence and latency, therapy failure/resistance, and immune evasion. the cell-to-cell mode of transmission is inherent to the pathogenesis of hsv- ; it is the main mode of spread in the epithelium and from epithelial cells to neurons during primary infection where it establishes latency and moves back through axons to epithelial cells during reactivation (dingwell et al., ; wang et al., ) . cell-to-cell transmission has also been implicated in persistence of hcv infection and establishment of chronic infection in the liver, in addition to resistance to direct-acting antiviral drugs and therapy failure (timpe et al., ; witteveldt et al., ; xiao et al., ; zeisel et al., ) . resistance to antivirals due to direct cell-to-cell spread has also been shown for influenza virus with resistance to oseltamivir treatment (kumar et al., ; mori et al., ; roberts et al., ) . the different modes of cell-to-cell transmission that hiv- utilizes contribute to the persistence of viral reservoirs (costiniuk and jenabian, ) and resistance to antiretroviral therapy (duncan et al., ; sigal et al., ) . understanding mechanisms of cell-to-cell spread is thus important for development of a deeper understanding of viral pathogenesis and for the development of potent antiviral therapies. cell-to-cell spread multiple approaches have been used in in-vitro cell culture models to study the different modes of virus transmission. experimental conditions were developed for blocking cell-free or cell-to-cell spread to determine the contribution of either in infection dynamics. addition of a viscous media such as methylcellulose or agarose to an infected cell culture suppresses diffusion of released virus particles and thus decreases spread by cell-free virus and not by direct cell-to-cell spread (el najjar et al., ; jin et al., ; martin et al., ; timpe et al., ) . another approach for blocking cell-free virus infection that provides more convincing experimental support is assaying spread in the presence of virus-neutralizing antibodies. while neutralizing antibodies are successful in completely, or almost completely, inhibiting cell-free infection, cell-to-cell transmission is generally resistant to neutralizing antibodies. spread in the presence of neutralizing antibodies has been used to demonstrate cell-to-cell transmission for several viruses including hcv- , herpesviruses, alphaviruses, coxsackievirus b , iav, hmpv and prrsv (brimacombe et al., ; favoreel et al., favoreel et al., , guo et al., ; jansens et al., ; martinez and kielian, ; roberts et al., ; sarfo et al., ) . in some cases however, such as hiv- and cmv, the effect of neutralizing antibodies can be inconsistent, as discussed above, and further validation of transmission modes requires additional investigation (dufloo et al., ; jacob et al., ) . in addition, some neutralizing antibodies can prevent cell-to-cell transmission by inhibiting establishment or stability of cell-cell contacts between an infected donor cell and an uninfected target cell. for instance, cell-to-cell transmission of hiv- depends on cell-cell contacts that form by the interaction of env protein in the donor cell with the cd receptor on the target cell; thus neutralizing antibodies against env protein can block cell-cell contact and cell-to-cell spread (chen et al., ; rudnicka et al., ) . it is therefore important to use neutralizing antibodies that do not interfere with the formation of cell-cell contacts in order to differentiate the susceptibility of cell-free and cell-to-cell transmission to neutralization. experimental assays to assess the role of direct contact between infected cells and neighboring cells in spreading infection include using a trans-well to physically separate donor and target cells and prevent cell migration, cell shaking to abolish the formation of cell-cell contacts or lowering cell density to prevent close cell contact. coculturing of donor infected cells with target cells is another widely used assay to identify cell-to-cell transmission. in this assay, infected target cells are harvested and cocultured with uninfected target cells under conditions that prevent infection by cell-free virus such as addition of neutralizing antibodies or blocking receptor binding (branscome et al., ; el najjar et al., ; martinez and kielian, ; xiao et al., ; zhong et al., ) . a fluorescent dye is usually used to differentiate target cells from donor cells, and quantification of cell spread is determined microscopically or by flow cytometry of a reporter virus or a labeled viral antigen. the cell coculture system has been used to identify viral and host factors that contribute to direct cell spread of several viruses. cell-to-cell transmission of hcv depends on viral envelope proteins and utilizes the same receptors used for cell-free infection including scavenger receptor bi, cd , epidermal growth factor receptor (egfr) and the tight junction proteins claudin- and occludin (catanese et al., ; fofana et al., ; lupberger et al., ; witteveldt et al., ; zahid et al., ) . cell-to-cell spread for other viruses such as hmpv and sinv occurs in a manner that is independent, or partially independent, of the receptor binding which is needed for cell-free entry (el najjar et al., ; martinez and kielian, ) . for studying spread of herpesviruses, two cell culture systems are primarily used: a dissociated model where rat superior cervical ganglia (scg) are plated and form a network of axons; and a compartmentalized system that provides isolation of the neuron cell body from the distal axon termini (curanovic et al., ; ch'ng et al., ). the latter system has been used to study the number and diversity of viral genomes after anterograde direct spread of hsv- and prv particles, with results showing limited virus transmission from neurons to epithelial cells (taylor et al., a) . experiments conducted in -dimensional cell culture models provide valuable information on the mechanisms of cell-to-cell spread of viruses; however, they do not take into account how the complexity and environment of the tissue impacts virus spread in vivo. to overcome some of these limitations, different -dimensional cell culture models have been utilized for studying spread for a number of viruses. a -d cell culture system using hiv- -infected primary human cd t lymphocytes in a -d scaffold showed the importance of cell motility and density to hiv- spread (imle et al., ) . studying measles virus in a -d model of primary human differentiated airway epithelial cells revealed a novel mechanism of rnp horizontal spread along apical f-actin rings (singh et al., ) . a neutralizing antibody-resistant mode of spread was recently described for hmpv in a similar -d model of differentiated human bronchial epithelial cells (kinder et al., ) . further studies in -d tissue models are necessary to elucidate how viruses actually spread within the host and identify antivirals that affect one mode of virus transmission but not the other. imaging fixed cells by fluorescence and electron microcopy has been widely used for showing virus transfer between cells. detection of viral proteins in intercellular connections or at sites of cell-cell contact by immunofluorescence (if) is usually added to the line of evidence to demonstrate cellto-cell spread. a number of studies showed localization of viral proteins in tnts, filopodia, and other intercellular projections for viruses of different families that spread by direct cell-to-cell transmission, including the herpesvirus bohv- , alphavirus sinv, prrsv and hmpv. localization of viral genomes within these cellular structures, as detected by fluorescence in situ hybridization (fish), indicated that not only viral antigens are localizing to intercellular connections but also genetic material, thus suggesting passage of virus particles or viral genomes across these structures (el najjar et al., ; favoreel et al., ; guo et al., ; martinez and kielian, ) . for hiv- , a vs was initially described by if showing clustering of receptor on the target cell and env protein on the effector cell mcdonald et al., ) , and transmission electron microscopy provided cross sectional views of the synapse (wang et al., ) . advances in microscopic imaging facilitated by the generation of fluorescently labeled recombinant viruses helped to further deliver mechanistic insights underlying cell-to-cell spread of viruses. live cell imaging provides a powerful tool for demonstrating the dynamics of virus transport between cells. combining if and time lapse microscopy with correlative fluorescence and scanning microscopy revealed dynamics of movement of single mlv particles on the surface of cytonemes that form by interaction of env protein with the receptor mcat ( jin et al., ; sherer et al., ) . live cell -d microscopy and expansion microscopy of influenza virus infected cells, in addition to scanning electron microscopy (sem), showed that the ends of tnt are continuous with the cytoplasm of cells and that there are bundled fibers inside the tnts (kumar et al., ) . generation of fluorescently labeled alphaherpesviruses with fluorophore tagged capsid, tegument, or glycoproteins allowed examination of egress and spread mechanisms. live imaging of neurons infected with different labeled viruses indicated that both capsids and enveloped particles move across axons and that the us membrane protein is needed for anterograde transport of progeny virus particles (taylor et al., b; wisner et al., ) . imaging alphaherpesviruses hsv- and prv, tagged with different fluorophores, allowed determination of the diversity and number of viral genomes following anterograde-directed spread from isolated neurites to epithelial cells in a compartmentalized cell culture model (taylor et al., a) . imaging techniques have also advanced our understanding of measles virus spread. time lapse microscopy with -d z-stack imaging of gfp-tagged measles virus, or more recently of a recombinant virus containing gfp/p fusion protein, in a model of airway epithelial tissue was used to reveal rapid spread. infectious centers and rnp were shown along the apical f actin network stained with the lifeact marker as mentioned in more detail in section . (singh et al., (singh et al., , . more information has been gathered in recent years on the formation of the vs mediating hiv- spread. analysis using ion abrasion sem and electron tomography provided -d visualization of the vss with invaginations forming in the donor cell and a projection extending from the target cell (felts et al., ) . kinetics of virus clustering and transfer were determined by quantitative -d live imaging (hubner et al., ; real et al., ) . recently, live imaging using a gfp-tagged env mutant virus and cells stably expressing mcherry-gag revealed that the env protein is first transferred across the vs followed by redistribution of gag to cell-cell contact sites (wang et al., ) . super resolution microscopy revealed an organized localization of n and m proteins of hmpv in budding filaments with n protein on the inside surrounded by the m protein (el najjar et al., ) . combining functional and imaging approaches is thus essential for elucidating the mechanisms underlying cell-to-cell spread of different viruses. in addition, mathematical models have been employed in recent years to provide better quantitation of the contribution of the different modes of spread in infection dynamics. such models account for the role of space and effects of the immune response on viral spread and utilize experimental and clinical data to assess modes of virus spread in vivo (goyal and murray, ; graw et al., ; graw and perelson, ; kumberger et al., ) . research in the last several decades has highlighted both the variety of mechanisms that viruses utilize for spreading to new target cells and the surprising commonality of many mechanisms. recent work suggests that these mechanisms may be important for pathogenesis, providing a more efficient means of propagating infection under many circumstances. future work dissecting the role of cell-to-cell spread in animal models will be critical to understand the importance of these mechanisms in overall pathogenesis. in addition, dissection of these mechanisms may provide important new targets for antiviral treatment. cell-cell transmission enables hiv- to evade inhibition by potent cd bs directed antibodies extracellular vesicles: vehicles of en bloc viral transmission casein kinase regulates vaccinia virus actin tail formation the formin fhod and the small gtpase rac promote vaccinia virus actin-based motility unique features of hiv- spread through t cell virological synapses cell-fusion events induced by α-herpesviruses the virological synapse facilitates herpes simplex virus entry into t cells the immune control and cell-to-cell spread of human t-lymphotropic virus type engagement of specific t-cell surface molecules regulates cytoskeletal polarization in htlv- -infected lymphocytes identification of host trafficking genes required for hiv- virological synapse formation in dendritic cells nonlytic viral spread enhanced by autophagy components the isolation and characterization of mutants of herpes simplex virus type that induce cell fusion the global phosphorylation landscape of sars-cov- infection hiv cell-to-cell spread results in earlier onset of viral gene expression by multiple infections per cell stem cell extracellular vesicles and their potential to contribute to the repair of damaged cns cells neutralizing antibody-resistant hepatitis c virus cell-to-cell transmission rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes different requirements for scavenger receptor class b type i in hepatitis c virus cell-free versus cell-to-cell transmission feline tetherin is characterized by a short n-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein a viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion f-actin dynamics transform filopodial bridges into intercellular nanotubes capable of distant cell communication predominant mode of human immunodeficiency virus transfer between t cells is mediated by sustained envdependent neutralization-resistant virological synapses phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses culturing primary and transformed neuronal cells for studying pseudorabies virus infection hantavirus gn and gc envelope glycoproteins: key structural units for virus cell entry and virus assembly varicella-zoster virus glycoprotein i is essential for growth of virus in vero cells membrane fusion mediated by herpesvirus glycoproteins: the paradigm of varicella-zoster virus they might be giants: does syncytium formation sink or spread hiv infection? cell-to-cell transfer of hiv infection: implications for hiv viral persistence actin-based motility of vaccinia virus compartmented neuron cultures for directional infection by alpha herpesviruses gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis a virus multiploid inheritance of hiv- during cell-to-cell infection differential identity of filopodia and tunneling nanotubes revealed by the opposite functions of actin regulatory complexes quantitation of human immunodeficiency virus type infection kinetics the herpes simplex virus ge-gi complex facilitates cell-to-cell spread and binds to components of cell junctions herpes simplex virus glycoproteins e and i facilitate cell-to-cell spread in vivo and across junctions of cultured cells deletion of the pseudorabies virus ge/gi-us p complex disrupts kinesin kif a and kif c recruitment during egress, and alters the properties of microtubule-dependent transport in vitro herpes simplex virus type induces filopodia in differentiated p neural cells to facilitate viral spread three-dimensional imaging of hiv- virological synapses reveals membrane architectures involved in virus transmission repulsion of superinfecting virions: a mechanism for rapid virus spread wip provides an essential link between nck and n-wasp during arp / -dependent actin polymerization hiv- cell-to-cell transmission and broadly neutralizing antibodies fusogenic reoviruses and their fusion-associated small transmembrane (fast) proteins avian reovirus-induced syncytium formation is independent of infectious progeny virus production and enhances the rate, but is not essential, for virus-induced cytopathology and virus egress high multiplicity hiv- cell-to-cell transmission from macrophages to cd + t cells limits antiretroviral efficacy loss of actin-based motility impairs ectromelia virus release in vitro but is not critical to spread in vivo macrophage cell-cell interactions promoting hiv- infection actin remodeling to facilitate membrane fusion human metapneumovirus induces reorganization of the actin cytoskeleton for direct cell-to-cell spread tunneling nanotubes (tnt) are induced by hiv-infection of macrophages: a potential mechanism for intercellular hiv trafficking the making of filopodia attachment and postattachment receptors important for hepatitis c virus infection and cell-to-cell transmission herpes simplex virus ge/gi must accumulate in the trans-golgi network at early times and then redistribute to cell junctions to promote cell-cell spread cytoskeletal rearrangements and cell extensions induced by the us kinase of an alphaherpesvirus are associated with enhanced spread herpesvirus interference with virus-specific antibodies: bridging antibodies, internalizing antibodies, and hiding from antibodies d visualization of hiv transfer at the virological synapse between dendritic cells and t cells a pathogenic picornavirus acquires an envelope by hijacking cellular membranes a novel monoclonal anti-cd antibody produced by genetic immunization efficiently inhibits hepatitis c virus cell-cell transmission tyrosine phosphorylation is required for actin-based motility of vaccinia but not listeria or shigella actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling infection of specific dendritic cells by ccr -tropic human immunodeficiency virus type promotes cell-mediated transmission of virus resistant to broadly neutralizing antibodies trans-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells viroporins customize host cells for efficient viral propagation inhibitory effect of individual or combinations of broadly neutralizing antibodies and antiviral reagents against cell-free and cell-to-cell hiv- transmission rhoa signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology modelling the impact of cell-to-cell transmission in hepatitis b virus modeling viral spread quantification of hepatitis c virus cell-to-cell spread using a stochastic modeling approach the tax-inducible actin-bundling protein fascin is crucial for release and cell-to-cell transmission of human t-cell leukemia virus type (htlv- ) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread intercellular transfer of mitochondria rescues virusinduced cell death but facilitates cell-to-cell spreading of porcine reproductive and respiratory syndrome virus cell-to-cell transmission of human immunodeficiency virus type in the presence of azidothymidine and neutralizing antibody human metapneumovirus: a new player among respiratory viruses virus-cell and cell-cell fusion identification of residues in the human respiratory syncytial virus fusion protein that modulate fusion activity and pathogenesis herpes simplex virus ge/gi extracellular domains promote axonal transport and spread from neurons to epithelial cells rotavirus nonstructural proteins: structure and function quantitative d video microscopy of hiv transfer across t cell virological synapses clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread spread of htlv-i between lymphocytes by virus-induced polarization of the cytoskeleton experimental and computational analyses reveal that environmental restrictions shape hiv- spread in d cultures neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus pseudorabies virus us -induced tunneling nanotubes contain stabilized microtubules, interact with neighbouring cells via cadherins and allow intercellular molecular communication bridging the gap: virus long-distance spread via tunneling nanotubes assembly of the murine leukemia virus is directed towards sites of cell-cell contact herpesviruses remodel host membranes for virus egress directed egress of animal viruses promotes cell-to-cell spread herpes simplex virus ge/gi sorts nascent virions to epithelial cell junctions, promoting virus spread retroviral spread by induction of virological synapses hiv- cell to cell transfer across an env-induced, actin-dependent synapse cell-cell spread of human immunodeficiency virus type overcomes tetherin/bst- -mediated restriction in t cells the regulated secretory pathway in cd (+) t cells contributes to human immunodeficiency virus type- cell-to-cell spread at the virological synapse african swine fever virus induces filopodia-like projections at the plasma membrane recombination: multiply infected spleen cells in hiv patients mechanisms of virus membrane fusion proteins respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) infections in -d human airway tissues expose an interesting dichotomy in viral replication, spread, and inhibition by neutralizing antibodies efficiency of cell-free and cell-associated virus in mucosal transmission of human immunodeficiency virus type and simian immunodeficiency virus glycoproteins ge and gi are required for efficient kif a-dependent anterograde axonal transport of alphaherpesvirus particles in neurons the tumor marker fascin is strongly induced by the tax oncoprotein of htlv- through nf-kappab signals protein a responsible for antibody-resistant spread of vaccinia virus is homologous to c-type lectin-like proteins influenza virus exploits tunneling nanotubes for cell-to-cell spread accounting for space-quantification of cell-to-cell transmission kinetics using virus dynamics models the nucleoprotein of lymphocytic choriomeningitis virus facilitates spread of persistent infection through stabilization of the keratin network absence of keratin restricts the course of infection with lymphocytic choriomeningitis virus strain mx antibody-sensitive and antibody-resistant cellto-cell spread by vaccinia virus: role of the a r protein in antibody-resistant spread in vivo hiv- cell-to-cell transmission promotes multicopy micro-compartmentalized infection measles virus spread between neurons requires cell contact but not cd expression, syncytium formation, or extracellular virus production actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells role of lipids in virus replication egfr and epha are host factors for hepatitis c virus entry and possible targets for antiviral therapy pseudorabies virus us directs axonal sorting of viral capsids virological synapse-mediated spread of human immunodeficiency virus type between t cells is sensitive to entry inhibition intercellular extensions are induced by the alphavirus structural proteins and mediate virus transmission imaging the alphavirus exit pathway divergent roles of beta-and gamma-actin isoforms during spread of vaccinia virus antigp antibodies fail to block early events of virological synapses but inhibit hiv spread between t cells connections matter-how viruses use cell-cell adhesion components filopodia: molecular architecture and cellular functions spontaneous electrical activity induced by herpes virus infection in rat sensory neuron cultures pseudorabies virus infection alters neuronal activity and connectivity in vitro recruitment of hiv and its receptors to dendritic cell-t cell junctions herpes simplex virus type glycoprotein e mediates retrograde spread from epithelial cells to neurites actin-related protein (arp ) and virus-induced filopodia facilitate human respiratory syncytial virus spread viruses roll the dice: the stochastic behavior of viral genome molecules accelerates viral adaptation at the cell and tissue levels hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes tamiflu-resistant but ha-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses jc polyomavirus uses extracellular vesicles to infect target cells hiv-infected t cells are migratory vehicles for viral dissemination characterization of cell death pathways in human immunodeficiency virus-associated encephalitis demonstration of respiratory syncytial virus in an autopsy series human t-lymphotropic virus, type , tax protein triggers microtubule reorientation in the virological synapse viruses that ride on the coat-tails of actin nucleation abl collaborates with src family kinases to stimulate actin-based motility of vaccinia virus viroporins: structure and biological functions rho, rac, and cdc gtpases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria biofilm-like extracellular viral assemblies mediate htlv- cell-to-cell transmission at virological synapses coxsackievirus b -induced cellular protrusions: structural characteristics and functional competence tunneling nanotubes as a novel route of cell-to-cell spread of herpesviruses effect of anti-v antibodies on cell-free and cell-to-cell human immunodeficiency virus transmission actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virusinduced cell fusion polyploid measles virus with hexameric genome length exosomemediated transmission of hepatitis c virus between human hepatoma huh . cells live imaging of hiv- transfer across t cell virological synapse to epithelial cells that promotes stromal macrophage infection variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and src family tyrosine kinases mechanisms of varicella-zoster virus neuropathogenesis in human dorsal root ganglia mode of transmission affects the sensitivity of human immunodeficiency virus type to restriction by rhesus trim alpha cellular entry and uncoating of naked and quasi-enveloped human hepatoviruses influenza a virus uses intercellular connections to spread to neighboring cells simultaneous cell-tocell transmission of human immunodeficiency virus to multiple targets through polysynapses multiple proviral integration events after virological synapse-mediated hiv- spread nanotubular highways for intercellular organelle transport requirement of interaction of nectin- alpha/hvec with afadin for efficient cell-cell spread of herpes simplex virus type collective infectious units in viruses why viruses sometimes disperse in groups?(dagger) vesicle-cloaked virus clusters are optimal units for inter-organismal viral transmission the ul tegument protein of herpes simplex virus is differentially required for the syncytial phenotype grb and nck act cooperatively to promote actin-based motility of vaccinia virus human papillomavirus type entry: retrograde cell surface transport along actin-rich protrusions rho gtpase activity modulates paramyxovirus fusion proteinmediated cell-cell fusion retroviruses use cd -mediated trans-infection of permissive lymphocytes to establish infection retroviruses can establish filopodial bridges for efficient cell-to-cell transmission cooperation between different rna virus genomes produces a new phenotype a new class of fusion-associated small transmembrane (fast) proteins encoded by the non-enveloped fusogenic reoviruses cell-to-cell spread of hiv permits ongoing replication despite antiretroviral therapy the nectin- /afadin protein complex and intercellular membrane pores contribute to rapid spread of measles virus in primary human airway epithelia cell-to-cell contact and nectin- govern spread of measles virus from primary human myeloid cells to primary human airway epithelial cells measles virus ribonucleoprotein complexes rapidly spread across well-differentiated primary human airway epithelial cells along f-actin rings new connections: cell-to-cell hiv- transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif the exit of vaccinia virus from infected cells virus activated filopodia promote human papillomavirus type uptake from the extracellular matrix herpes simplex virus ge/gi and us proteins promote transport of both capsids and virion glycoproteins in neuronal axons inefficient human immunodeficiency virus replication in mobile lymphocytes membrane nanotubes physically connect t cells over long distances presenting a novel route for hiv- transmission hiv- infection kinetics in tissue cultures high-voltage electron microscope study of the release of vaccinia virus from whole cells wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a slam(cd )-independent mechanism alphaherpesvirus axon-to-cell spread involves limited virion transmission visualization of an alphaherpesvirus membrane protein that is essential for anterograde axonal spread of infection in neurons subversion of the actin cytoskeleton during viral infection viral extracellular biofilms: a novel spreading trick of viruses? efficient capture of antibody neutralized hiv- by cells expressing dc-sign and transfer to cd + t lymphocytes human t-cell leukemia virus type p protein increases cellular conduits and virus transmission pathology of human metapneumovirus infection: insights into the pathogenesis of a newly identified respiratory virus human immunodeficiency virus type envelope gp -induced partial t-cell receptor signaling creates an f-actin-depleted zone in the virological synapse virus budding and the escrt pathway functionally distinct transmission of human immunodeficiency virus type mediated by immature and mature dendritic cells herpes simplex virus type glycoprotein e is required for efficient virus spread from epithelial cells to neurons and for targeting viral proteins from the neuron cell body into axons sequential trafficking of env and gag to hiv- t cell virological synapses revealed by live imaging how to get out: ssrna enveloped viruses and membrane fission arp / -mediated actin-based motility: a tail of pathogen abuse anterograde transport of herpes simplex virus capsids in neurons by both separate and married mechanisms cd is dispensable for hepatitis c virus cell-to-cell transmission in hepatoma cells hepatitis c virus cell-cell transmission and resistance to direct-acting antiviral agents dynamic monitoring of membrane nanotubes formation induced by vaccinia virus on a high throughput microfluidic chip the cytoplasmic domain of varicella-zoster virus glycoprotein h regulates syncytia formation and skin pathogenesis disruption of adherens junctions liberates nectin- to serve as receptor for herpes simplex virus and pseudorabies virus entry the postbinding activity of scavenger receptor class b type i mediates initiation of hepatitis c virus infection and viral dissemination host-targeting agents for prevention and treatment of chronic hepatitis c-perspectives and challenges binding of herpes simplex virus glycoprotein d to nectin- exploits host cell adhesion cell-to-cell transmission can overcome multiple donor and target cell barriers imposed on cell-free hiv glycoprotein gi of pseudorabies virus promotes cell fusion and virus spread via direct cell-to-cell transmission this work was supported in part by nih grants ai and ai to r.e.d. and fondecyt inicio grant to n.c.m. images were created using biorender.com. key: cord- -kg dmgq authors: darden, dijoia b.; hawkins, russell b.; larson, shawn d.; iovine, nicole m.; prough, donald s.; efron, philip a. title: the clinical presentation and immunology of viral pneumonia and implications for management of coronavirus disease date: - - journal: crit care explor doi: . /cce. sha: doc_id: cord_uid: kg dmgq this review will briefly examine the clinical presentation and important immunology of viral pneumonia with a focus on severe acute respiratory syndrome coronavirus (coronavirus disease ). data sources, study selection, data extraction, and data synthesis: the most relevant, original and review literature were assessed for inclusion in this review. sources included the centers for disease control and prevention, world health organization, and pubmed. conclusions: pneumonia is a leading cause of hospitalization and death worldwide, with viral etiologies being very common. given the rapidly emerging pandemic associated with the novel severe acute respiratory syndrome coronavirus causing coronavirus disease , it is important to review the clinical presentation and immunologic changes associated with viral pneumonia. symptoms of viral pneumonia include common respiratory tract infection symptoms of cough, fever, and shortness of breath. immunologic changes include up-regulation of airway pro-inflammatory cytokines and pathogen- and damage-associated molecular patterns contributing to cytokine and genomic changes. coronavirus disease clinical presentation is typical of viral pneumonia with an increased prevalence of early pulmonary infiltrates and lymphopenia. principles of early coronavirus disease management and isolation as well as potential therapeutic approaches to the emerging pandemic are discussed. objectives: this review will briefly examine the clinical presentation and important immunology of viral pneumonia with a focus on severe acute respiratory syndrome coronavirus (coronavirus disease ). data sources, study selection, data extraction, and data synthesis: the most relevant, original and review literature were assessed for inclusion in this review. sources included the centers for disease control and prevention, world health organization, and pubmed. conclusions: pneumonia is a leading cause of hospitalization and death worldwide, with viral etiologies being very common. given the rapidly emerging pandemic associated with the novel severe acute respiratory syndrome coronavirus causing coronavirus disease , it is important to review the clinical presentation and immunologic changes associated with viral pneumonia. symptoms of viral pneumonia include common respiratory tract infection symptoms of cough, fever, and shortness of breath. immunologic changes include up-regulation of airway pro-inflammatory cytokines and pathogenand damage-associated molecular patterns contributing to cytokine and genomic changes. coronavirus disease clinical presentation is typical of viral pneumonia with an increased prevalence of early pulmonary infiltrates and lymphopenia. principles of early coronavirus disease management and isolation as well as potential therapeutic approaches to the emerging pandemic are discussed. key words: coronavirus; immunology; influenza virus; severe acute respiratory syndrome; viral pneumonia p neumonia is the leading infectious cause of hospitalization among adults and children in the united states ( ) . according to the world health organization (who), lower respiratory tract infection is among the top causes of death globally ( ) . the centers for disease control and prevention (cdc) etiology of pneumonia in the community study estimated prevalence of pneumonia-related hospitalizations among adults older than to be - times higher than those to years old ( ) . viral infections are the leading cause of community-acquired pneumonia (cap) and are an important source of morbidity and mortality. severe acute respiratory syndrome coronavirus (sars-cov- ) is a newly discovered virus causing coronavirus disease (covid- ) that is responsible for an emerging pandemic. given the rapid spread of this virus and its association with severe pulmonary disease, the purpose of this review is to provide an overview of the presentation and immunology of viral pneumonia, principles of early management, and application to covid- . according to the cdc, the prevalence of cap is highest among adults to years old ( ) . hospitalization among adults is highest in elderly patients (≥ yr) and those with preexisting obstructive lung disease or other cardiopulmonary disorders ( , ) . the most common cause of community-or hospital-acquired pneumonia in adults is viral with the most frequently detected pathogen being human rhinovirus, followed by influenza ( - % and - %, respectively) ( ) ( ) ( ) ( ) ( ) . other commonly detected causes of viral pneumonia include adenovirus, conventional coronaviruses, human metapneumovirus (hmpv), respiratory syncytial virus (rsv), and parainfluenza. the prevalence of viral respiratory illness is temporal in north america, with peaks of influenza, hmpv, and rsv normally seen in the winter months ( table ) ( ) . the clinical presentation of viral pneumonia does not differentiate between the specific viral causes of respiratory infection. the common clinical presentation of acute viral respiratory infection includes cough, dyspnea, fever, and pleuritic chest pain. viral etiologies of lower respiratory infection are less likely to cause sputum production, and if present, tends to be watery or scant. in contrast, sputum production tends to be mucopurulent when due to bacterial pneumonia ( , ) . clinical signs of viral respiratory illness include fever, rales (crackles) on auscultation, hypoxemia, and tachycardia. these four signs together have a positive predictive value of . %, with fever as the strongest clinically predictive sign of a viral respiratory infection versus that of bacterial etiology ( ) . typically, patients with viral pneumonia also will present with a normal leukocyte count and bilateral pulmonary infiltrates on chest radiograph ( ) . severe viral pneumonia can manifest as sepsis and respiratory distress requiring intensive care ( ) . in many moderate to severe cases of pneumonia, hypoxemia occurs from impaired alveolar gas exchange ( ) , often necessitating mechanical ventilation. biopsies in pneumonia are not routinely performed due to the lack of diagnostic, prognostic, and treatment value. however, since influenza has caused the most viral respiratory epidemics to date, a number of studies have examined infected patient's lung biopsy specimens ( ) . biopsies obtained during influenza infection reveal a wide range of pathologies, including alveolar edema and exudate, interstitial inflammatory infiltration, and ulceration of bronchial mucosa to type ii cell metaplasia ( , ) . in autopsy specimens from h n influenza patients, the respiratory tract exhibited tracheitis, bronchitis, diffuse hemorrhagic alveolar damage, and inflammatory infiltration of alveolar ducts and alveoli ( , ) . the host response to severe viral lung infection occurs secondary to immune dysregulation leading to lung injury and the systemic inflammatory response. there have been many studies on the immunologic changes associated with influenza a virus (iav). however, little is known about other respiratory viral illnesses in adults. therefore, much of our discussion on the immunology of viral pneumonia will focus on iav studies. during a respiratory infection, airway epithelial cells, natural killer (nk), and cd t-cells release interferon-gamma (inf-γ) to limit viral replication ( , ) . there is additional release of interleukin (il)- and il- , important mediators of tissue damage and associated with disease progression, respectively ( ) . high levels of il- , tumor necrosis factor (tnf)-α, inf-γ, and il- have been found in postmortem human lung tissue after severe iav ( ) . although there seems to be a difference in cytokine response based on the cause of respiratory infection, there are mixed results in the utility of plasma cytokine levels for prediction of pneumonia etiology ( , ) . in a recent single-center study, differences in admission plasma levels of il- , il- , il- a, and inf-γ were observed between different etiologies of cap, with inf-γ most elevated in viral cap ( ) . conversely, a similar study demonstrated, admission plasma cytokine levels were not statistically different based on etiology (bacterial vs viral vs mixed bacterialviral vs unknown etiology) ( ) . other studies noted that serum transforming growth factor-beta (tgf-β) levels predicted viral pneumonia, as opposed to other etiologies of cap, where tgfβ had negative correlations with the sequential organ failure assessment score in patients that progressed to sepsis ( , ) . therefore, although the specific cytokine profile elicited by particular viruses is unknown, it is clear that, as with most etiologies of sepsis, an elevation of both pro-and anti-inflammatory cytokines are responsible for the host septic and systemic inflammatory response syndrome response in all severe viral cases of pneumonia ( , ( ) ( ) ( ) . as with many other responses to infection, it is pertinent to recognize the role of pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps) in viral respiratory infection. pattern recognition receptors on respiratory epithelial cells, such as toll-like receptors (tlrs), detect evolutionarily conserved microbial ligands, or pamps ( , ) . viral pamps are typically viral envelope proteins or nucleic acids motifs within the dna or rna genomes of the virus, which are critical for structure and function ( ) . the recognition of viral pamps leads to transcription and release of type i interferons ( ) which effect decreased expression of viral proteins and replication, enhance antigen presentation and nk cell function, and augment adaptive immune responses. additional recognition of host cell constituents from damaged or dying cells, recognized as damps, are thought to control the magnitude of the immune response ( ) ( ) ( ) . together, pamps and damps play a major role in the initiation of both the innate and adaptive immune response to viral lung infection ( , , ( ) ( ) ( ) ( ) . viruses can be the primary cause of pneumonia, present in conjunction with bacterial pneumonia, and/or contribute to increased susceptibility to secondary bacterial infection. in addition to influenza, other viruses, such as rhinovirus, can cause severe pneumonia requiring mechanical ventilation, however, this usually occurs in the elderly and immunocompromised ( , ) . severe pneumonia associated with noninfluenza viruses is also significantly associated with bacterial coinfection ( , ( ) ( ) ( ) , most commonly due to staphylococcus aureus, streptococcus pneumoniae, or haemophilus influenzae ( , ) . a pattern of dysregulated inflammation caused by viral respiratory infection leads to this increased susceptibility to secondary bacterial pneumonia or coinfection. most research on viral-bacterial respiratory coinfection has been focused on elucidating the pathophysiology of influenza viruses given its high propensity to cause pandemics and higher mortality when compared with the other viruses ( ) . influenza a causes a reduction in murine alveolar macrophages and dysregulation of remaining macrophages and neutrophils, one of the body's primary defense mechanisms against bacterial pathogens ( ) ( ) ( ) . additionally, prior infection with influenza virus attenuates bacterial induced release of il- leading to decreased innate t cell-mediated bacterial clearance ( ) . replication of iav in respiratory epithelium impairs mucociliary clearance, allowing for increased bacterial colonization ( , , ) . additionally, there is evidence of sustained desensitization of tlr ligands following viral infection, leading to decreased chemokine release and nuclear factor kappa b activation in macrophages ( , ) . this in turn results in attenuated neutrophil recruitment, further decreasing the ability to reduce bacterial load in secondary bacterial infection ( , ) . although cap remains a significant source of morbidity and mortality, very little work has been done establishing genomic, epigenetic, or transcriptomic changes specifically associated with viral pneumonia ( ) . in particular, no clear polymorphism definitively raises the risk of viral pneumonia, limiting personalized medicine for predictive models. in one of the few studies of transcriptomics in viral pneumonia, microarray analysis and ingenuity pathway analysis (qiagen, redwood city, ca) was performed on critically ill patients with h n influenza a pneumonia. the most severely ill group of patients demonstrated impaired expression of numerous genes participating in adaptive immune responses (e.g., diminished antigen presentation, b-cell development, t-helper cell differentiation, and apoptosis), suggesting impaired adaptive immunity in severe viral pneumonia ( ) . in terms of epigenetics, many postulate that longterm epigenetic changes following severe pneumonia are responsible an increased likelihood of later infections and death, although specific epigenetic changes are yet to be identified ( ) . natural infection with viral causes of pneumonia does not induce long-term protective immunity due to an evolutionary advantage allowing viruses to evade host immune defenses via antigenic shift and drift. antigenic drift occurs with small point mutations in the viral genome leading to minor changes in key viral epitopes, while antigenic shift is a major change in a key gene leading to a complete exchange of a key epitope ( ) . antigenic shift often leads to influenza epidemics secondary to vaccine strain-circulating strain mismatches. antigenic shift is the molecular mechanism by which novel influenza strains emerge and is the cause of pandemics such as the h n pandemic ( ) ( ) ( ) ( ) ( ) . influenza vaccines rely on conserved antigens such as ectodomain of influenza m protein, m e, or hemagglutinin stalk domains. hemagglutinin globular head specific antibodies confer immunity since it interferes with virus attachment to host cell receptors; however, they are also one of the most variable viral antigens ( ) . additional adjuvants are important in vaccine formulations to induce desired immune responses that would not be triggered with the antigen alone ( ) . the need for adjuvants in vaccinations confers an additional important role for damps and pamps in viral immunity. one recent study used pamp tlr agonist and commonly used pharmaceutical additive to induce the release of damps to improve immunogenic response to the seasonal influenza vaccine ( ) . prevention of viral pneumonia is mainly limited to influenza vaccines since the formalin-inactivated rsv vaccine in the s failed secondary to adverse events ( ) . however, oral adenovirus vaccination has been used in military populations with -fold reduction of respiratory illnesses ( , ) . production of this vaccine was stopped in but was reintroduced in , leading to a dramatic and sustained decrease of acute respiratory distress outbreaks among u.s. army trainees ( , ) . additionally, there is still ongoing work to develop a vaccine to prevent rsv infection. recently, one study reported protective immunity against rsv with a molecularly adjuvanted adenovirus serotype based rsv oral vaccine in a rat model ( ) . however, two recent randomized control failed to establish an effect of anti-rsv monoclonal antibodies and recurrent wheeze of early childhood or asthma ( ) ( ) ( ) ( ) . since the covid- caused by the novel coronavirus known as sars-cov- began its rapid spread in wuhan, china, in november , researchers have responded swiftly to help thwart the pandemic by quickly establishing studies to better understand the virus. sars-cov- is a novel beta-coronavirus that likely originated in bats. the virus uses a glycosylated spike protein to bind to and enter the human host cell predominantly via angiotensin-converting enzyme receptors that are highly expressed in type alveolar cells ( ) . the clinical presentation of covid- can be indistinguishable from other viral causes of pneumonia and include fever ( - %), dry cough ( - %), and fatigue or myalgia ( - %) ( , ) . the median age of confirmed covid- cases is in the th decade of life with a slight male predominance. twenty-five percent of patients have severe symptoms requiring intensive care treatment of which % develop respiratory failure requiring mechanical ventilation. chest radiograph imaging of these patients reveals bilateral patchy infiltrates and ct imaging shows ground-glass infiltrates. patients typically present with laboratory findings of prolonged prothrombin time, elevated lactate dehydrogenase, and lymphopenia ( % of patients) ( ) . however, it is unclear if the lymphopenia is related to direct cytotoxic effect of the virus or underlying chronic conditions ( , ) . there are limited publications on the autopsy results of patients who have died from covid- . however, pathologic samples show hyaline membrane formation, interstitial mononuclear inflammatory infiltrates, and multinucleated giant cells. there are also high levels of pro-inflammatory cytokines, such as il- and tnf-α. as with other causes of severe viral pneumonia, a "cytokine storm" occurs which also contributes to the high morbidity and mortality ( , ) . the most important aspect of early management of viral spread has been early isolation of those presenting with concerning symptoms, history, and high likelihood of exposure to prevent spread of the disease to those in immunocompromised states, the elderly, and/or those with comorbid conditions. a chest radiograph along with throat and mid-turbinate nasal swabs for respiratory viral panel (reverse transcriptase-polymerase chain reaction) are needed for proper diagnosis of covid- . among hospitalized patients, negative pressure rooms and airborne-droplet-contact precautions are important for prevention and further spread between patients and hospital care-workers ( ) . currently, there is no approved drug or vaccination for the treatment or prevention of sars-cov- viral pneumonia. there are many trials underway attempting to attenuate the disease with remdesivir, il- receptor blockers, il- , and antiretrovirals such as lopinavir-ritonavir ( ) . the new england journal of medicine recently published a randomized controlled trial evaluating the efficacy of lopinavirritonavir versus standard care alone in the treatment of adult hospitalized patients with severe covid- . there were no differences in hospital mortality, time to clinical improvement, or viral rna levels. although the median time to improvement was day shorter with lopinavir-ritonavir on intention-to-treat analysis, % of patients had adverse events requiring treatment discontinuation. therefore, it was concluded that there was no benefit observed with lopinavir-ritonavir treatment versus standard treatment of severe covid- patients ( ) . historically, hydroxychloroquine, an anti-malarial and antiinflammatory agent, has shown some promise in reducing mortality from sars and, therefore, is currently being studied for covid- ( ) . in one very limited study from france (n = per group, nonrandomized), hydroxychloroquine was associated with reduced viral load and reduced duration of viral detection which was further attenuated by the addition of azithromycin ( ) . research is already underway to create a vaccine to protect against sars-cov- . taking advantage of the similarities in structure between sars-cov (responsible for the sars epidemic) and sars-cov- (responsible for covid- ), studies have mapped several epitopes to be targeted for a potential vaccine ( , ) . who estimates an approximately -month timeframe for covid- vaccine availability. until such time that effective therapies and vaccines become available, public health efforts should continue to focus on mitigating the spread of sars-cov- through well-established infection control strategies ( ) . this can be aided in the hospital with admission of sars-cov- positive patients into negative pressure rooms with contact precaution protocols requiring personal protective equipment such as gowns, gloves, fit-tested n respirators, and face shields. additionally, rules limiting the people entering the isolation room and requiring logging of healthcare workers involved in covid- patient care should be followed to effectively monitor patient contact and limit spread. all equipment (monitors, etc.) in the isolation room should be designated for the case patient only. physicians should limit potential spread by recognizing any necessary aerosol-generating procedures and preparing accordingly (e.g., for intubation using controlled measures including paralytics, video laryngoscopy, n masks). although fomites are suspected as the main source of transmission, there is also possible fecal-oral transmission; therefore, hand washing is a mainstay of control/prevention ( ) . although viral pneumonia is common, the specific inflammatory and immunosuppressive effects it has on the host is still largely unknown. covid- has brought viral pneumonia and subsequent host pathology to the forefront of medical care and research. sars-cov- spread worldwide in a matter of months to cause a pandemic not seen since influenza in . our highly interconnected global society creates ample opportunity for the rapid spread of novel viruses. since these types of viral pandemics have occurred multiple times historically (e.g., influenza in , middle east respiratory syndrome in , and sars in ) and will continue to occur in the future, research into immunomodulative therapies for patients afflicted with viral 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of covid- : results of an open-label non-randomized clinical trial preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus review of the clinical characteristics of coronavirus disease (covid- ) world health organization: coronavirus disease (covid- ) technical guidance: infection prevention and control/wash. key: cord- - v tl gi authors: arias, armando; emmott, edward; vashist, surender; goodfellow, ian title: progress towards the prevention and treatment of norovirus infections date: - - journal: future microbiol doi: . /fmb. . sha: doc_id: cord_uid: v tl gi noroviruses are now recognized as the major cause of acute gastroenteritis in the developed world, yet our ability to prevent and control infection is limited. recent work has highlighted that, while typically an acute infection in the population, immunocompromised patients often experience long-term infections that may last many years. this cohort of patients and those regularly exposed to infectious material, for example, care workers and others, would benefit greatly from the development of a vaccine or antiviral therapy. while a licensed vaccine or antiviral has yet to be developed, work over the past years in this area has intensified and trials with a vaccine candidate have proven promising. numerous antiviral targets and small molecule inhibitors that have efficacy in cell culture have now been identified; however, further studies in this area are required in order to make these suitable for clinical use. research in the area of norovirus vaccines and antivirals has increased in intensity in the past years. this is at least partly due to better surveillance mechanisms, improving our understanding of the disease burden, but also due to the availability of assays to study the development of the immune response and the efficacy of small molecule inhibitors. while the efficient culture of human noroviruses (hunovs) in immortalized cells has yet to be achieved [ ] , the development of a norovirus replicon [ ] , which allows the generation of cell lines stably replicating norwalk virus rna, has facilitated many small molecule inhibitors to be tested in vitro. the discovery of murine norovirus (mnv) [ ] , which replicates efficiently in immortalized macrophage cells and has both reverse genetics systems and small animal models available [ ] , has also enabled the examination of the immune responses to noroviruses as well as the efficacy of inhibitors in vitro and in vivo. noroviruses are members of the caliciviridae family of small positive-sense rna viruses and in a similar manner to other rna viruses, they replicate using an error prone mechanism that generates a high degree of genetic diversity. noroviruses are divided into five genogroups (gs; gi-gv), with only gi, ii and iv causing disease in humans [ ] . the error prone replication results in rapid evolution and the generation of new antigenic variants leading to increased diversity and the rapid emergence of new strains capable of evading any herd immunity [ ] . despite these difficulties, substantial efforts have been placed on the generation of control measures, treatments and vaccines, with recent developments in these areas reviewed below. hunovs are one of the major causes of foodborne gastroenteritis [ ] . norovirus outbreaks are frequently associated with bivalve shellfish, such as oysters, or with freshly prepared produce such as salads or fruit, generally due to contamination of water with infected fecal matter, or from an infected food handler [ ] . oysters are particularly problematic as they are often eaten raw, and they filter and trap large numbers of particles, serving to concentrate viruses. outbreaks from contaminated oysters display distinct seasonality and, as such are amenable to testing, detection and forecasting measures [ ] . in terms of decontamination methods, high-pressure inactivation ( mpa) has shown to effectively neutralize infectivity in oysters seeded with hunov in volunteer studies, with the added advantage of not involving chemical treatment of the food [ ] . other methods including γirradiation, thermal inactivation, steam-ultrasound, uv radiation, chloride or ozone disinfection, and electron beam irradiation have also been tested with varying degrees of success [ ] . however, the vast majority of such studies are conducted with norovirus surrogates such as feline calicivirus or mnv, as testing decontamination procedures for hunov is difficult owing to the lack of an available cell culture system to detect any remaining infectivity. they often have limited efficacy against noroviruses with some reports suggesting that their use is in fact a risk factor for norovirus outbreaks in hospitals, although this is currently controversial [ ] . simple mechanical removal of the virus through adherence to proper hand washing techniques appears most effective with the additional incorporation of alcoholbased sanitizers into a hand washing protocol offering minor improvements [ ] . noroviruses remain viable on contaminated surfaces for extended periods of time, and can transfer between surfaces with relative ease [ ] . effective decontamination of noroviruscontaminated surfaces is therefore of vital importance. in the home, the cdc recommends decontamination with diluted solutions containing sodium hypochlorite (bleach). studies investigating different cleaning regimens for hospitals have shown that detergent-based cleaning is ineffective alone; however, combined detergent and bleach may be effective. for the decontamination of larger areas following outbreaks newer approaches such as use of hydrogen peroxide vapor are under investigation and show promise, although they have only been tested against hunov surrogates to date [ ] . control measures are usually implemented for containing and slowing norovirus outbreaks particularly in closed or semiclosed environments such as hospitals, care homes or military bases. common control procedures during an outbreak include quarantine of infected individuals, enhanced environmental decontamination and enhanced hand hygiene [ , ] . adherence to control measures are often problematic with only % compliance of staff reported during one study [ ] . whilst control measures are clearly important for limiting a norovirus outbreak, further studies are needed to determine their efficacy and apply the appropriate procedures in each occasion. there are no commercially available prophylactics against norovirus that could be of use during an outbreak. one strategy currently being explored is the use of glycosylated hydrogels [ ] . these are crosslinked polymers that can swell to many times their original size upon the addition of water. the incorporation in the hydrogel of human blood group antigens (hbgas), which are the receptor molecules for hunov [ ] , enables the trapping of viral particles. hunov bound to hydrogel would then pass through the gastrointestinal tract and be excreted as normal. a more recently proposed approach involves providing passive immunization by administering antinorovirus antibodies prepared in chicken eggs (igy) [ ] . the development of effective vaccines has been delayed by the inability to propagate hunov in cell culture, preventing the use of viral neutralization assays to monitor the effectiveness of antibody responses [ ] . the recent establishment of a receptor-blocking assay whereby the ability of antisera to prevent the interaction of recombinant norovirus virus-like particles (vlps) with soluble hbga is examined and has proven to be a suitable alternative to an in vitro neutralization assay [ ] . norovirus vaccine development has also been limited by the fact that the immune response to hunovs is not well understood [ ] . for instance, infection in volunteers with norwalk virus resulted in a lack of long-term protection against reinfection, suggesting that the immune system is unable to generate a durable response. in addition, given the substantial norovirus strain diversity, it may be difficult to generate a broadly crossreactive vaccine capable of protecting against all norovirus strains. the cost of vaccine development for hunov is high, although a recent study has indicated that it would result in substantial cost savings, ≤us$ . million over years [ ] . despite the difficulties, there is a strong case indicating the feasibility of a norovirus vaccine [ ] . importantly, the expression of the norovirus major capsid protein vp in eukaryotic cells leads to assembly into vlps, which are antigenically and morphologically similar to native noroviruses. the inoculation of norovirus vlps in vivo results in strong humoral and cellular immune responses [ ] . neutralizing antibodies generated during infection or immunization with vlps are able to block the binding of viral capsids to their hbga receptors [ ] . large-scale systems for the preparation of hunov vlps have been developed, including expression in plants such as tobacco and potato, vlps from which are immunogenic in mice [ ] . the route of inoculation is one of the main factors influencing the efficacy of a vaccine. intranasally inoculated vlps induced a protective immune response in volunteers subsequently challenged with norwalk virus, leading to a % reduction in the occurrence of gastro enteritis in vaccinated volunteers [ ] . high levels of specific iga antibodies against hunov were detected, further supporting this route of inoculation to induce a robust mucosal protection. the advantages of an approach based on intranasal delivery are its ease of administration and the stimulation of mucosal dendritic cells facilitating a local immune response [ ] . in fact, a strong mucosal iga response in gastrointestinal and respiratory tracts has been associated to increased protection against hunov [ ] . studies involving an intramuscular route of vaccination in chimpanzees have also provided positive results for norovirus vaccination. animals inoculated with norwalk virus vlps (gi), were protected against subsequent challenge with norwalk virus, while animals vaccinated with vlps from a gii norovirus were not [ ] . these results highlight the need for the development of efficient bivalent and broadly cross-reactive vaccines. in summary, the development of vaccines against hunov is clearly achievable; however, it is probable that more efficient vaccine formulations are required to overcome the problems associated with the large strain diversity. while promising, vaccines may not be suitable for the treatment of immunocompromised patients in which long-term secretion is common, or for the control of rapidly evolving outbreaks. in both cases, the use of antiviral approaches are likely to be more appropriate. antiviral strategies against hunovs can target many aspects of the virus life cycle; viral proteins or cellular proteins directly, or processes required for virus replication (figure , tables & ). the development of such therapies requires an in-depth knowledge of the norovirus life cycle (figure ), yet the inability of hunov to be efficiently propagated in cell culture has resulted in a limited understanding of this process. a growing body of literature exists on mnv and other members of the caliciviridae family, where parallels can be drawn [ ] . studies on other positive-strand rna viruses may also provide potential insights into antiviral approaches that may be applicable to hunov. hunov interacts with hbgas and, although binding to hbga is not sufficient to enter cultured cells, it is thought that this interaction is critical for virus internalization and subsequent infection [ , ] . hbgas are complex carbo hydrate molecules present in the surface of red blood cells, mucosal epithelia and also in different body fluids (i.e., milk and saliva). hunov capsids interact with different families of hbgas including abo, secretor and α , -sialylated carbohydrates of the type chain (e.g., sialyl-lewis x). individuals harboring a single polymorphism in the fut gene are less susceptible to hunov infection, further supporting the hypothesis that hbgas are the receptor molecules for hunov [ , ] . the fut gene encodes for a α , -fucosyl transferase that catalyzes the fucosylation of hbgas and is responsible for the secretor blood type [ ] . nonsecretor individuals present a polymorphism in the fut gene resulting in an enzyme unable to fucosylate h-type backbones in hbgas. although the interaction with hbga is required for virus entry, it is thought that other cellular cofactors are required as overexpression of fut does not rescue infectivity in cells that are competent for hunov replication [ ] . recent investigations suggest that hunov binding to cells and internalization in vivo can also occur independently of hbga, suggesting the participation of other receptor molecules [ ] . in particular, it has been demonstrated that hunov capsids interact with heparan sulphate, a cell membrane glycosaminoglycan, which might have a role in cell entry [ ] . by contrast, mnv uses the ganglioside gd a as an attachment ligand for infection of permissive cells, although the relevance of this molecule for hunov has yet to be determined [ ] . the processes following hunov capsid internalization are largely unknown owing to the absence of efficient cell culture systems supporting infection. studies with the related norovirus mnv demonstrated that virus internalization is dependent on cholesterol and dynamin in a clathrin-and caveolae-independent pathway [ , ] . once the viral genome (a positive strand rna molecule - kb in length) is released in the cytoplasm, viral proteins are synthesized ( figure ). the hunov genome contains three open reading frames (orfs). orf encodes a polyprotein that, after proteolytic processing, results in the production of six or seven mature nonstructural proteins (ns - ). these include ns pol , the viral rna-dependent rna polymerase (rdrp) responsible for viral rna synthesis, as well as ns pro , the viral protease that catalyzes the proteolytic processing of orf . translation of structural proteins vp (orf ) and vp (orf ) occurs from a subgenomic rna produced during virus replication ( figure ). the process of viral protein translation in noroviruses relies on the recruitment of eukaryotic translation initiation factors to the virus encoded vpg protein (ns ) covalently linked to the ′ end of the viral rna. a specific interaction between vpg and eif and/or eif e has been demonstrated and may contribute to viral translation initiation [ , ] . infection [ , ] . mnv replication complexes can be found associated with double membrane vesicles, originating in the perinuclear region colocalizing with early and late secretory pathway components [ ] . viral replication takes place adjacent to the microtubule organizing center, and utilizes microtubules to position the replication complex, as their chemical disruption reduces viral replication [ ] . for hunov there is limited information regarding intracellular rearrangements associated to virus replication although it has been suggested that the replication complex is associated to membranes that originated from the disassembly of golgi [ ] . based on the extensive knowledge on the adhesion of hunov capsid to different carbohydrates on the surface of the cell [ , ] , multiple approaches targeting viral entry have been proposed. hunovs maintain highly conserved residues in the hbga interaction surface; however, this conservation is only maintained among members of the same genogroup [ ] . in gii viruses, the conserved structural region involved in the interaction with α , -fucose present in hbga is located in the outer dimeric capsid interface; while in gi viruses, the interacting surface with hbga molecules comprise residues from a single capsid monomer [ ] . given the large degree of conservation of capsid interacting surfaces, antiviral approaches have been focused on the design of specific molecules against them ( figure ). examples of carbohydrate analogs with potential antiviral activity are citrate and other glycomimetics since they resemble the molecular structure of fucose ( figure ) [ ] . the identification of novel antiviral compounds targeting the capsid has also been attempted with a novel approach based on library screening, x-ray crystallography and nuclear magnetic resonance [ ] . this method has been instrumental in the identification of two new antiviral candidates with high affinity for the fucose binding pocket of norwalk virus. standard screening studies involving > synthetic small molecules and enzyme immune assay have also been applied successfully with different compounds identified to effectively inhibit hunov capsid binding to at least one of the members of the hbga abo family [ ] . since hunov capsid binds heparan sulphate, another possible field of investigation would be the use of heparan sulphate analogs such as heparin, suramin and other heparan derivatives [ ] . the observation that suramin also targets the viral polymerase (discussed later), further supports its use as a potential therapeutic approach. one feasible possible approach yet to be followed would be the identification of therapies targeting α , -fucosyl transferase encoded by the fut gene with the aim of transiently reducing hbga fucosylation. nonsecretor individuals are naturally less sensitive to infection [ ] , supporting the hypothesis that transient reduction in the fucosylation may reduce susceptibility to infection. vpg-host factor interactions implicated in viral translation-genomic and subgenomic norovirus rnas recruit host factors for their translation via the viral vpg protein, which is covalently bound to the ′ end of the viral rna [ , ] . the use of europe pmc funders author manuscripts specific drugs aimed to disrupt these interfaces could provide an attractive antiviral therapy. hunov vpg is known to interact with eif and components of the eif f complex, thus a possible approach would consist of targeting either of these interactions with small molecules that disrupt their interaction with the norovirus vpg protein but not host cell factors. hippuristanol is an encouraging example of the existence of drugs that specifically affect virus translation with lower toxicity for the cell. it is a marine compound identified during the screening of a panel of small molecules with affinity for the c-terminus of eif a. hippuristanol inhibits translation of both mnv and feline calicivirus (fcv) and appears to be less toxic for cells than for the virus [ ] . hippuristanol has been recently used to control tumor growth in mice, supporting its use in vivo [ ] . other approaches targeting translation initiation include the development of drugs against host factor interacting surfaces in vpg that could now be facilitated by the recent description of mnv and fcv vpg protein structures [ ] . viral protease inhibitors-norovirus orf translation produces a large polypeptide that is cleaved to release the various mature nonstructural proteins in a process carried out by the viral ns protease (and its precursors). the recent resolution of the hunov ns pro crystal structure has allowed the identification of new inhibitors based on the specific recognition of the peptide substrate by the protease [ ] . a series of products, including bisulfite adducts, dipeptide and tripeptide aldehydes, ketoamides and ketoheterocycles have been synthesized and shown to elicit a strong inhibitory activity against ns pro in vitro and reduced virus replication in cell culture [ , ] . the inhibition of protease activity is an approach widely used for other viral infections [ ] , which in the case of noroviruses, results in reduced levels of the mature nonstructural proteins required for viral replication. targeting the viral rna: rnai & phosphorodiamidiate morpholino oligomers -targeting the norovirus rna genome directly has also been investigated as a method of regulating virus replication. phosphorodiamidate morpholino oligomers (pmos) inhibit protein expression of a target molecule by annealing in a watson-crick conformation, causing the steric blockade of protein translation. peptide-conjugated pmos (ppmos) directed against the first aug region in norovirus orf gene are effective in inhibiting hunov and mnv replication in cell culture [ ] . pmos are similar to dna oligonucleotides, but they are soluble in water and highly resistant to degradation making them suitable for treatments in vivo. the use of sirna molecules to target replication is more efficient than ppmos; ppmos and sirnas targeting the equivalent genomic sequence in fcv showed that sirna elicited inhibition -fold larger than ppmos [ ] . these promising approaches based on sirna or shrna molecules may, however, require technical improvements to achieve efficient delivery in vivo [ ] . interferon-type i and ii interferons elicit a robust anti viral response against hunov and mnv, which appears to be due to direct inhibition of virus translation [ , ] . norovirus replication inhibition is stronger when interferon treatment is combined with ribavirin (rbv), as discussed below [ ] . given that the combination of rbv and interferon is currently used for the clinical treatment of hcv infections, it is likely that the use of the same combination may be effective against noroviruses in vivo. however, to date, the europe pmc funders author manuscripts therapeutic use of rbv or interferon for the treatment of norovirus infection in humans is yet to be described. targeting initiation of replication by vpg-norovirus genome replication requires the protein primer vpg to initiate viral rna synthesis [ ] . preceding the initiation of viral rna replication, vpg must be guanylated in a step carried out by the virus polymerase ns pol or its precursor ns -ns [ ] . this guanylated-vpg product is then used as a primer for viral rna synthesis. accordingly, it may be possible to design specific nucleoside analogs to compete with gtp to inhibit vpg-nucleotydylylation, or to be incorporated but prevent the subsequent elongation of viral rna synthesis. for example, in picornaviruses, -fluorouracil triphosphate functions as an inhibitor of vpg-primed rna synthesis [ ] . the -fluorouracil triphosphate is bound to vpg with higher affinity than uridine ′-triphosphate, the natural nucleotide typically incorporated, and this binding inhibits the formation of long vpg-primed rna polymers, suggesting that its antiviral activity is partly due to the blocking of viral rna synthesis initiation [ ] . inhibition of viral rna synthesis by nucleoside analogs-typically, the rdrp of rna viruses are selected as key targets of many antiviral compounds, including nucleoside and non-nucleoside analogs, as they play a central role in the virus life cycle. several nucleoside compounds have activity against noroviruses in cell culture, opening up their possible application as therapeutic compounds. rbv, a purine analog that has been found to possess antiviral activity against a vast number of different viruses [ ] , also works against hunov and mnv in cell culture [ ] . rbv is phosphorylated by cellular enzymes into rbv mono-, di-and tri-phosphate and exerts its broad antiviral effect through various mechanisms: competitive inhibition of inosine monophosphate dehydrogenase, which reduces the intracellular concentrations of guanine nucleotides; inhibition of viral rnadependent rna polymerases; inhibition of mrna cap formation; enhancement of antiviral immune responses; and lethal mutagenesis of viral quasi species as a result of incorporation of rbv monophosphate into viral rna (reviewed in [ ] ). for mnv and the novovirus replicon, rbv antiviral activity is associated with a decrease in gtp levels since the complementation with gtp reduces its antiviral activity. mycophenolic acid, a nonnucleoside inosine monophosphate dehydrogenase inhibitor, also affects norovirus replication further supporting that norovirus is affected by imbalances in the ntp pools. there is no evidence however, for increased mutation frequencies in noro virus genomes after rbv treatment, suggesting it does not elicit antiviral activity through lethal mutagenesis [ ] . a strong inhibitory activity on mnv replication in cell culture was observed using ′-cmethylcytidine ( cmc) [ ] , a drug that was initially developed to treat hcv. recent studies have shown that cmc and other derivatives are also efficient in the control of hunov replication in cell culture [ ] . concerns over the use of cmc have been raised owing to the observation that some toxicity occurred in patients treated for hcv [ ] . new derivatives of cmc with reduced toxicity in patients are currently under investigation [ ] , which could also hold promise against norovirus infections. a recent study with mnv has also supported the use of favipiravir, a nucleoside analog with some homology to rbv, to treat norovirus infections [ ] . favipiravir is similar to rbv in that it is effective against a broad range of viruses and increased mutation frequencies associated have also been reported [ ] . other nucleoside analogs, ′-arauridine and ′deoxyuridine, have also been found to inhibit the viral polymerase activity in vitro [ ] ; however their efficacy in cells or in vivo remain to be tested. non-nucleoside analogs targeting the viral polymerase-apart from nucleoside analogs, where inhibition is directed to the catalytic site of the viral polymerase, other nonnucleoside compounds have been found to inhibit the norovirus rdrp, in particular, suramin and nf [ ] . enzymatic assays have confirmed that suramin and nf are inhibitors of hunov and mnv rdrps, with ic values in the nanomolar range. importantly, suramin is a drug currently used in the clinical treatment of sleeping sickness caused by trypanosoma [ ] . in addition to its capacity to inhibit viral rna replication, suramin has been found to inhibit hunov capsid binding to heparan sulphate [ ] . the styrylchromones are a new class of flavonoid compounds found to elicit antiviral activities against a broad spectrum of rna viruses, including mnv [ ] . evidence indicates that they might be targeting the viral rdrp, although the precise mechanism of action is not yet known. inhibition of viral ntpase activity-the norovirus ntpase (ns ) shares homology with other viral ntpases previously described, such as hcv ns and picornavirus c. these molecules are classified in the superfamily iii of rna helicases and it is believed that they catalyze the hydrolysis of nucleoside triphosphates to unwind the viral nucleic acids during replication, although no evidence for helicase activity has been reported [ , ] . nonetheless, the inhibition of viral ntpase activities results in the concomitant inhibition of viral rna synthesis. although no inhibitors have been identified against the norovirus ns , thiazolobenzimidazoles inhibit the replication of several different picornaviruses, by targeting the viral ntpase c [ ] . the untranslated regions in rna virus genomes normally recruit multiple host factors to the viral rna genome that play important roles in viral translation and replication. in noroviruses the untranslated regions are extremely short but the coding and noncoding regions in genomic and subgenomic rna are known to fold into highly-ordered secondary structures that interact with multiple host factors including proteins la, ptb, pcbp, ddx and various hnrnps among others [ , ] . although the precise role of these proteins in the norovirus life cycle is yet to be determined, they are probably involved in viral rna replication and genome circularization, a process that is thought to be required for many rna viruses [ ] . so far, no antinorovirus strategies have been investigated based on targeting these proteins, but as small molecule inhibitors exist for some of them, further studies in this area are warranted. for example, the identification of small inhibitory molecules targeting ddx has opened the possibility of targeting norovirus infections in vivo as specific downregulation of ddx protein levels in cells results in the inhibition of mnv [ ] . supporting this possibility, ddx inhibitors have shown antiviral activity against hiv in cell culture [ ] . ddx is a multifunctional host cell rna helicase europe pmc funders author manuscripts implicated in the life cycle of a number of viruses [ ] . ddx contributes to the innate immune response, but also to host cell translation initiation via interaction with eif f and eif [ ] . the la protein interacts with hunov rna and reduction of the cellular levels of la also affects norovirus replication in cells [ , ] . downregulation of cellular la protein levels by small molecule inhibitors has been recently found to elicit antiviral activity against hbv [ ] , supporting its testing against noroviruses. la is an rna binding protein originally identified as an autoantigen in diverse autoimmune syndromes and is typically involved in the maturation and translation of some cellular mrnas [ ] . in rna viruses, la participates in the regulation of inositol-requiring enzyme substrate-driven translation from a variety of different positive-strand rna viruses; however, its role in norovirus replication has yet to be elucidated. we have also identified ptb as an important factor associated to norovirus rna and its binding to a pyrimidine-rich tract in the ′ terminal stem-loop contributes to virulence in the host [ ] . downregulation of ptb protein levels in cell culture also results in decreased mnv replication [ ] . whilst the function of ptb remains to be fully elucidated, work with fcv, a related member of the caliciviridae, indicated that ptb plays a negative role in viral translation, possibly regulating the shift between translation and replication [ ] . hnrnp a has also been reported to interact with mnv rna [ ] . the hnrnps are typically involved in the metabolism of cellular precursor mrnas [ ] . antitumor drugs targeting hnrnp a , such as camptothecine and derivatives like -nitrocamptothecine [ ] , could be tested for the treatment of norovirus infections. inhibition of ubiquitinases & cellular stress response-the cellular deubiquitinase (dub) usp has recently been shown to be involved in hunov and mnv replication, and its specific downregulation or inhibition results in reduced virus replication in cell culture and in vivo [ ] . usp interacts with the inositol-requiring enzyme , a central protein in the activation of the unfolded protein response (upr) as a result of cellular stress. usp has been also identified to interact with norovirus ′ genomic rna extremity, although whether this is a direct rna-protein interaction or one mediated via an intermediate interacting protein remains to be determined [ ] . a small molecule inhibitor of several dubs including usp has been found to inhibit both mnv and novovirus replication, and this inhibition is associated to the activation of upr in an inositol-requiring enzyme dependent mechanism. the authors have proposed that the inhibition of cellular dubs leading to the subsequent upr constitute a novel target approach to block viral infections [ ] . membrane rearrangement & trafficking host factors-hunov ns - protein, p , interacts with vap-a, a snare-binding protein with an important role in cellular vesicle transport regulation. it is believed that this interaction is responsible for localizing ns - to intra cellular vesicles in cells and preventing normal intracellular protein trafficking [ ] . expression of hunov ns - is sufficient to block the expression of membrane proteins at the cell surface. therapies based on disrupting the interaction between these proteins or regulating the membrane rearrangements have yet to be explored but may prove a tractable approach worth considering. for example, botulinum toxin type b is a drug that targets vamp- and has been approved for the treatment of cervical dystonia [ ] ; however, the issue of toxicity and the ability to deliver to the site of virus replication remains a significant obstacle to be overcome. the socio-economic impact of norovirus infection is now well established, thus the case for the development of effective vaccines and antiviral approaches is strong. whilst there are many inhibitors that have proven efficacy against noroviruses in cell culture and at least one that appears to work in the mouse model for mnv, none have made it through to clinical use for the treatment or prevention of norovirus infection. as with all therapies targeting intestinal pathogens, there is a significant problem associated with the delivery of the inhibitor to the site of replication, as well as stability in this rather harsh environment. subsequently, there are a number of significant hurdles that must be overcome for the use of these inhibitors in a clinical setting. an area that has yet to be fully explored is the use of inhibitors that are currently in clinical use for other rna viruses. rbv is widely used for the treatment of viral infections and has been shown to be effective in cell culture against hunov, yet clinical use in the treatment of chronic norovirus infections has yet to be described. given the conserved nature of rna synthesis, the viral rna polymerase provides a particularly attractive target as numerous nucleoside and non-nucleoside inhibitors are under trial for other rna viruses such as hcv. clearly further studies in this area are required; these inhibitors hold great promise for the treatment of numerous rna virus infections, including noroviruses. as discussed above, a major limitation to the development of effective control measures has been the lack of a fully permissive cell culture system and small animal model for hunovs. the momentum generated by researchers in the field has resulted in numerous developments in these areas, yet a licensed vaccine or clinically approved antiviral remains elusive. the development of an in vitro culture system and an animal model that recapitulates all aspects of the disease observed in humans would prove invaluable and should remain a high priority. a better understanding of the correlates of protection would also enable better vaccine design and could lead to the generation of a broadly protective vaccine that may at least produce short-term immunity in particularly vulnerable or regularly exposed individuals. until such a time, the use of effective quarantine and hygiene measures remain the only alternative for the control of norovirus outbreaks and provide a way of minimizing the impact of these important pathogens. a case for norovirus therapeutic approaches ■ noroviruses are a major cause of gastroenteritis. ■ infection results in > , hospitalizations and deaths in the usa per annum. ■ economic losses exceed us$ billion per annum in the usa alone. noroviruses are a very diverse group of viruses that replicate using an error prone mechanism, facilitating the regular emergence of new antigenic variants. ■ chronic long-term shedding of virus is common in the immunocompromised. ■ noroviruses are stable in the environment and difficult to remove. ■ current control measures involve quarantine and increased hygiene practices. ■ alcohol hand sanitizers are generally poorly effective against noroviruses. vaccines ■ expression of the major capsid protein in eukaryotic cells produced virus-like particles that are antigenically identical to infectious norovirus. immunization with virus-like particles produces a protective immune response, which is genogroup specific. the development of broadly crossprotective immune responses has yet to be achieved. the norovirus life cycle is poorly understood owing to an inability to culture human norovirus in immortalized cells. ■ recent work has developed human norovirus replicons and identified murine norovirus as the only norovirus that can replicate efficiently in cell culture. ■ numerous antivirals targeting many aspects of the virus life cycle have now been identified but very few have been tested in animals. targeting the viral rna polymerase or protease holds promise given the effectiveness in other viral systems. environmental transmission of norovirus gastroenteritis ranking the disease burden of pathogens in food sources in the united states using attribution data from outbreak investigations and expert elicitation norovirus and medically attended gastroenteritis in us children norovirus gastroenteritis in immunocompromised patients laboratory efforts to cultivate noroviruses stable expression of a norwalk virus rna replicon in a human hepatoma cell line the identification of murine norovirus, which was used as a surrogate model for human noroviruses and subsequently providing insights in the molecular biology model systems for the study of human norovirus biology viral shape-shifting: norovirus evasion of the human immune system emergence of salsa and guacamole as frequent vehicles of foodborne disease outbreaks in the united states detection and forecasting of oyster norovirus outbreaks: recent advances and future perspectives critical review of norovirus surrogates in food safety research: rationale for considering volunteer studies comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and gii. norovirus hand hygiene regimens for the reduction of risk in food service environments hydrogen peroxide vapour decontamination of surfaces artificially contaminated with norovirus surrogate feline calicivirus author manuscript; available in pmc europe pmc funders author manuscripts europe pmc funders author manuscripts a review of nosocomial norovirus outbreaks: infection control interventions found effective adherence to selfquarantine recommendations during an outbreak of norovirus infection trapping norovirus by glycosylated hydrogels: a potential oral antiviral drug norovirus-host interaction: multi-selections by human histo-blood group antigens evaluation of anti-norovirus igy from egg yolk of chickens immunized with norovirus p particles norovirus virus-like particle vaccines for the prevention of acute gastroenteritis binding of norwalk virus-like particles to abh histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice norwalk virus vaccines: challenges and progress the potential economic value of a human norovirus vaccine for the united states norwalk virus-like particles as vaccines norovirus vaccine against experimental human norwalk virus illness recombinant norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses an intranasally delivered toll-like receptor agonist elicits robust systemic and mucosal responses to norwalk virus-like particles chimpanzees as an animal model for human norovirus infection and vaccine development norwalk virus rna is infectious in mammalian cells norovirus binding to intestinal epithelial cells is independent of histo-blood group antigens genogroup ii noroviruses efficiently bind to heparan sulfate proteoglycan associated with the cellular membrane ganglioside-linked terminal sialic acid moieties on murine macrophages function as attachment receptors for murine noroviruses murine norovirus- cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin-and cholesterol-dependent pathway endocytosis of murine norovirus into murine macrophages is dependent on dynamin ii and cholesterol the genome-linked protein vpg of the norwalk virus binds eif , suggesting its role in translation initiation complex recruitment calicivirus translation initiation requires an interaction between vpg and eif e norwalk virus nonstructural protein p forms a complex with the snare regulator vap-a and prevents cell surface expression of vesicular stomatitis virus g protein mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway mouse norovirus utilizes the cytoskeleton network to establish localization of the replication complex proximal to the microtubule structural basis for norovirus inhibition and fucose mimicry by citrate targeting norovirus infection-multivalent entry inhibitor design based on nmr experiments library screen for inhibitors targeting norovirus binding to histo-blood group antigen receptors caliciviruses differ in their functional requirements for eif f components effects of hippuristanol, an inhibitor of eif a, on adult t-cell leukemia structures of the compact helical core domains of feline calicivirus and murine norovirus vpg proteins inhibition of norovirus cl protease by bisulfite adducts of transition state inhibitors broad-spectrum antivirals against c or c-like proteases of picornaviruses, noroviruses, and coronaviruses inhibition of norovirus replication by morpholino oligomers targeting the ′-end of the genome antiviral strategies to control calicivirus infections interfering with disease: a progress report on sirna-based therapeutics interferons and ribavirin effectively inhibit norwalk virus replication in replicon-bearing cells type i and type ii interferons inhibit the translation of murine norovirus proteins protein-primed and de novo initiation of rna synthesis by norovirus dpol nucleotidylylation of the vpg protein of a human norovirus by its proteinase-polymerase precursor protein -fluorouracil in lethal mutagenesis of foot-and-mouth disease virus mechanisms of action of ribavirin against distinct viruses inhibition of norovirus replication by the nucleoside analogue ′-c-methylcytidine antiviral activity of nucleoside analogs against norovirus nm ), alone or with peg-interferon, compared with peg-interferon/ribavirin (pegifn/rbv) retreatment in patients with hcv- infection and prior non-response to pegifn/rbv: one year results antiviral efficacy upon administration of a hepdirect prodrug of ′-c-methylcytidine to hepatitis c virus-infected chimpanzees t- ) inhibits in vitro norovirus replication t- (favipiravir) induces lethal mutagenesis in influenza a h n viruses in vitro structure-based inhibition of norovirus rnadependent rna-polymerases suramin: clinical uses and structureactivity relationships e)- -styrylchromones as potential anti-norovirus agents viral and cellular rna helicases as antiviral targets polypeptide p of a norwalk-like virus is a nucleic acid-independent nucleoside triphosphatase picornavirus non-structural proteins as targets for new anti-virals with broad activity ptb, and pab proteins bind to the (′) untranslated region of norwalk virus genomic rna ■■ proteomics identification of over different host factors that interact with norovirus genomic and subgenomic rna extremities animal virus schemes for translation dominance toward the discovery of novel anti-hiv drugs. secondgeneration inhibitors of the cellular atpase ddx with improved anti-hiv activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation viruses and the human dead-box helicase ddx : inhibition or exploitation? the role of the dead-box rna helicase ddx in mrna metabolism a novel inhibitor of human la protein with anti-hbv activity discovered by structure-based virtual screening and in vitro evaluation the la protein-rna complex surfaces functional analysis of rna structures present at the ′ extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence polypyrimidine tract binding protein functions as a negative regulator of feline calicivirus translation turning on a fuel switch of cancer: hnrnp proteins regulate alternative splicing of pyruvate kinase mrna camptothecin (cpt) directly binds to human heterogeneous nuclear ribonucleoprotein a (hnrnp a ) and inhibits the hnrnp a / topoisomerase i interaction antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response botulinum neurotoxins in the treatment of refractory pain this work has been supported by a wellcome trust senior fellowship (wt ma) granted to i goodfellow.the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. future microbiol. author manuscript; available in pmc january . europe pmc funders author manuscriptsno writing assistance was utilized in the production of this manuscript. the norovirus infectious particle interacts with hbga and hs expressed on the cell surface. the attached virion is then internalized and disassembled, leading to the release of the viral genome in the cytoplasm. vpg covalently attached to the ′ end of the viral genome recruits different hfs, including components of eif and eif f, required for cap-independent protein translation, permitting the expression of orf . orf is proteolytically cleaved by the viral protease ns pro (and its precursors). mature nonstructural proteins released after proteolysis promote the replication of the viral rna; vpg acts as primer of viral rna synthesis, remaining covalently attached to the viral genome after replication; the viral rna-dependent rna polymerase ns pol catalyze the synthesis of viral rna in a process assisted by the helicase-homologous viral ntpase (ns ). the binding of several host factors (hfs) such as la protein, ddx , hnrnp a and ptb to the viral genome is critical for the efficient virus replication. the replication of a subgenomic rna leads to the generation of shorter viral rna products that are translated in the major and minor capsid proteins vp and vp (orf and orf , respectively). newly synthesized genomes are encapsidated in mature particles that are released into the extracellular environment. hbga: histo blood group antigen; hf: host factor; hs: heparan sulfate; orf: open reading frame. key: cord- -k oku h authors: dong, hui-jun; zhang, rui; kuang, yu; wang, xiao-jia title: selective regulation in ribosome biogenesis and protein production for efficient viral translation date: - - journal: arch microbiol doi: . /s - - - sha: doc_id: cord_uid: k oku h as intracellular parasites, viruses depend heavily on host cell structures and their functions to complete their life cycle and produce new viral particles. viruses utilize or modulate cellular translational machinery to achieve efficient replication; the role of ribosome biogenesis and protein synthesis in viral replication particularly highlights the importance of the ribosome quantity and/or quality in controlling viral protein synthesis. recently reported studies have demonstrated that ribosome biogenesis factors (rbfs) and ribosomal proteins (rps) act as multifaceted regulators in selective translation of viral transcripts. here we summarize the recent literature on rbfs and rps and their association with subcellular redistribution, post-translational modification, enzyme catalysis, and direct interaction with viral proteins. the advances described in this literature establish a rationale for targeting ribosome production and function in the design of the next generation of antiviral agents. initiation, as the first stage of translation, can be carried out by either cap-dependent or cap-independent mechanism in eukaryotic cells (kapp and lorsch ) . the majority of the eukaryotic genome is translated via cap-dependent translation initiation, mainly through the s preinitiation complex (pic) binding to the mrna ′-end cap structure and scanning along the mrna to the aug initiation codon to initiate translation (haimov et al. ) . in contrast to the cap-dependent mechanism, the cap-independent mechanism is translated by s pic (or a single s unit in some specific mrna templates) directly binding to the internal ribosome entry site (iress) in the genome intergenic region (kapp and lorsch ) . the number and types of translation initiation factors required for ires-mediated translation initiation can vary significantly among specific mrna species. for example, eif e is not required for ires-mediated picornavirus genome translation (avanzino et al. ) , and ires elements present in the hepatitis c virus (hcv) genome require fewer translation initiation factors to initiate translation (torrecilla et al. ; kerr et al. ) . ribosomes are apparatuses that catalyze protein synthesis. the eukaryotic s ribosome is composed of two subunits: the s subunit contains the decoding center, which monitors the complementarity of transfer rna (trna) and messenger rnas (mrna) and is composed of ribosomal proteins (rps), and the s subunit, which catalyzes the formation of peptide bonds and is composed of rps (khatter et al. ; barna ) . ribosome biogenesis is a complex process, which takes place mainly within the nucleoli of eukaryotes, and requires more than ribosomal biogenesis factors (rbfs) (tafforeau et al. ) . rps are coordinated with ribosomal rna (rrna) to synthesize, mature, assemble, and export into the cytoplasm, and they are crucial participants in the cellular process of translation from mrna (lafontaine and tollervey ; moore ) . ribosome-mediated translational regulation can be explained by two different models simsek et al. ) . the ribosome-concentration hypothesis communicated by erko stackebrandt. proposes that the changes in cellular ribosome abundance may be a major driver of changes in translation of mrna pools. the specialized ribosomes hypothesis is based on the finding that some rps differ in composition and specifically regulate a subset of specific mrna (such as rpl for hox mrna) translation (shi and barna, ; xue and barna, ; simsek et al. ; briggs et al. ; genuth and barna, ) . rbfs control ribosome biogenesis to participate in the cellular process of mrna translation. several viruses have evolved sophisticated mechanisms that utilize cellular translational machinery to obtain efficient viral protein synthesis and viral replication (hilton et al. ; narayanan et al. ; li et al. a, b) . the ribosome itself (primarily ribosomal proteins), and the ribosomal biogenic processes that are exploited by viruses to facilitate their own replication, should be considered as targets in the development of antiviral agents, as depicted in table and as summarized in this study. ribosome biogenesis is critical for cell proliferation and stress response. the synthesis of viral proteins depends on the host ribosome, and ribosome biogenesis has implications for viral infection through the effects of rbfs on viral transcription, proliferation, and antiviral immune responses, as well as the effects of viral proteins on ribosomal biosynthesis. ribosomal rna transcription, catalyzed by rna polymerase i (pol i), plays a critical role in ribosome biogenesis. transcriptional activation of rrna is also closely associated with viral infection. however, restricting ribosome biogenesis by interfering with rrna accumulation-triggered ribosomal stress stimulated the viral replication of human cytomegalovirus (hcmv); the process inhibited the expression of innate immune-related genes, such as the high-mobility group box (hmgb ). this reveals that rrna accumulation and/or ribosome biogenesis regulate innate immune responses to restrict virus reproduction and regulate inflammation (bianco and mohr ) . similarly, ribosomes are required for the protein synthesis of host cells and viruses, but the biogenesis factor rbfs can also impact the proliferation of virus and cell-intrinsic immune responses. at this intersection of apparently competing processes, it is intriguing to find rbfs that are required specifically for the viral protein biosynthesis, but do not affect global translation. nop (gltscr /pict- ), for example, shares homology with the yeast s ribosomal protein nop p, which acts as an essential ribosome biogenesis factor (sydorskyy et al. ; thomson et al. ) . a previous study by the present author showed that nop , expressed as a set of discrete globular structures within the nucleolus, is migrated to the cytoplasm upon infection by herpes simplex virus (hsv- ) (meng et al. ) . furthermore, the cytoplasmic translocation of nop is required for efficient viral replication, which occurs via depression of the activity of innate immune receptor rig-i and decrease of the phosphorylation level of eif α to facilitate the production of viral proteins without affecting global protein synthesis (meng et al. (meng et al. , . similarly, both nucleolar proteins of ribosomal rna processing homolog b (rrp b) and nucleolin are involved in ribosomal biogenesis (chamousset et al. ) . rrp b associated with pre- s ribosomal subunits was translocated to the nucleoplasm upon viral infection of influenza virus, where rrp b improves viral rna-dependent rna polymerase (rdrp) activity, which is responsible for virus transcription and replication (su et al. ) . nucleolin, which is thought to control rna metabolism and ribosome biogenesis (cong, et al. ; allain ) , is prevented from entering the nucleus in poliovirusinfected cells (waggoner ) ; this allows stimulation of ires-mediated translation of viral transcripts (izumi ) . upstream binding factor (ubf) plays a major role in the regulation of rrna synthesis and associates with viral replication of adenovirus; the antibody against ubf reduces viral replication (lawrence et al. ) . in mammalian cells, there are two sets of ribosome particles, which reside in the cytoplasm and the mitochondria. some viruses manipulate mitochondrial biogenesis to support replication. it has been reported that the protein levels involved in mitochondrial ribosome biogenesis are significantly up-regulated by infection with human cytomegalovirus (hcmv). the inhibition of mitochondrial translation with chloramphenicol or knockdown of ribosome biogenesis factor mrm abolished the hcmv-mediated increase in mitochondrial coding proteins and significantly impaired viral growth (karniely et al., ) . the main function of the nucleolus is in ribosome biogenesis, regulation of the cell cycle, and the response to cellular stress. many viral proteins, such as the nucleocapsid protein of coronavirus (chen et al. ; cawood et al. ; dove et al. ; emmott et al. ; hiscox et al. ; you et al. ) , the matrix protein of newcastle disease virus (ndv) (peeples et al. ) , and the non-structural protein of influenza virus (melén et al. ) , transport to the nucleolus to regulate ribosome biosynthesis, change the nucleolar morphology, or affect the function of the nucleolus. during nucleocytoplasmic shuttling, viral proteins can recruit nucleolar rbfs to modulate ribosomal biosynthesis for efficient viral replication. the multifunctional nucleolar phosphoprotein nucleophosmin (npm) plays a lead role in ribosome biogenesis to stimulate rna pol i-dependent transcription (li and hann ) and regulates sars-cov, ibv, hiv- , hcv recruited by viral proteins to facilitate viral replication chen et al. ; zhou et al. ; goh et al. ; bortz et al. the export of ribosomal proteins and mature ribosomes to the cytoplasm (yu et al. ) . the hbx oncoprotein of hepatitis b virus (hbv) directly interacted with the c-terminal domain of npm to stimulate viral transcription (ahuja et al. ) . several rna helicases have been demonstrated to facilitate ribosome biogenesis, involving the processing of ribosomal rna (rrna) as well as its assembly into functional ribonucleoprotein complexes (bleichert et al. ). ddx promotes the synthesis and maturation of rrna and ultimately increases ribosome output and proliferation (jalal et al. ) . many viral proteins, such as coronavirus nsp (chen et al. ), rev of human immunodeficiency virus (hiv- ) (zhou et al. ), ns b of hepatitis c virus (hcv) (goh et al. ) , and np protein of influenza virus (bortz et al. ) have evolved to hijack ddx in order to facilitate viral replication. studies on the regulation or manipulation of viral protein in ribosome biosynthesis provides new insights into efficient viral replication. the ribosome was once considered to be a large protein synthesis machine that merely translated mrnas into proteins. in recent years, however, the role of ribosomes in the regulation of initiation and selection of mrnas has been the subject of increased attention. most rps are essential for translation and viral replication, and regulate translation of viral mrnas as constituents of the ribosome, although a few represent the defense signaling of host cells (zhou et al. ) . (the role of rps in activating an immune response is beyond the scope of the present paper.) of particular interest are the rps that are non-essential to the function of the ribosome, such as the large ribosomal subunit (rpl , rpl ) and the small ribosomal subunit (rps , rps ) (jack et al. ; karlas et al. ; xue et al. ; lee et al. ). in the process of coevolution with hosts, viruses have evolved different strategies to inhibit host translation or induce host translation shutoff while protecting their own protein synthesis. many viruses globally interfere with host translation by impairing cap-dependent ribosome recruitment to host mrnas. for example, vesicular stomatitis virus (vsv) inhibits the translation of host mrnas in infected cells, but allows the translation of its own capped mrnas (whitlow et al. ; wertz and youngner ) . rpl promotes efficient translation of vsv mrnas in a cap-dependent manner, but is not required for global protein synthesis (lee et al. ) . this may represent an endogenous specialized translation function for rpl . influenza a virus (iav) viral mrnas, on the other hand, are not preferentially translated compared to their host counterparts, and the extensive translation of viral proteins is the result of viral takeover of the mrna pool in the cell (bercovich-kinori et al. ) . ribosome footprints and mrna read density analysis reveal that rps are not strongly affected by iav infection but are enriched for genes involved in pathways that the virus may depend on (bercovich-kinori et al. ) . moreover, iav encodes c -type zinc finger peptide (zfp) motif-containing viral protein (such as matrix protein m ) for replication and virus budding (fernandez-pol et al. ) ; rps also contains a zfp motif, and has recently been reported to facilitate translation and viral replication (fernandez-pol et al. ; fernandez-pol ) . when rps is eliminated, the replication and infectivity of iav is abolished, but it is dispensable for global protein synthesis (karlas et al. ) . research on the essential viral and cellular zfp motif-containing proteins has implications for the prevention and therapy of viral diseases, and these proteins should be considered as targets for novel antiviral compounds. in eukaryotes, certain rps control selectivity during ires-driven translation. for example, rpl and rpl control the selective translation of a specific mrna by promoting translation primarily at the initiation stage (kondrashov et al. ; jiang et al. ; xue et al. ) . several viruses whose genomes lack a ′-end cap structure use capindependent mechanisms for translation initiation (daijogo and semler ; martinez-salas et al. ) . these viruses have in fact evolved to hijack specific rps to achieve optimal viral protein synthesis; they facilitate translation of viral transcripts of ires-containing viruses. the rps include rps (landry et al. ), rps (fukushi et al. ; bhat et al. ) , rps (huang et al. ) , rps (fukushi et al. ) , and rack (majzoub et al. ) , as well as rpl a , rpl (wood et al. ) , and rplp / (campos et al. ) . of these, rack (majzoub et al. ) and rps (huang et al. ) are specifically required for ires activity in various types of viral iress. rpl a regulates translation from the hcv and cricket paralysis virus (crpv) iress but not from the encephalomyocarditis virus (emcv) ires . in other words, rpl a is an example of an rp that plays a specific role in promoting translation of particular classes of viral iress. rps is essential for initiation from the hcv and crpv ires (landry et al. ). rps deletion in yeast or mammalian cells has minimal effects on cellular protein synthesis, which implies that this ribosomal protein may be selectively required for viral ires-mediated translation (jack et al. ; hertz et al. ) . similarly, rack is not required for cellular iress or global synthesis (majzoub et al. ) . overall, rps specifically required for protein synthesis in particular viruses but not for global synthesis may provide an effective target for methods to fight viral infection. some of the related functions of rps are ribosome-dependent, as when rps participate in viral protein biosynthesis, and other functions, typically involved in the regulation of infection in host cells, are independent of the ribosome. alterations in rp characteristics for optional translation are implicated in interaction with viral proteins, post-translational modification, and redistribution, directly or with the assistance of rna helicase. some viruses have developed to hijack specific rps using viral proteins to achieve optimal viral translation. ibdv vp is a multifunctional protein playing a key role in virus assembly and pathogenesis. ribosomal protein l (rpl ) ) and ribosomal protein l were identified as interacting partners of vp protein and as being involved in the regulation of ibdv replication. rpl interacts with nucleocapsid protein of rice stripe tenuivirus (rsv), and silencing rpl significantly reduces viral mrna translation and replication of rsv (li et al. a, b) . rpl interacts with the vp to participate in viral infection of white spot syndrome virus (wssv), and the addition of anti-rpl antibody inhibits wssv infection in litopenaeus vannamei . among flaviviruses, the envelope protein of west-nile virus (wnv), dengue virus (denv), and yellow fever virus (yfv) bind with rpsa, which serves as the viral receptor (zidane et al. ) . as regards alphaviruses, rpsa includes different binding sites for venezualian equine encephalitis virus (veev) and sindbis virus (sinv) (jamieson et al. ; malygin et al. ). hiv- tat inhibits cell proliferation via an interaction with rps and increases the level of rps in the nucleus, thereby disrupting mitotic spindle formation during hiv- infection (kim and kim ) . fmdv vp inhibits the antiviral response of rpsa by interacting with rpsa to promote fmdv replication (zhu et al. ) . binding of viral proteins to specific rps plays a crucial role in viral infection. effective inhibition of the binding between rps and viral proteins can be a potential target for antiviral design. post-translational modifications such as phosphorylation, ubiquitination, acetylation, methylation, or o-glcnacylation activate, deactivate, or modify rp's functions. over , modifications of human rps have been reported; these modifications impact rp activity and subsequently modify the global rate of translation by modulating initiation, elongation, and termination rates (emmott et al. ) . one wellstudied modification on the ribosome is phosphorylation following activation or inhibition of intra and extracellular signaling cascades in response to stimuli occurring in physiological or pathological conditions (emmott et al. ) . phosphorylation of rps is perhaps the most widely studied ribosomal protein phosphorylation among the rps involved in virus infection; it is induced by various viral infections decker ; banham et al. ; beaud et al. ). in addition, an early study revealed that phosphorylation of rpl was induced by herpes simplex virus- (hsv- ) (simonin et al. ) . rpsa, rps and rps appear specifically phosphorylated in cells early after infection with vaccinia virus (kaerlin and horak ; buendia et al. ) . efficient phosphorylation of the ribosomal proteins correlates well with possible translational mechanisms, ensuring efficient expression of early and late genes of vaccinia virus. it has been shown that phosphorylation of the rack a protein on two residues-ser- and thr -by an atypical serine/ threonine protein kinase wnk (with no lysine ) negatively regulates rack a function in the glucose responsiveness pathway by influencing its protein stability (urano et al. ) . rack a proteins are phosphorylated by tyrosine, depending on diverse environmental stresses (sabila et al. ). viral infection leads to activation of ribosomal protein phosphorylation, which may be closely related to effective viral translation. the change of phospho-rp level may affect viral translation. however, despite reports on the increasing phosphorylation of rps induced by viral infection (emmott et al. ; diaz et al. ) , the details of the role of this modification in virus infection remain poorly understood. another extensively studied post-translational modification on the ribosome is ubiquitylation, or covalent bonding of ubiquitin to substrate proteins for disposal by the proteasome (degradative ubiquitylation) or alteration of the function of the protein (regulatory ubiquitylation) (haas et al. ; thrower et al. ) . degradative ubiquitylation is important for ribosome-independent function. in fact, excess rps that are not incorporated into the ribosome are degraded by the proteasome (lam et al. ; genuth and barna, ) . several rps have been shown to be dynamically ubiquitylated in response to cellular conditions; rps , rps , and rps , for instance, undergo regulatory ubiquitylation during the unfolded protein response (upr) in yeast, fruit fly, and human cell lines (higgins et al. ) . notably, the ubiquitination of rps was found to be specifically required for viral replication and synthesis of poxvirus proteins (digiuseppe et al. ). in addition to ubiquitin, other related proteins have been found to modify the ribosome. rpl , for instance, is the principal target of ufm conjugation, a ubiquitin-related modification (ufmylation), and ufmylated rpl is highly enriched on er membranebound ribosomes and polysomes (walczak et al. ; wang et al. ) . ufm is also conjugated to rps , rps , and rpl (simsek et al. ) . the overlap of ufmylation and ubiquitylation on rps and rps also occurs, and this suggests that certain rps have combinatorial modifications. o-glcnacylation modification was also found to be activated in response to adverse stress to protect cellular proteins from damage hart , ; slawson et al. ). in the stress response, o-glcnac-modified proteins are prominent components of stress granules, including small and large ribosomal subunit proteins (rps , , and , and rpl , a, , and the ribosomeassociated protein rack (ohn et al. ) . although the effect of rp o-glcnacylation on viral infection has been as yet little studied, we can to some extent understand rp o-glcnacylation under viral infection through other forms of cellular stimulation, and start to develop an understanding of the possible role of post-translational modification of rps in antiviral strategy. although rps are all synthesized in the cytoplasm, they assemble into functional subunits in different subcellular compartments (kressler et al. ; li ) . sometimes, ribosomal proteins change their localization under virus infection to exercise functions outside the ribosome. rpl , for instance, is translocated from the nucleoli to the nucleoplasm and colocalized with icp in infection by hsv- . the location change of rpl plays a role in the regulatory functions expressed by icp (leopardi and roizman, ) . in another example of similar function, after denv infection, rpl is redistributed to the perinuclear region for regulation of viral replication (cervantes-salazar et al. ) . in eukaryotic cells, acidic ribosomal protein rplp binds two p /p protein heterodimers to form a pentameric p-stalk (ballesta et al. ; naganuma et al. ). eukaryotic rplp and rplp proteins also exist in free form in the cytoplasm, and the exchange between the ribosome-bound rplp and rplp proteins and the cytoplasmic pools is thought to regulate the activity of the ribosome (lee et al. ) . this property may lead to changes in the localization of rplp / in the ribosome and cytoplasmic pools under stress conditions. viral infection is one of numerous possible stress stimuli, so viral infection may cause rplp / to produce this subcellular compartment exchange to affect virushost translation regulation. under nucleolar stress induction, rplp accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein (deryło et al. ) . a ribosome-free pool of rplp has been shown to be a population of proteins released from pre-existing ribosomes. the presence of rplp on the ribosome seems to be affected in stressed cells, and it might be considered as a regulatory element responding to environmental fluctuations. in addition, the mitochondrial rps (mrps) are encoded by nuclear genes and then imported into mitochondria for assembly; they are responsible for the translation of mitochondrial mrnas (christian and spremulli ) . mitochondrial rpl acts as a critical regulator of the stress response and generates a cytosolic isoform in a stress-dependent manner. the cytosolic rpl is incorporated into the s ribosome and facilitates ribosome engagement in heat-shock protein (hsp) mrna translation to escape from the shutoff of global protein synthesis during stress (zhang et al. ) . accumulating evidence has revealed the roles of rps belonging to the large s subunit in regulating selective translation of specific mrnas, although they are primarily involved in catalyzing peptide-bond formation (wilson et al. ) . a previous study by the present author identified rpl as a critical regulator of ires-driven translation during foot-and-mouth disease virus (fmdv) infection, but found that it is not essential for cellular global translation (han et al. ) . our results support a model whereby the viral iress recruit helicase ddx downstream of the rpl to facilitate ires-driven translation and viral replication. specifically, the depletion of ddx disrupts binding of rpl to the fmdv ires, whereas the reduction in rpl expression impairs the ability of ddx to promote ires-driven translation directly. this work was the first to identify a connection between ddx and rps in modulating viral ires-dependent translation (han et al. ) . ddx is well known, however, to play roles in various key aspects of rna metabolism, including transcriptional regulation, splicing, mrna export, ribosome biogenesis, and translational regulation (chuang et al. ; rocak and linder ; guenther et al. ) . improved knowledge about these rna helicases and their relation to translation initiation could have important implications for the understanding of selective translation of viral mrna, and thus for the development of effective antivirals. the strategy of targeting a host factor, instead of the virus directly, could circumvent the danger of resistance in mutation-associated variations and the evolution of new viral variants after prolonged use of otherwise-promising antiviral drugs. after years of research on ribosomes, the ribosome has emerged as a major player in translational regulation, and its place in the list of potential antiviral therapeutic targets has been reinforced. targeting the ribosome, either biogenesis or function, may provide an efficient and focused approach for design of antiviral agents. as our understanding of the function of rbfs and rps in viral translation has evolved, the design of therapeutic strategies for rbfs and rps has been explored. recent structural analysis of prokaryotic and eukaryotic ribosomes has demonstrated that certain molecules, including some antibiotics and chemical inhibitors of translation, can bind to the ribosome (svetlov et al. ; tereshchenkov et al. ) . cx- , for example, is a ribosomal biogenesis inhibitor that disrupts rna pol i-mediated transcription (drygin et al. ) , and inhibits viral dna synthesis and virus production in the early and late stages of the hcmv infection (westdorp and terhune ) . toyocamycin is a small molecule inhibitor of rio kinase activity (kiburu et al. ) ; rio is an essential ribosome biogenesis factor required for maturation of s ribosomal subunit (angermayr et al. ) . toyocamycin inhibits ribosome biogenesis to produce antiviral effects (ríman et al. ) . notably, translation of ires-containing viruses (such as hcv, htlv- , crpv, poliovirus, and adenovirus) is dependent on rps (landry et al. ; hertz et al. ; olivares et al. ) , and its activity is highly sensitive to the antibiotic edeine (olivares et al. ) . picolinic acid (pa) and fusaric acid (fu) disrupt the zfp motifs of rps or crucial viral proteins to produce antiviral effects (yu et al. ; everett et al. ; wakefield and brownlee ; love et al. ) . ricin is an rplp protein inhibitor (may et al. ) ; rplp / is an essential host factor for flavivirus replication (campos et al. ) . rack is essential for the translation of many viruses, including hcv, drosophila c (dcv), cricket paralysis (crpv) (majzoub et al. ) , and vaccinia viruses (jha et al. ) . thus, rack inhibitors may be developed as novel antiviral therapeutics. the drug sd- and analogues show high efficacy in inhibition of virus proliferation (ullah et al. ) . exploring strategies for targeting rbfs and rps is a natural path for further study in this field. once the function of rbfs and rps in viral translation is understood, targeting inhibitors will need to be identified, designed, and screened. to accelerate the process of drug discovery for both novel targets and existing targets that suffer from drug resistance, basic platforms for drug design and screening are required. in a recent study, structure-based screening of two million commercially available compounds was used to screen for small molecules to target the rack y phosphorylation site. (as noted, host rack protein is an attractive target for developing broad antiviral drugs.) dozens of small compounds were identified that could potentially bind to the experimentally determined functional site of the rack a protein; the drugs show high efficacy in inhibition of the proliferation of viruses that require rack for translation (ullah et al. ) . this approach can be used to study the ribosomal proteins that are essential for the translation of many viruses, and eventually to design broad-spectrum antiviral drugs. furthermore, depletion of regulatory rbfs and rps is not essential for basic ribosomal function in experiments, making them potential targets for the design of small molecule antiviral drugs. rna interference (rnai) and strategies based on crispr/cas are also promising approaches in targeting of rbf and rp genes. of course, projecting forward to the practical application of antiviral drugs, the adverse cell response caused by targeting of rbf and rp genes must be studied in detail. deciphering the functional details of translation of viral mrna will require the in-depth scrutiny of rps. a number of technological efforts have been made over the last decade to facilitate exploration of rp biogenesis and functions. ribosome profiling is an emerging technique that allows the distribution of ribosome units on each transcript to be obtained, enabling systematic probing of translation and increased sensitivity and efficiency. this method, in conjunction with rna-seq, has been used to probe the complex viral replication of several viruses irigoyen et al. ). using high-coverage tandem mass tag (tmt) mass spectrometry, the relative monosome and polysome fractions of each rp were compared. a thorough examination of each rp revealed that some rps (such as rps and rpl ) are more abundant in monosomes than in polysomes (slavov et al. ) . tmt technology has been extended to the investigation of potential heterogeneity of rps. mass spectrometry has also been improved to address directly and more accurately the possibility of variability in rp stoichiometry. this novel technology, called selected reaction monitoring (srm)-based proteomics, uses the spiking of samples with known amounts of labeled peptides derived from rps as a standard for absolute quantification. using this technology, absolute quantification of rps isolated from polysomes was assessed and rps, rpl a, rpl , rps , and rps , were identified as being substoichiometric in murine esc ribosomes ). based on this technology, the heterogeneity in ribosome composition within a single cell type and a single polysome profile fraction was revealed for the first time. these approaches provide a starting point in the attempt to identify and quantify each rp expression profile in cells challenged with viruses, and at different infectious stages. in a recent study, both the small and large ribosomal subunits in mouse escs were endogenously tagged, and affinity enrichment for each of the tagged ribosomal subunits was performed to define the intersection of the two separate ribosomal subunit datasets. this has led to the identification of ribosome-associated proteins (raps), which fall into unexpected functional categories, such as energy metabolism, cell cycle, and key protein and rna modification enzymes (simsek et al. ). this method may facilitate identification of a series of proteins (such as raps) that are specifically recruited to ribosomes with the assistance of rps, and act as potential targets for antiviral therapeutic design. technical limitations have thus far impeded the study of selective translation mediated by rps in viral infection, and considerable research will be required before clinical application of viral therapeutics based on the targeting of rps. historically, the ribosome has been viewed as a complex ribozyme, and ribosome activity has been considered to be highly regulated. although the concepts of ribosome heterogeneity and specialization at the level of core rps are still in infancy, solid data have been published in various areas, offering convincing evidence that the ribosome plays a constitutive role as well as a regulatory function in translation. moreover, constitutive components of the ribosome may perform more specialized activities by virtue of their interactions with specific mrna regulatory elements, such as iress. these findings add numerous layers of subtlety to the conventional interpretation of translational regulation. viruses depend on host cell structures and translation systems to complete their life cycle. in order to survive in the host cell and achieve rapid replication and proliferation, viruses have developed various mechanisms to allow the selective translation of viral mrna and repress cellular mrna translation. inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread by fostering viral protein synthesis and crippling host antiviral responses. in this process, some rps are activated to promote the translation of specific transcripts, and thus the virus can still synthesize its own proteins to optimize their replication and spread when host translation is shut off. in this review, we focus on the important role of rp production and function in viral or host translation processes that support viral replication and infection. understanding the research on the specialized rbfs and rps utilized by a virus can deepen the understanding of selective translation, and expand the depth and breadth of the field of virus-host interactions. this research, data, and theoretical evidence will facilitate the identification of antiviral targets and the eventual design of antiviral drugs, and advance the development of therapeutic strategies to produce optimal antiviral agents for effective control of viral diseases. the hbx oncoprotein of hepatitis b virus engages nucleophosmin to promote rdna transcription and cellular proliferation molecular basis of sequence specific recognition of pre-ribosomal rna by nucleolin yeast rio p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression cellular cap-binding protein, eif e, promotes picornavirus genome restructuring and translation the large ribosomal subunit stalk as a regulatory element of the eukaryotic translational machinery phosphorylation of ribosomal proteins by the vaccinia virus b r protein kinase ribosomes take control ribosomal protein s /sa kinase purified from hela cells infected with vaccinia virus corresponds to the b r protein kinase and phosphorylates in vitro the viral ssdna-binding protein a systematic view on influenza induced host shutoff nucleolar trafficking of the mouse mammary tumor virus gag protein induced by interaction with ribosomal protein l the beta hairpin structure within ribosomal protein s mediates interplay between domains ii and iv and regulates hcv ires function ribosome biogenesis restricts innate immune responses to virus infection and dna the long unwinding road of rna helicases host-and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins subtractional heterogeneity: a crucial step toward defining specialized ribosomes ribosomal protein phosphorylation in vivo and in vitro by vaccinia virus rplp and rplp are essential flavivirus host factors that promote early viral protein accumulation cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein dengue virus ns protein interacts with the ribosomal protein rpl : this interaction is required for viral translation and replication in huh- cells rrp b targets pp to mammalian cell nucleoli and is associated with pre- s ribosomal subunits ribosomal protein s interacts with the latency-associated nuclear antigen of kaposi's sarcomaassociated herpesvirus interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell interaction between sars-cov helicase and a multifunctional cellular protein (ddx ) revealed by yeast and mammalian cell two-hybrid systems ribosomal protein l interacts with viral protein vp and regulates the replication of infectious bursal disease virus characterization of the interaction between hantavirus nucleocapsid protein (n) and ribosomal protein s (rps ) mechanism of protein biosynthesis in mammalian mitochondria requirement of the dead-box protein ded p for messenger rna translation interaction of nucleolin with ribosomal rna genes and its role in rna polymerase i transcription mechanistic intersections between picornavirus translation and rna replication phosphorylation of ribosomal protein s in avian sarcoma virus-transformed chicken embryo fibroblasts the ul protein, a component of the ribosomal p-stalk, is released from the ribosome in nucleolar stress alteration of ribosomal protein maps in herpes simplex virus type infection znf plays distinct roles in interferon-stimulated gene expression and poxvirus protein synthesis changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus targeting rna polymerase i with an oral small molecule cx- inhibits ribosomal rna synthesis and solid tumor growth elucidation of the avian nucleolar proteome by quantitative proteomics using silac and changes in cells infected with the coronavirus infectious bronchitis virus ribosome stoichiometry: from form to function a novel arrangement of zinc-binding residues and secondary structure in the c hc motif of an alpha herpes virus protein family conservation of multifunctional ribosomal protein metallopanstimulin- (rps ) through complex evolution demonstrates its key role in growth regulation in archaea, eukaryotic cells, dna repair, translation and viral replication essential viral and cellular zinc and iron containing metalloproteins as targets for novel antiviral and anticancer agents: implications for prevention and therapy of viral diseases and cancer ribosomal protein s interacts with the internal ribosomal entry site of hepatitis c virus heterogeneity and specialized functions of translation machinery:from genes to organisms cellular rna helicase p relocalization and interaction with the hepatitis c virus (hcv) ns b protein and the potential role of p in hcv rna replication the helicase ded p controls use of near-cognate translation initiation codons in ' utrs ubiquitin-activating enzyme mechanism and role in protein-ubiquitin conjugation dikstein r ( ) cap-dependent, scanning-free translation initiation mechanisms ribosomal protein l promotes ires-driven translation of fmdv in helicase ddx -dependent manner ribosomal protein s dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting the unfolded protein response triggers site-specific regulatory ubiquitylation of s ribosomal proteins translational control in murine hepatitis virus infection the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus rps a enhances ebv-encoded lmp -mediated proliferation and invasion by stabilizing of lmp attenuation of s ribosomal subunit abundance differentially affects host and hcv translation and suppresses hcv replication high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling nucleolin stimulates viral internal ribosome entry site-mediated translation rrna pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells redundant role of dead box proteins p (ddx ) and p /p (ddx ) in ribosome biogenesis and cell proliferation crystal structure of the human laminin receptor precursor trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase s ribosomal protein l regulates beta-casein translational elongation and secretion in bovine mammary epithelial cells identification and characterization of ribosomal proteins phosphorylated in vaccinia-virus-infected hela cells the molecular mechanics of eukaryotic translation genome-wide rnai screen identifies human host factors crucial for influenza virus replication human cytomegalovirus infection upregulates the mitochondrial transcription and translation machineries increased phosphorylation of ribosomal protein s in hamster fibroblasts transformed by polyoma virus and simian virus phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus molecular analysis of the factorless internal ribosome entry site in cricket paralysis virus infection structure of the human s ribosome interaction of rio kinase with toyocamycin reveals a conformational switch that controls oligomeric state and catalytic activity effect of hiv- tat on the formation of the mitotic spindle by interaction with ribosomal protein s ribosome-mediated specificity in hox mrna translation and vertebrate tissue patterning driving ribosome assembly the function and synthesis of ribosomes analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins rps is essential for translation initiation by the dicistroviridae and hepatitis c viral iress nucleolar protein upstream binding factor is sequestered into adenovirus dna replication centres during infection without affecting rna polymerase i location or ablating rrna synthesis solution structure of the dimerization domain of ribosomal protein p provides insights for the structural organization of eukaryotic stalk a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas functional interaction and colocalization of the herpes simplex virus major regulatory protein icp with eap, a nucleolar-ribosomal protein regulation of ribosomal proteins on viral infection nucleophosmin is essential for c-myc nucleolar localization and c-myc-mediated rdna transcription ribosomal protein l is an essential factor that promote rice stripe virus accumulation in small brown planthopper porcine reproductive and respiratory syndrome virus infection induces both eif α phosphorylation-dependent and -independent host translation shutoff white spot syndrome virus vp interact with ribosomal protein l of litopenaeus vannamei the crystal structure of hepatitis c virus ns proteinase reveals a trypsin-like fold and a structural zinc binding site rack controls ires-mediated translation of viruses c-terminal fragment of human laminin-binding protein contains a receptor domain for venezuelan equine encephalitis and tick-borne encephalitis viruses picornavirus ires elements: rna structure and host protein interactions the p /p proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells characterization of the interaction between the hiv- gag structural polyprotein and the cellular ribosomal protein l and its implication in viral nucleic acid remodeling influenza a h n subtype virus ns protein targets into the nucleus and binds primarily via its c-terminal nls /nols to nucleolin and fibrillarin multifunctional viral proteinγ manipulates nucleolar protein nop for optimal viral replication of hsv- targeting nuclear proteins for control of viral replication how should we think about the ribosome? the n-terminal regions of eukaryotic acidic phosphoproteins p and p are crucial for heterodimerization and assembly into the ribosomal gtpaseassociated center severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells a functional rnai screen links o-glcnac modification of ribosomal proteins to stress granule and processing body assembly the untranslated region of the human t-cell lymphotropic virus type mrna enables cap-independent translation initiation nuclear entry and nucleolar localization of the newcastle disease virus (ndv) matrix protein occur early in infection and do not require other ndv proteins influence of toyocamycin on rna synthesis in chick embryo cells noninfected and infected with strain mc avian leukosis virus dead-box proteins: the driving forces behind rna metabolism tyrosine phosphorylation based homo-dimerization of arabidopsis rack a proteins regulates oxidative stress signaling pathways in yeast ribosome protein l is essential for epstein-barr virus nuclear antigen function translating the genome in time and space: specialized ribosomes, rna regulons, and rna-binding proteins heterogeneous ribosomes preferentially translate distinct subpools of mrnas genome-wide phosphorylation of ribosomal protein l after herpes simplex virus type infection the mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity differential stoichiometry among core ribosomal proteins o-glcnac cycling: how a single sugar post-translational modification is changing the way we think about signaling networks a nucleolar protein, ribosomal rna processing homolog b (rrp b), enhances the recruitment of cellular mrna in influenza virus transcription kinetics of drugribosome interactions defines the cidality of macrolide antibiotics nop p is a novel nucleolar s ribosomal subunit biogenesis protein the complexity of human ribosome biogenesis revealed by systematic nucleolar screening of pre-rrna processing factors binding and action of amino acid analogs of chloramphenicol upon the bacterial ribosome nop p is required for late s ribosome subunit maturation and nuclear export in yeast recognition of the polyubiquitin proteolytic signal silencing of hepatitis c virus replication by a non-viral vector based on solid lipid nanoparticles containing a shrna targeted to the internal ribosome entry site (ires) host targeted antiviral (hta): functional inhibitor compounds of scaffold protein rack inhibit herpes simplex virus proliferation arabidopsis receptor of activated c kinase phosphorylation by with no lysine kinase viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells rna-binding properties of influenza a virus matrix protein m ribosomal protein rpl is the principal target of ufmylation cloning of mouse genomic ribosomal protein l gene and analysis of its promoter the association of ribosomal protein l (rpl ) with infectious bursal disease virus viral protein vp enhances viral replication ufmylation of rpl links translocation-associated quality control to endoplasmic reticulum protein homeostasis interferon production and inhibition of host synthesis in cells infected with vesicular stomatitis virus impact of rna polymerase i inhibitor cx- on viral kinase-dependent and -independent cytomegalovirus replication new mrnas are preferentially translated during vesicular stomatitis virus infection the structure and function of the eukaryotic ribosome hepatitis c virus 'x region interacts with human ribosomal proteins specialized ribosomes: a new frontier in gene regulation and organismal biology rna regulons in hox ' utrs confer ribosome specificity to gene regulation deciphering poxvirus gene expression by rna sequencing and ribosome profiling subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein specific disulfide formation in the oxidation of hiv- zinc finger protein nucleocapsid p nucleophosmin is essential for ribosomal protein l nuclear export o-glcnac a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress cell signaling, the essential role of o-glcnac translational control of the cytosolic stress response by mitochondrial ribosomal protein l ddx facilitates hiv- replication as a cellular co-factor of rev ribosomal proteins: functions beyond the ribosome foot-and-mouth disease virus capsid protein vp interacts with host ribosomal protein sa to maintain the activation of mapks signal pathway and promote virus replication the folded and disordered domains of human ribosomal protein sa have both idiosyncratic and shared functions as membrane receptors acknowledgments this work was supported by the national natural science foundation of china ( ) and a cau-grant for the prevention and control of immunosuppressive disease in animals (cau-g-pcida) of the china agricultural university. conflict of interest the authors declare that they have no conflict of interest. key: cord- - g ih zl authors: bax, adriaan; bax, christina e; stadnytskyi, valentyn; anfinrud, philip title: sars-cov- transmission via speech-generated respiratory droplets date: - - journal: lancet infect dis doi: . /s - ( ) -x sha: doc_id: cord_uid: g ih zl nan the relative humidity (rh) of % in which our measurements were conducted is percentage points below the cdc guidelines for healthcare facilities ( %- %) and within the rh reported for common office buildings ( %- %). we assume that the authors were more concerned with the effect of rh on the rate of evaporation from respiratory droplets. again, while the laws of physics dictate that higher rh increases the time needed for evaporation of water, and thereby decreases the time needed for a droplet's fall to ground, to first order this time scales with ( -rh) and has no significant impact for any of the small droplets observed in our work. for example, droplets of micron diameter will fully dry out at % rh in ca ms, many orders of magnitude faster than the time needed to fall to ground if they were to remain fully hydrated. "the duration of recorded speech was s, but the results were artificially extrapolated to min." as clearly described in the results section and figure of our pnas article, light scattering observations resulted from seconds of recorded speech. as explicitly stated in the discussion, these results were used to estimate the number of potential virions emitted in one minute of speaking. there is nothing artificial about normalizing measured results to standard units. for example, we could have stated "an average of virions per second over a period of seconds" but such a number would suggest a precision higher than warranted, considering the wide variation in viral load and the fractional uncertainty in the diameter of the fully hydrated particles. instead, our reported " per minute" provides an order of magnitude estimate. "in the . min preceding the beginning of the speech, we counted at least instances where flying particles were observed" indeed, even when using a high-efficiency particulate air filter, infiltration of particles from outside sources contributed to our low background particle count rate. as indicated in figure a of our pnas article, the decay of observed particles returns to this low background of . particles per frame (for the green curve; smallest particles). this was explicitly stated in the figure legend and was used when fitting the decay curves. "the authors used fluorescent green light to illuminate particles" nowhere did we state or suggest that fluorescent light was involved in any of our measurements. the laser used in our study generated coherent green light with a wavelength of nm. laser light is not fluorescent. our measurements recorded green light scattered from particles passing through the light sheet. "no report of the loudness, measured in decibels, was found in either manuscript, although in the videos it seems that in some cases the study participant was shouting, so the claim of normal speech is dubious." evidently, abbas and pittet failed to read the legend for figure in our pnas paper which reports that the speaker used "a loud (maximum dbb at a distance of cm; average dbb)." notably, the average loudness ( db) is consistent with the cdc's definition of conversational volume ( db). the "shouting" we assume the authors refer to is when the speaker had the mouth covered by a washcloth, which was used to illustrate that even when shouting, the number of detectable speech-generated droplets remains close to background levels when wearing a mouth cover. "the authors were mistaken when stating that high viral loads were found in asymptomatic patients while referring to the study by wolfel and colleagues. only one patient reported being asymptomatic in the severe acute respiratory syndrome coronavirus (sars-cov- ) outbreak in bavaria, germany, and that patient was not included in wolfel and colleagues' study, which included only hospitalised patients." evidently, abbas and pittet misread or misunderstood the results reported by wölfel et al. wölfel et al. report the high viral loads of nine individuals. this patient population was a subset of the covid- cases reported at the end of january in bavaria, germany by boehmer et al. we thank abbas and pittet for referring us to boehmer et al., whose report provides further evidence that presymptomatic spread occurs, with at least one patient, but probably five additional patients, being infected by a presymptomatic carrier. moreover, it is important to note that wölfel's patients were hospitalized not for the severity of symptoms, but preemptively on the basis of a positive covid- test. in fact, / patients ( %) never had a cough (figure a,d,f,i) and wölfel et al. state that "the clinical courses in the patients under study-all of whom were young-to middle-aged professionals without notable underlying disease-were mild". indeed, patient no. did not exhibit symptoms during his hospitalization ( fig. and table ). "the presence of a fan at the bottom of the black box during the speech and for s after the end of speech does not represent real-life conditions" in "real-life" conditions, exhaled air emerges with high humidity at a temperature near ºc and rapidly mixes with room air, as demonstrated by schlieren images. speaking into an enclosure creates thermal and humidity gradients that are nominally eliminated by operating the internal fan for a short period of time during and after speaking. using the fan to achieve a more homogeneous distribution of droplets prior to the actual decay time measurement does not influence the rate at which droplet nuclei subsequently disappear from view. the dispersion of speech droplets achieved by operating the fan for a short period of time is not unlike that produced by air currents generated when a person walks past a speaker. to suggest that use of the fan does not represent real-life conditions is as perplexing as it is irrelevant. we stated that transmission by asymptomatic covid- carriers is plausible. there is now an abundance of evidence for this observation in the scientific literature, as acknowledged by who on july , . while the nuances between presymptomatic and asymptomatic transmission, or even oligosymptomatic transmission, had not yet been extensively discussed within the context of transmission when our work was submitted and published, the key point remains that disease carriers with no symptoms, that is, subjects who are by definition unlikely to be coughing or sneezing, may be transmitting the virus via speaking. the number of speech droplets observed in our studies, and in particular for the fraction that is sufficiently small to remain airborne for many minutes, is far higher than was previously considered by the medical community. multiple studies have shown that the oropharyngeal viral load in asymptomatic or presymptomatic patients is similar to that of symptomatic patients, , with infectivity appearing to peak prior to onset of symptoms. , [ ] [ ] [ ] "second, the authors assumed an average viral load in saliva of × ⁶ copies per ml on the basis of a prospective study wherein viral load was measured in sputum. thus, they assume that viral load in sputum is the same as in saliva." wolfel et al. report throat viral loads as high as × copies per throat swab. considering a throat swab to contain ca - µl of oral fluid, the viral load was as high as ~ × copies per ml. wolfel et al. also explicitly state "there were no discernible differences in viral loads or detection rates when comparing naso-and oropharyngeal swabs (fig. b) ". sputum consists of "lower respiratory tract secretions along with nasopharyngeal and oropharyngeal secretions, cellular debris, and microorganisms". as mentioned above, speech droplets originate from oral fluid, both at the vocal folds (mostly sputum) and at the front of the oral cavity (mostly saliva). oropharyngeal swabs represent an intermediate location. vowel sounds have been associated with high levels of small speech droplets and are minimally modulated by other narrow passages before entering the atmosphere. these droplets therefore are generated at the same physical location as cough droplets. it is important to note that the viral load of a disease carrier, that is, whether it is high or low, equally impacts the probability of disease transmission through the airborne, large-droplet, and fomite pathways. the relative probability of transmission through these pathways is primarily defined by the likelihood that secreted virions reach the respiratory tract of a bystander, not by the viral load of the droplets. only if abbas and pittet wish to argue that the fecal route dominates disease transmission does the absolute viral load of respiratory fluid secretions become relevant. "the group also assume that every rna copy detected is a potentially infectious virion" nowhere did we state or assume that every rna copy detected is a potentially infectious virion. viability of excreted virions will modulate all pathways equally. indeed, as highlighted in figure f and g of wolfel et al., the ability to culture virus from respiratory secretions rapidly decreases after onset of symptoms whereas viral loads decrease substantially slower, indicating that a progressively smaller fraction of virions is viable in culture as the infection progresses. however, the viability of virions modulates all transmission pathways equally. surfing the covid- scientific wave the size and the duration of air-carriage of respiratory droplets and dropletnuclei on air-borne infection -study ii droplets and droplet nuclei guidelines for environmental infection control in health-care facilities indoor air humidity, air quality, and health -an overview mechanisms of airborne infection via evaporating and sedimenting droplets produced by speaking the airborne lifetime of small speech droplets and their potential importance in sars-cov- transmission what noises cause hearing loss visualizing speech-generated oral fluid droplets with laser light scattering virological assessment of hospitalized patients with covid- investigation of a covid- outbreak in germany resulting from a single travel-associated primary case: a case series a schlieren optical study of the human cough with and without wearing masks for aerosol infection control substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov- ) who confirms there's 'emerging evidence' of airborne transmission of the role of particle size in aerosolised pathogen transmission: a review sars-cov- viral load in upper respiratory specimens of infected patients viral rna load in mildly symptomatic and asymptomatic children with covid- temporal dynamics in viral shedding and transmissibility of covid- temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study physiology of airway mucus clearance modality of human expired aerosol size distributions size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities key: cord- -mwy t ny authors: gu, li; qu, jiuxin; sun, bing; yu, xiaomin; li, hui; cao, bin title: sustained viremia and high viral load in respiratory tract secretions are predictors for death in immunocompetent adults with adenovirus pneumonia date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: mwy t ny the predictors for fatal adenovirus (adv) pneumonia among immunocompetent adults are unclear. laboratory-confirmed, hospitalized adv pneumonia adults were prospectively enrolled in beijing chao-yang hospital from march to june . clinical data and serial whole blood and respiratory tract secretions from such patients were collected. quantitative real-time polymerase chain reaction was performed to quantify the viral load. a total of adv pneumonia cases were consecutively enrolled, and four of them were fatal. ten cases were caused by adv- , three by adv- and one by adv- . there were no differences in age, gender or underlying diseases between the patients in the fatal cases and surviving cases. at admission (on day – after illness onset), the patients in fatal cases presented higher initial viral loads in respiratory tract secretions ( . ± . vs . ± . log( ) copies/ml, p = . ). all patients in fatal cases presented with viremia on day – ( % vs . %, p = . ). a higher initial viral load in the respiratory tract and sustained viremia (more than weeks) may be predictors for fatal clinical outcomes. severe adenovirus (adv) infections causing significant acute respiratory distress syndrome have raised concerns for immunocompetent adults [ ] . most severe cases were previously reported to be associated with adv- , , and [ , ] . a new strain, adv- (formerly known as adv- a), has been a major adv pneumonia pathogen in immunocompetent adolescents and adults in china since [ ] . our previous study identified a fatal adv- infection case with a patient who presented with systemic infection and high-level viremia [ ] . however, even though adv- as well as adv- , , and can cause severe cases, those strains are not necessarily associated with bad outcomes, and the pathogenesis of fatality is still unknown. we have already ascertained that adv can be detected in whole blood specimens of severe cases, but we still lack the knowledge about the viral shedding history and the relationship between the viral clearance in the respiratory tract and viremia duration and clinical outcome. in this study, we focus on the dynamic virological changes in whole blood and respiratory tract secretions to see if lethal cases experienced a high viral load and longer duration of viral shedding compared to the non-lethal cases. we hypothesize that sustained virus shedding in the blood and/or respiratory tract can appear in severe immunocompetent adult cases, and it may be a risk factor for fatal outcome. the study was reviewed and approved by the institutional review board of beijing chao-yang hospital (the project approval number is -ke- ). written informed consent was provided by all adults and the parents of patients aged less than years. adults with communityacquired pneumonia (cap) (age yrs) admitted to beijing chao-yang hospital from march to june were prospectively included. patients with hiv infection or neutropenia; receiving immunosuppressive chemotherapy or steroids equivalent to prednisone > mg/d for days; who were pregnant or breast feeding women; or who were known or suspected to have active tuberculosis were excluded. methods of etiological evaluation followed the standard for adults suspected with cap [ ] [ ] [ ] . in short, sputum or respiratory tract aspiration, blood and urine were collected at admission and submitted to the infectious disease and clinical microbiology laboratory. microbiological methods were based on the following tests: ( )sputum specimens for gram stain and cultures considered valid only if microscopy showed > neutrophils and < epithelial cells per low field microscopy; ( )urine specimens for the rapid detection of s. pneumonia and legionella pneumophila antigen; ( ) blood culture; ( ) sputum and tracheal aspiration for virus real-time polymerase chain reaction (pcr) detection, including rhinovirus, influenza a and b, respiratory syncytial virus a and b, adenovirus, parainfluenza - , coronavirus oc and e, and metapneumovirus; and ( ) sputum for mycoplasma pneumoniae pcr detection. only subjects with positive adenovirus results who were negative for other etiologies during the study period were enrolled. the results of pcr testing for respiratory viruses were reported to clinicians within hours after sputum collection. for those with positive adv pcr testing, serial whole blood and respiratory tract samples were collected until death or discharge. in this study, we tried to collect the same type of respiratory tract samples from each patient to carry out serial viral load testing, to lower bias from different types of samples. for patients with mechanical ventilation, we collected serial tracheal aspirations, and for patients without mechanical ventilation, we collected sputum samples. we also collected data regarding age, gender, co-morbidities, clinical symptoms, vital signs, antimicrobial treatment, chest radiographic findings, and laboratory results. recorded complications included the following: use of mechanical ventilation and extracorporeal membrane oxygenation (ecmo), and second bacterial or fungal infection. patients were also followed up to discharge or death. respiratory tract samples, including sputum and tracheal aspirations, were processed by equal volume % trypsin digestion. the viral dna was extracted from milliliter (ml) digested respiratory sample and ml whole blood sample using a qiaamp dna mini kit (qiagen, valencia, ca, usa). first, we amplified the entire hexon genes of the samples by pcr. the primer sequences are listed in table [ ] . then, the adv type was determined using blast (http:// blast.ncbi.nlm.nih.gov/blast.cgi). regarding the quantification of adv in the samples, we used a commercial fq-pcr kit (daan gene, cat. #da-b , guangzhou, china) targeting the dna polymerase gene [genebank: kf . ] two-tailed independent samples t-test or mann-whitney u-test (on condition of non-normal distributions) was used to compare continuous variables between the two groups. for the categorical data, univariate analysis was performed using the chi-square test or fisher's exact test. significance was fixed at p value < . . data analysis was performed using spss . (spss inc; chicago, il). from march to june , a total of admitted cases were confirmed with adenovirus as the only pathogen of pneumonia; four of the patients died in the icu. the mean age was years old. males predominated in number over females, with a sex ratio of approximately : . only two patients had co-morbidities, one with tuberculous pleuritis for months and one with congenital heart disease. there were no differences in age, gender or underlying diseases between fatal cases and surviving cases. there are three types of adv found in this study, adv- (n = ), adv- (n = ) and adv- (n = ). the adv- ratio in the fatal group was similar to that in the surviving group ( % vs %, p = . ) ( table ) . clinical features: comparison between surviving and fatal cases most clinical symptoms and signs noted in the surviving cases and fatal cases had no differences, except for the pneumonia severity index (psi) score and dyspnea. the fatal cases had a higher psi score ( . ± . ) and dyspnea incidence ( / ) compared to the psi score ( . ± . ) and dyspnea incidence ( / ) in surviving cases (p = . and . , respectively) ( table ). all of the fatal cases described bilateral involvement on chest radiography, and % noted pleural effusion (table ) . for the surviving cases, % ( / ) noted bilateral involvement, and % ( / ) described pleural effusion. there were no significant differences between the two groups in bilateral involvement or pleural effusion. the adv load tracking in the respiratory tract samples and whole blood the mean time from disease onset to initial pcr test for respiratory tract samples and whole blood was . days, and there were no significant difference between the fatal group ( . ± . days) and surviving group ( . ± . days). we measured the adenoviral load in respiratory tract samples and whole blood, expressed as log dna copies per ml samples. for initial viral load in respiratory tract samples on day - after disease onset, the results showed that, compared to the surviving cases, the fatal cases had a higher initial viral load ( . ± . vs . ± . , p = . ) (fig a) . however, for the initial viral load in whole blood, there was no significant difference between the two groups ( . ± . vs . ± . , p = . , fig b) . we also investigated viral shedding duration evaluated by positive ratio on day - and on day - after illness onset. for respiratory tract samples, there was no significant difference of viral positive ratio between the two groups either on day - ( % vs %) or on day - after onset of disease ( % vs %, p = . ) (fig c) . viremia was common at admission (on - day after onset of illness), as noted in % ( / ) of the fatal cases and . % ( / ) of the surviving cases (p = . ). on day - after onset of illness, however, it persisted in % ( / ) of the fatal cases with . % ( / ) of the surviving cases (p = . ) (fig d) . for the surviving severe patients, we found that the clinical manifestation recovered gradually with a downward trend in viral load in respiratory tract and whole blood samples. as shown in fig , a -year-old male with acute respiratory distress syndrome (ards) had a high initial viral load ( . copies/ml) in tracheal aspiration. his condition improved, with the sputum viral load going down on day , and ecmo was withdrawn on day . with the viral load in blood going down to negative on day , the clinical condition was significantly improved, and the patient did not need oxygen therapy; the chest x-ray was also remarkably resolved. as shown in fig , the respiratory tract viral load of a -year-old male with ards also had a high initial viral load ( . copies/ml) in tracheal aspiration. the viral load in tracheal aspiration gradually decreased during the first days, and the lung infiltrate absorbed partially. however, the viral load trended up again and the patient died. he also maintained a viral load in whole blood before death. antibiotics were given to all of the patients empirically before and after confirmed diagnosis. there is currently no formally approved antiviral therapy for the treatment of severe lifethreatening adenovirus infection in china. acyclovir, ganciclovir or ribavirin is commonly chosen by the physician to treat adenoviral infection. in this study, all of the fatal patients were administered antiviral drugs-one was treated with ganciclovir, two with acyclovir and one with ribavirin. in surviving patients, % were treated with antiviral drugs-three with ganciclovir, one with acyclovir and one with ribavirin. all of the fatal patients ( / ) were complicated with ards and admitted to the icu. they needed mechanical ventilation, and three of them received ecmo to maintain oxygenation. for surviving patients, one of them ( / ) was admitted to the icu due to ards, and had mechanical ventilation and ecmo; one patient with respiratory failure was treated with non-invasive ventilation, and one patient was treated for myocarditis. the myocarditis patient presented with peak levels of creatine kinase isoenzyme (ck-mb) and cardiac troponin i (ctni) on day - after disease onset, and eight days later, with viral load going down to negative, ck-mb and ctni went down to normal levels in parallel (fig ) . for the four fatal patients, the times of death were on days , , and after disease onset, respectively. there was no significant difference in length of stay in-hospital between the two groups ( . ± . days vs . ± . days, p = . ) ( table ). there were three patients complicated with bacterial or fungal infections among the fatal cases. the first patient presented with consecutive aspergillus fumigatus in tracheal aspirate cultures, the second patient had a cavity present on chest ct, and the third patient had acinetobacter baumannii in tracheal aspirate and pleural effusion cultures. there were only two patients complicated with superinfections in surviving patients. one patient (a -year-old male) presented with a typical crescent sign and halo sign with chest ct follow-up; voriconazole was administered to resolve this condition (fig ) . another patient with ecmo presented with acinetobacter baumannii in tracheal aspirates. we investigated the relationship between the virological factors and clinical outcomes in a cohort of hospitalized adults with adv pneumonia. our results suggest that a higher initial viral load ( copy/ml) in the respiratory tract samples on day - after disease onset is a predictor for fatal clinical outcome. we also reported that viremia is common and sustained viremia for days or more may be associated with mortality the pathogenesis of mortality in adv pneumonia is still unknown. virological factors, e.g., a new strain with new genetics, viral load, slow virus clearance and systemic infection with viremia likely play key roles for severe adv [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, no study has evaluated the viral shedding history among immunocompetent adults with adv pneumonia. this study first monitored the consecutive viral load in respiratory tract samples and whole blood samples. our previous clinical study demonstrated that on day - after disease onset, the peak stage of illness presented for patients with shortness of breath or severe dyspnea [ ] . again, in this study, we showed that the viral load on day - could also provide an insight into the severity of illness. evidence even proved that a higher level of viral load in respiratory tract samples on day - after disease onset was significantly associated with fatal outcome. we have noted that viremia is quite common on day - after disease onset, when out of ( . %) patients had viremia. adenovirus viremia has been found in hematopoietic stem cell transplantation recipients and associated with adv disease [ ] . compared with previous reports of viremia and clinical outcomes, another novel finding is that we demonstrated that fatal outcomes could be predicted by sustained viremia, but not by viremia itself. in this study, we showed that % ( / ) of patients in fatal cases presented with viremia on day - after disease onset, compared with % (p = . ) of the patients in surviving cases. in one case, as shown in fig , even though the patient presented with a higher viral load ( . copies / ml) in tracheal aspiration, which may be associated fatal outcome, his clinical manifestation recovered gradually with a downward trend in the viral load in respiratory tract and whole blood samples. compared to this case in fig , the patient described in fig not only had a higher viral load ( . copies/ml) in tracheal aspiration but also presented with sustained elevated viral copies, especially in whole blood. shike et al. also reported a -monthold infant with systemic infection by adenovirus who had high-level viremia and showed reduction in viral load paralleling her clinical recovery [ ] . therefore, in severe cases, dynamic monitoring of viral shedding, especially in whole blood, could help predict the clinical outcome. patients might have bad outcomes if the viral load in whole blood does not present a significant downward trend around two weeks after disease onset. there is currently no formally approved antiviral therapy for the treatment of severe lifethreatening adenovirus infection in china. cidofovir is considered the medicine of choice for severe infection in immunocompromised patients. cidofovir is not available in most hospitals in china, including our hospital. acyclovir, ganciclovir or ribavirin is usually prescribed in china. in this study, antiviral drugs were administered in all of the fatal cases-one patient was treated with ganciclovir, two with acyclovir and one with ribavirin. in surviving patients, % were treated by antiviral drugs-three with ganciclovir, one with acyclovir and one with ribavirin. the choice of the antiviral medicine was decided by the patient's physician. as none of these three medicines have been confirmed to be effective for adv infection, the relationship between viral shedding and clinical outcomes in this study was not associated with anti-adenoviral treatment effect. our study has two limitations. as adv was the most common infection type ( / , . %) in this study, results might be more significant in adv -associated pneumonia and might not be generalizable to other types of adv pneumonia. in our previous study, adults infected with adv were years older and presented with higher psi scores compared with adults infected with other serotypes [ ] . another limitation of this descriptive work may be the small number of analyzed patients, especially in the group of fatal cases (n = ). more cases are needed to confirm our findings. in conclusion, our data provide new insight into the virology of adv pneumonia. a higher initial viral load ( copy/ml) in the respiratory tract on day - after disease onset and sustained viremia for weeks or more may be associated with fatal clinical outcomes. two fatal cases of adenovirus-related illness in previously healthy young adults-illinois severe adenovirus pneumonia in immunocompetent adults: a case report and review of the literature severe pneumonia due to adenovirus serotype : a new respiratory threat? emergence of community acquired adenovirus type as a cause of community-onset pneumonia severe community-acquired pneumonia caused by adenovirus type in immunocompetent adults in beijing viral and mycoplasma pneumoniae community-acquired pneumonia and novel clinical outcome evaluation in ambulatory adult patients in china outbreak of acute respiratory disease in china caused by b species of adenovius type adenovirus viremia and disease: comparison of t celle depleted and conventional hematopoietic stem cell transplantation recipients from a single institution quantitation of adenovirus genome during acute infection in normal children we thank drs. ran li, chen ma, yudong yin, lin wu, and yiqun guo for their contributions to specimen collection.notation of publication: this work has been accepted as an oral presentation at the third isirv-avg conference on influenza and other respiratory virus infections: advances in clinical management on june th - th , at the keio plaza hotel, tokyo, japan. conceived and designed the experiments: bc. lg jxq bs xmy.analyzed the data: lg hl.contributed reagents/materials/analysis tools: jxq bc.wrote the paper: lg jxq bc. competing interests: the authors have declared that no competing interests exist. key: cord- - afgpunj authors: owino, collins oduor; chu, justin jang hann title: recent advances on the role of host factors during non-poliovirus enteroviral infections date: - - journal: j biomed sci doi: . /s - - -y sha: doc_id: cord_uid: afgpunj non-polio enteroviruses are emerging viruses known to cause outbreaks of polio-like infections in different parts of the world with several cases already reported in asia pacific, europe and in united states of america. these outbreaks normally result in overstretching of health facilities as well as death in children under the age of five. most of these infections are usually self-limiting except for the neurological complications associated with human enterovirus a (ev-a ). the infection dynamics of these viruses have not been fully understood, with most inferences made from previous studies conducted with poliovirus. non-poliovirus enteroviral infections are responsible for major outbreaks of hand, foot and mouth disease (hfmd) often associated with neurological complications and severe respiratory diseases. the myriad of disease presentations observed so far in children calls for an urgent need to fully elucidate the replication processes of these viruses. there are concerted efforts from different research groups to fully map out the role of human host factors in the replication cycle of these viral infections. understanding the interaction between viral proteins and human host factors will unravel important insights on the lifecycle of this groups of viruses. this review provides the latest update on the interplay between human host factors/processes and non-polio enteroviruses (npev). we focus on the interactions involved in viral attachment, entry, internalization, uncoating, replication, virion assembly and eventual egress of the npev from the infected cells. we emphasize on the virus- human host interplay and highlight existing knowledge gaps that needs further studies. understanding the npev-human host factors interactions will be key in the design and development of vaccines as well as antivirals against enteroviral infections. dissecting the role of human host factors during npev infection cycle will provide a clear picture of how npevs usurp the human cellular processes to establish an efficient infection. this will be a boost to the drug and vaccine development against enteroviruses which will be key in control and eventual elimination of the viral infections. non-polio enteroviruses belong to the genus enterovirus (consisting of species); family picornaviridae [ ] and have been identified in different parts of the world affecting human population [ ] . major outbreaks of nonpolio virus associated infections have been recently reported in asia pacific, europe, canada and united states of america (usa). the peak of these infections is coming at a time when the world is nearing eradication of poliomyelitis, with just small number of cases reported in some parts of the world [ ] . the burden of these infections has been felt in children under the age of five; most of whom are just beginning their early years at school. most of these infections are known to be selflimiting but severe neurological complications and even death has been reported in some cases. the focus of this review is to highlight the known role of human host factors and processes during the selected npev infections. a brief introduction on the epidemiology and pathogenesis of the selected non-polio viruses are described. the viral-host process/protein interactions are then discussed, followed by the existing gaps that need to be addressed in future. the ability of various npev viruses to usurp various cellular processes such as; cell cycle division, autophagy as well apoptosis, necroptosis and pyroptosis for efficient replication are also highlighted. the state of antiviral therapy research against these viruses is briefly discussed and existing gaps highlighted. the future perspectives and areas of concern are also emphasized. enterovirus a (ev-a ) was first isolated from fecal and throat swab samples from patients with central nervous system complications in california [ ] . since then, ev-a has been linked with outbreaks of foot, hand and mouth disease (hfmd); often a self-limiting infection characterized with and severe forms characterized with acute flaccid paralysis and brainstem encephalomyelitis [ ] [ ] [ ] [ ] . coxsackievirus a (cv-a ), also plays a major role in hand, foot and mouth disease (hfmd) epidemics. renal failure has also been reported in two hfmd cases due to cv-a infection [ , ] and more recently one case of acute kidney injury secondary to ev-a infection was reported by xu and colleagues [ ] . hfmd outbreaks have been reported in different parts of asia pacific; often with neurological complications in children under the age of five especially in preschool centers as observed in singapore [ ] . for example, between to there were about . million probable cases of hfmd and about fatal cases reported in mainland china alone with high economic costs [ ] . this year, cases of encephalitis/ neurological complications as a result of ev-a virus infection have been reported in colorado, united states of america [ ] . a - yearly cyclic pattern of hand, foot and mouth disease outbreaks have been reported in asia pacific region [ ] . the drivers of seasonality of npev in usa was studied recently by pons-salort and coworkers and identified the month of july and september to be the peak of these infections [ ] . these outbreaks always result in the overburdening of the health care systems, pain and loss of lives in severe cases of the disease. even though recent mathematical modelling findings using data from singapore showed high incident rates with limited disability-adjusted life years (dalys) compared to other infectious diseases prevalent in the south east asian countries [ ] , hfmd has a potential threat to global health. analysis of samples earlier collected for poliovirus surveillance studies in seven western african countries identified several npevs circulating in the region with echoviruses being the dominant strain [ ] . this study also identified least described types such as ev-a , ev-b , cv-a as well as ev-d among others to be circulating in this region [ ] . the identification and molecular characterization of npevs in west africa points to the global diversity of these viruses and calls for a stronger surveillance system for better management and control. recently, minor outbreaks of hfmd have been attributed to other coxsackieviruses such as cv-a and cv-a . even though the magnitude of their effects during outbreaks is not as large as that of ev-a and cv-a ; there is need to understand pathogenesis of the infections as well as quantifying their burden for easy disease monitoring. coxsackievirus a (cv-a ) was isolated in usa in and has been recognized as one of the causative agents of hand, foot and mouth disease in different parts of the world including usa, europe (finland, spain) and asia pacific (taiwan, japan, china, thailand and vietnam among other countries in the region) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the emergence of cv-a as a player in the hfmd outbreaks eventually complicates vaccine and antiviral therapy development against hfmd. cv-a and ev-a have been broadly studied; however little success has been achieved in vaccine and therapy development thus the emergence of cv-a points to the urgent need of understanding its infection dynamics. coxsackievirus a and a have been linked to sporadic outbreaks of atypical hfmd infections in china and france [ ] [ ] [ ] [ ] [ ] . between and , cv-a and cv-a contributed to about . and . % cases of hfmd correspondingly in china [ ] . with time, cv-a has become one of the major causative agents of both severe and mild cases of hand, foot and mouth disease in china between to ; accounting for approximately . % of mild and . % of severe cases in [ ] . there is a high possibility of virulent strains of hfmd viruses emerging as frequent recombination of enteroviruses a have been reported [ , ] . these viruses have a potential of causing major outbreaks with potential threat to global health. enterovirus d (ev-d ); first identified from throat swabs of children suffering from respiratory infections in and named as "fermon virus" by schieble and coworkers [ ] . since then, severe outbreaks of respiratory infections as a result of enterovirus d infections have been reported in taiwan, usa, canada and in europe among other endemic regions [ , [ ] [ ] [ ] . the link to acute flaccid paralysis and acute flaccid myelitis further exacerbates ev-d infections [ ] . several research studies have demonstrated the infection dynamics of this viral infection; for example, the ability of ev-d to infect neuronal cells has been reported by brown and colleagues. using neuronal cell line; sh-sy y confirming its neurotropism in line with the observed acute flaccid myelitis/ paralysis in patients [ ] . systemic and molecular diversity studies of ev-d in lyon france, showed a diversification pattern for this virus [ ] . the establishment of an experimental mouse model by hixon and colleagues for studying the effects of ev-d provides much needed animal model for better understanding of the infection cycle of this virus [ ] . establishing the ev-d human host cell interactions will provide an insight into the pathogenesis of the infection and eventually be vital in the design of antivirals and vaccines against the virus. there is necessity to extensively understand the molecular mechanisms of these viruses including the infection paradigms which will be key in the development of vaccines and antiviral therapy as well as players in molecular epidemiology. host factors/processes involved in npev attachment, entry and internalizatione viral tissue tropism depends solely on cellular receptors which are responsible for attachment and entry of the virus particles into the host cells. human host proteins act as receptors for viral attachment and eventual entry into the cells playing a role in the tissue tropism for various viral infections. several receptors have been identified for various picornaviruses with poliovirus receptors being the first ones to be identified in this family. with the recent reemergence of enteroviral infection outbreaks, there is need to document all the recent findings in the entry process of these viruses; pointing to the eventual gaps that needs further research. interplay between viral proteins and human host proteins play a major role in the attachment, entry and internalization of viral infections. specific viruses use a confined set of receptors on the cell membrane for entry into susceptible cells, eventual uncoating of the virus. this process is vital for the eventual reproduction of viral genome and for continuity of the viral life cycle. among the picornaviruses, poliovirus is the most extensively studied and several studies on non-polio enteroviruses have relied on these studies. a few host factors have been identified as possible receptors for the npevs, but the dynamics of the eventual attachment, entry and internalization is not yet fully understood. clathrin-mediated endocytosis as an entry pathway for ev-a virus was identified through sirna screens targeting key genes involved in the process of endocytosis cytoskeletal dynamics, and endosomal trafficking [ , ] . since then it has always been known that clathrin mediated endocytosis is the major route of ev-a entry into susceptible cells. however, inhibition of the clathrinmediated endocytosis pathways by chlorpromazine (cpz) or dynasore (dns) did not inhibit ev-a entry into the a cells, thus pointing to a combination of pathways involved in the viral entry [ ] . among the picornaviruses, poliovirus and rhinovirus receptors were identified in ; being the first enterovirus receptors to be described. greve and his colleagues identified intercellular adhesion molecule (icam- ) as a rhinovirus receptor [ ] while cd was described as a poliovirus receptor by mendelshon and colleagues [ ] . some ev-a receptors have been identified; but these putative receptors have not been able to fully explain the diverse nature of symptoms observed in hand, foot and mouth disease cases. ev-a receptors include; human scavenger receptor class b member (scarb ); a known to not only function as an attachment receptor but also as an uncoating receptor during ev-a infection [ ] . scarb receptor is ubiquitously expressed in different parts of the body including neuronal cells. scarb is a transmembrane receptor and a known βglucocerebrosidase (β-gc) receptor responsible for transport from endoplasmic reticulum to lysosome and is also key in lysosome maintenance [ ] . scarb was also identified as an attachment receptor for human enterovirus species a and coxsackie a virus [ ] . several cell types are known to express scarb , including the neurons thus may be directly linked to the neurological complications associated with ev-a infections; even though this has not been validated. at acidic and neutral conditions, the scarb undergoes conformational changes leading to the opening up of lipid transfer channel mediating ejection of hydrophobic pocket from the virion a process important for viral uncoating [ ] . p-selectin glycoprotein ligand- (psgl ) a membrane protein expressed on white blood cells where it is responsible for inflammation, tethering or rolling of leukocytes at the vascular endothelial has also been described as a receptor for ev-a responsible for the viral entry into blood cells [ ] [ ] [ ] . psgl- has a high avidity for ev-a virus compared to scarbr yet its associated with low infection effeciency due to its inability to induce viral uncoating [ ] . sialyated glycans were also elucidated to be playing a role in ev-a infection of the dld intestinal cells [ ] . another attachment receptor; heparan sulfate glycosaminoglycan was also identified by tan and colleagues pointing to the number of binding options available for ev-a virus [ ] . a recent study by tseligka and coworkers confirmed the importance of heparan sulfate during ev-a infection [ ] . this explains the wide range of symptoms associated with ev-a infections from mild infections to neurological complications in some cases. yang and colleagues identified the interaction between ev-a viral protein (vp ) and human annexin protein thereby enhancing ev-a infection [ ] . cell surface vimentin has also been described as attachment receptor for ev-a pointing to the presence of array of receptors responsible for the viral entry into the cells [ ] . using glycoproteomic approach, su and colleagues identified cell surface nucleolin to be aiding in ev-a attachment and entry by interacting with viral protein [ ] . cell surface prohibitin was recently identified as the first possible host factor that interacts with ev-a during viral entry into neuronal cells thereby aiding in the neuropathies associated with ev-a infections [ ] . fibronectin; a high molecular weight glycoprotein joins the list of the wide array of ev-a receptors to be discovered recently by qiao and colleagues [ ] . this study postulates that ev-a may be binding to the fibronectin protein through its vp structural protein. a recent genome-wide rnai screening by yueng and colleagues identified human tryptophanyl-trna sythetase (hwars) as an entry factor for ev-a as well as cv-a and ev-d [ ] . the results from this study proposed an interesting view as the hwars are not anchored on the membrane surface where it may be acting as a receptor; thus, there is need for further studies to unravel the exact mechanism of action of these proteins. as suggested by perlman and gallagher [ ] in their commentary review on the findings from yueng's group, we support the need to further evaluate mechanisms of the three known ev-a entry receptors to find out if there is any interactions or if they are all needed for effective entry of the virus into susceptible cells. possible mode of action for this new perspective in ev-a infection has been extensively reviewed in the commentary issue by perlman and gallagher [ ] . given that ev-d and cv-a viruses do not depend on psgl and scarb receptors for entry into cells, the findings of this study will be key in understanding the pathogenesis of these viruses upon validation of the exact mechanism of action. this was the first report linking interferon gamma to inducing viral entry into the cells. the continued research aiming at documenting the array of receptors for ev-a and other picornaviruses will provide vital information in design of antiviral therapies and vaccines. completely mapping out all the essential host proteins acting as functional receptors for ev-a will provide a rich niche for design and development of vaccines and therapy against infections associated with it. the existing ev-a and cv-a receptors have not been able to completely explain the pathogenesis of hand, foot and mouth disease. human psgl for example seems to only facilitate a small number of enteroviral entry into the cells, while scarb has been shown to support an array of the viruses. this points out to the need of a more concerted efforts to identify and establish all the possible functional entry receptors for ev-a . the recently identified hwars needs to be further be validated to determine the efficiency in supporting entry of the enteroviruses reported from this study. much need to be done going forward to fully understand the pathogenesis of the hand, foot and mouth disease. with a full map of entry receptors or factors, we will be able to design antiviral therapy able to block the entry pathway of the viruses thus limiting viral infections. this will be important in the design of antivirals against enteroviruses associated with the hand, foot and mouth disease. sialic acid as well as intercellular adhesion molecule- (icam ) have been identified as receptors for enterovirus d (ev-d ) facilitating entry into susceptible cells [ , ] . the coxsackievirus-adenovirus receptor (car) protein was the first receptor to be identified for coxsackie b virus subgroups a, c, d e and f [ , ] . thereafter, other receptors for coxsackievirus a and coxsackievirus a variant (cv-a v) responsible for acute hemorrhagic conjunctivitis (ahc) have been described. icam- was identified as an uncoating receptor for cv-a ; sialic acid as an attachment receptor for cv-a v [ ] . low density lipoprotein receptor (ldlr) was purified by hofer and coworkers from hela cell culture supernatant and classified as minor rhinovirus receptor [ ] . very low lipoprotein receptor was also identified to be a receptor of the human rhinovirus (hrv ) [ ] . intercellular adhesion molecule- (icam- ) was also observed to be aiding infection of mouse cells by coxsackievirus a and rhinovirus thereby acting as its receptor [ , ] . another host factor; kremen was recently shown to play a role in the entry of coxsackievirus a (cv-a ); serotype a enterovirus [ ] . this study also showed that kremen played a major role in entry of other serotype a enteroviruses; a , a , a , a , a and a [ ] . interestingly sequence analysis of these viruses using the enteroviral structural protein p showed that they cluster together on the phylogenetic tree. studies on another enterovirus; rhinovirus c (rv-c), associated with severe respiratory diseases, wheezing and asthmas in children has been limited by the inability to grow in cell cultures. however a recent study identified human clathrin related family member (cdhr ) as a functional receptor for the rv-c [ ] . receptors for both the major group of rhinoviruses a and b have been described. major group of rhinovirus a and b (rv-a and rv-b) binds to the intercellular adhesive molecule (icam- ) [ ] while the minor group binds to the low density lipoprotein for efficient entry into the cells [ , ] . identification of receptors for the enteroviruses enables us to understand the pathogenicity of these epidemiologically important group of viruses. attachment, adsorption and entry of viruses into the cells are the key initial stages for establishing efficient viral infections. there is need to understand the infection-mics of the rhinoviruses with a goal of developing antivirals or vaccines towards this group of viruses. for echoviruses; decay accelerating factor (daf); cd known to regulate complement system within cells was also shown to be a receptor for a number of echoviruses and coxsackie b viruses [ ] [ ] [ ] . known npev receptors are summarized in table below. clearly dissecting the human host cell factors-npev interactions will provide a rich niche of interaction map that will be key in the design of antiviral therapy against this group of epidemiological importance. understanding the mechanisms involved in viral entry as well as the host cell factors acting as receptors will provide important information on the development of viral entry inhibitors. given that most of these viruses use an array of host factors/mechanisms to infect the host cell, as blocking of known entry inhibitors do not completely inhibit viral entry into the cells. this supports the need to clearly elucidate and map out all host factors involved in the viral attachment and eventual entry. this interaction between human host factors and viral proteins for eventual entry into the cells plays the key role in the viral tissue tropism. we therefore suggest that more concerted efforts need to be put in place to identify all possible entry mechanisms of these viruses with an aim of developing npev entry inhibitors into the cells thereby limiting viral infection. this can only be fruitful if we eventually identify all the host factors needed for the npev entry into cells. the recent technological advances have been essential in high throughput genome-wide screens aimed at discovery of the interplay between human host factors and the steps involved in viral infection. these techniques have revolutionized the identification of human host factors involved in viral infections with much success so far. cherry and panda presented techniques for sirna genome-wide screens, detailing all the basic steps involved [ ] . several studies have used the sirna genome-wide screens to identify the role of human host factors during enteroviral infections. wu and colleagues performed a sirna genome-wide screen which identified several human host factors necessary for ev-a virus infection [ ] . this study identified susceptible host factors and resistant host factors involved in ev-a infection; ngly and cdk and aurkb respectively pointing to an important interaction between viral proteins and human host cell factors. a small sirna screen targeting human membrane trafficking genes identified vasolin-containing protein (vcp-p ) as an important protein essential after pv viral replication and it interacts and colocalizes with bc/ c as well as ab/ b in poliovirus infected cells [ ] . ev-a through a pro and c pro have been shown to target endoplasmic reticulum proteins thereby leaving the erad proteins tethered within the er lumen [ ] . ev-a a pro specifically inhibits synthesis of herp and vimp at the translational level, while c pro cleaves ubc e at q g, q s, and q g thereby interfering with the erad processes [ ] . this study proposed that ev-a may be interfering with the er membranes and hijacks erad component; p to improve its replication [ ] . pharmacological inhibition of myristoyltransferases resulted in a decreased myristoylation of cxb virus structural proteins through reduction of vp acylation [ ] . inhibition of myristolyation by sirna knockdown and use of myristic acid analogues prevented cleavage between vp and vp as well as reduction in viral rna synthesis [ ] . these studies brings forth a new mechanism of myristoylation in picornaviral protein cleavage and processing of vp thus providing an alternative target for possible antivirals against these viruses [ ] . rna viruses have evolved with the human host cells to devise mechanisms of protecting themselves from the hostile environments within the host. these interactions result in the protection of the viral rna integrity for an efficient infection and eventual establishment of disease as reviewed by barr and fearns [ ] . it is a common belief that rna viruses can remodel their host cell intracellular membranes to form double membranous structures; replication organelles that acts as a replication site for their genome. however, the mechanism of host cell remodeling has not been fully explicated. the sequential events leading to the formation of replication organelles are not yet fully identified. there is need to elucidate the role of the human host factors especially the lipid transfer proteins within the endoplasmic reticulum. it has been postulated that enteroviruses usurp the lipid transfer at the membrane to aid in the formation of the replication organelles [ ] . stoeck and his colleagues showed that hepatitis c virus (hcv); positive stranded rna virus usurps lipid transfer protein neimann pick type c (npc ) within the late endosomes where it leads in localization of cholesterol leading to the formation of the double membrane structures essential for the formation of the replication organelle [ ] . it will be important to elucidate the role of other known lipid transport proteins including steroidogenic acute regulatory protein (star) and oxysterol-binding protein-related protein a and b (osbpl a) in the formation of replication organelle during npev viral infections. hsu and colleagues showed how viruses usurp host processes and proteins to reorganize host membranes to form replication organelles via the reorganization of the secretory pathways [ ] . this study showed how enteroviruses and flaviviruses exploit host machinery; arf and gbf resulting in recruitment of phosphatidylinositol- phosphate(pi p) lipid augmented organelles vital for their replication [ ] . specifically, this study showed that enterovirus rna polymerase binds pi p thus illustrating the importance of phosphoinositide lipids during viral genome replication. zhang and colleagues elucidated that arf and gbf ; vesicular proteins colocalizes with phosphatidylinositol- -kinase iiiβ(pi piiiβ) leading to accumulation of pi p thus pointing to their essential role during hcv virus infection [ ] . this far, it has been shown that enteroviruses recruit pi piiiβ via the a viral protein for efficient viral genome replication. a study by dorobantu and colleagues highlighted that the recruitment of pi piiiβ to the replication organelle does not depend on the interactions of gbf /arfa and acyl coenzyme a [acyl-coa]-binding protein domain (acbd ) during coxsackievirus b replication [ ] . thus, the mechanisms of recruiting the pi p leading to subsequent formation of replication complex remains unclear. furthermore, studies by xiao and coworkers showed that ev-a a protein facilitates the interaction between acbd and pi piiiβ at the replication sites [ ] . contrary to previous studies showing that pi piiiβ recruitment is independent of acbd during rhinovirus infection, this particular study points to a selective recruitment strategy of pi piiiβ facilitated by a protein to the replication sites during ev-a infections [ ] . a study by banerjees recently identified that picornaviral cd protein plays a crucial role as a master regulator during hijacking of the host cell phospholipid biosynthetic pathways; eventually resulting in proliferation of the membranes at the specific point [ ] . this study demonstrated that cd viral protein alone is sufficient to induce pi p, phosphatidylinositol- , -bisphosphate (pip ) and phosphatidylcholine (pc) synthesis during picornaviral infections [ ] . to this end, there is need to illustrate the mechanisms used by this viral protein to recruit an array of these cell membrane biogenesis lipids. to find out whether the formation of the replication organelle is conserved among the enteroviruses, melia and colleagues studied the architecture of the replication organelles formed during encephalomyocarditis virus; a picornavirus in the genus cardiovirus [ ] . this study postulated that the endoplasmic reticulum might be the likely donor organelle for the formation of the replication organelle during emcv infection [ ] . the common belief that enteroviruses replication and evasion of the innate immune system signaling is aided by the formation of the membranous web was recently challenged by melia and colleagues [ ] . using a known pi piiiβ inhibitor; bf (identified in an earlier screen by van der schaar and colleagues [ ] ), this study showed that a mutant coxsackievirus (cv-b a-h y) was able to replicate within the golgi apparatus in the absence of replication organelles [ ] . to this end, the clear steps involved in the formation of the double membranous structures required for the formation of the enteroviruses replication organelles remain unresolved. there is need to dissect the exact mechanisms involved in the formation of replication complex; a mechanism without which the replication of the viral genomes becomes compromised. this might be an opening towards the development and or design of antivirals targeting this exact mechanism. for example, the mechanisms of the cell remodeling during rna virus infection has been mined by a recent study by nguyen and coworkers [ ] . this study identified fatty acid synthase and ceramidase as potential inhibitory target against rhinoviruses [ ] , highlighting the possibility of targeting lipid transfer during the replication organelle formation for possible therapeutics. translation of viral proteins upon release into the cytoplasm is cap-independent thus human host proteins binds to the viral type internal ribosome entry site (ires) for efficient replication. some nuclear factors relocate to the cytoplasm during enteroviral infections where they bind to the internal ribosome entry sites (ires); acting as internal ribosome entry sites transacting factors (itafs) thereby recruiting ribosomes to the site for protein translation. rna binding protein; heterogenous nuclear ribonucleoprotein (hnrnp)a is known to shuttle from nucleus to cytoplasm during enteroviral infections [ , ] . lin and colleagues demonstrated that this rna binding protein(rbp) is an itaf and binds to 'utr of ev-a and sindbis virus during viral infection thus enhancing viral protein translation [ ] . tolbert and coworkers demonstrated that hnrnp a binds specifically to the stem loop ii of the ev-a ires [ ] . a follow-up study by the same group demonstrated that hnrnp a induces conformational changes upon binding to the stem loop ii of the ev-a ires leading to the enhanced viral protein translation [ ] . hnrnp a has also been linked to regulation of replication in other viruses such as hepatitis c virus [ ] , human cytomegalovirus where it interacts with immediate early gene protein [ ] , dengue virus [ ] and human papillomavirus type l [ ] among other viruses. far upstream element binding protein (fbp ) was described by lin and colleagues to be an itaf and a negative regulator of ev-a ires dependent replication [ ] . a follow-up study from the same group showed that ev-a induces proteasome, autophagy and caspase activity mediated cleavage of fbp into a positive regulator of viral protein synthesis [ ] . fbp ; another nuclear protein was also demonstrated to translocate to the cytoplasm during ev-a infection where it binds to the viral ires there by recruiting ribosomes to the sites for enhanced viral protein synthesis; thus, acting as a positive itaf [ ] . studies by zhang and coworkers described nuclear factor cellular factor -kda src-associated protein in mitosis (sam ) as an ev-a positive itaf; upon translocation into the cytoplasm [ ] . human host factors-viral protein studies identified nuclear factor; adenosine-uridine (au)-rich element rna binding factor (auf ) is targeted for cleavage by cv-b viral c protease upon translocation to the cytoplasm for enhanced stability of the ires dependent viral rna production [ ] , similar antiviral observations were made for poliovirus, coxsackievirus and human rhinovirus [ ] . rozovics and colleagues reported a cd dependent cleavage of auf during poliovirus and rhinovirus infections enhances rna replication [ ] . interestingly, the replication of another picornavirus; emcv was not affected by messenger rna decay protein: auf as observed in other enteroviruses, suggesting a variance in the restriction mechanism of this nuclear factor [ ] . investigating the role of auf in ev-a infections, lin and colleagues showed that it relocates to the cytoplasm during infection where it binds to the viral ires and restricts viral rna production [ ] . auf is the only nuclear factor which has shown an effect on the replication of other picornaviruses; pointing to its possible global role during these viral infections, offering a possible target for in the development of antivirals against enteroviruses. other host factors described to be involved in picornaviral translational activity include; misshapen nck-related kinase (mink) in ev-a [ ] , heterogenous nuclear ribonucleoprotein c [ ] , la autoantigen in hepatitis c cap-independent translation [ ] , polypyrimidine tractbinding protein (ptb) and poly(rc)-binding protein (pcbp) for ires dependent translation of poliovirus [ ] , double stranded rna binding protein (drbp ) acting as a negative ires regulator for rhinovirus [ , ] , as well as ploy(rc) binding protein and boosting poliovirus and rhinovirus ires dependent translation [ ] . the mode of action of enterovirus ires is not fully understood as seems to be a myriad of host nuclear factors involved in the cap-independent viral replication. there is need for further research to help identify all the host factors involved in enteroviral ires dependent rna production. identifying host factors that binds to the ires during enterovirus cap-independent viral translation will be key in understanding the viral replication cycle. neuronal cell death as a result of enteroviral infections have been observed in some cases of hfmd [ , ] and the mechanism linked to programmed cell death. for a long it has been a common belief that apoptosis and necrosis are the major players in programmed cell death (reviewed [ ] ). other mechanisms including pyroptosis and necroptosis have been described to play a role in complementing apoptosis in restricting viral infections [ ] [ ] [ ] [ ] . the process of caspace- induced pyroptosis was first described in salmonella enterica serovar typhimurium bacteria [ ] ; and has been elucidated to be used by other species of bacteria to escape inflammasome and stimulate cell death (reviewed [ ] ). pyroptosis; inflammatory programmed cell death, has been linked to cell death during ev-a infections in neuronal cell lines [ ] . aim mediated inflammation had been linked to the pyroptosis during ev-a infections as it was up-regulated as well as aim downstream stimulated genes such as card , caspase- and il- β during viral infection in neuronal cell lines (sk-n-sh) [ ] . yogarajah and coworkers recently identified radical s-adenosylmethionine domain containing (rsad ) and absent in melanoma (aim ) to be modulating ev-a and cv-a infections of the neuronal cells [ ] . consistent with previous findings from the same research group; the upregulation of aim resulted in reduced viral replication [ ] . the results from this study points to mechanisms involved in the neuronal complications observed in the fatal cases of ev-a infections which are not observed during cv-a infections. this observation is postulated to be as a result of differential stimulation of host factors during viral infections by the viral 'non-tranlsated regions [ ] . involvement of pyroptosis during viral infection has been reported for other viruses including; encephalomyocarditis virus (emcv) [ ] , rhinovirus [ ] and adenoviruses [ ] . viruses are known to target various host cellular factors for effective and efficient replication. several viruses have been shown to target human host cell cycle; arresting the cell division thereby avoiding competition from the dividing cells for their efficient genome replication. dna viruses have been shown to have the ability of entering the cell cycle s phase and arresting cycle for viral replication; for example simian virus [ ] , human papillomavirus and viral protein e interacts with p [ ] as well as herpes simplex virus ability to block cell cycle is reviewed in details by flemington and colleagues [ ] , have been shown to usurp the cell cycle for efficient viral replication process. infectious bronchitis virus (ibv); a coronavirus was shown by li and colleagues as well as dove and coworkers to induce cell cycle arrest during the s and g( )/m phases for improved viral replication [ , ] . influenza a virus replication has been shown to interact with cell division factors resulting to arrest of the cell cycle division at the g /g phase [ ] . arrest of cell cycle at g phase by human immunodeficiency virus- (hiv- ) viral protein r (vpr) through blocking of p cdc /cyclin b complex stimulation [ , ] . coronaviruses; severe acute respiratory syndrome and mouse hepatitis virus (mhv) are able to capture cell cycle at g /g phase for efficient genome replication [ ] [ ] [ ] . among enteroviruses, the cell cycle arrest has been reported for ev-a , cv-a , ev-d and recently for cv-a viruses. targeting the cell cycle host factors helps the viruses to replicate within the cells with limited competition from actively dividing cells. completely understanding the how viruses take advantage of the cellular processes/ proteins to establish efficient infection and genome replication is vital in the development of vaccines and antiviral therapy against these viruses. disruption of cell cycle division at s phase has been reported during ev-a infection thereby blocking the entry of the cells into g /m phase through viral rna dependent rna polymerase d non-structural protein [ ] . this study showed that ev-a mediates cell cycle through increasing transcription of cyclin e , promoting proteasomal degradation of cyclin a and eventual phosphorylation of cyclin dependent kinase (cdk ) thus regulating expression of these key cyclin regulators [ ] . the same study also showed that another picornavirus; coxsackievirus a infection as well mediates cell cycle division disruption at s phase [ ] . factors that control cell cycle and differentiation; aurora b kinase (aurkb) and cyclin dependent kinase (cdk ) were identified by wu and colleagues as ev-a restriction factors [ ] . ev-d mediates synchronization of cell division at the g /g but not at s phase thus promoting viral replication while cell cycle arrest at g /m phase inhibited viral replication [ ] . this observation is contrary to cv-a and ev-a where cell arrest at s phase promoted viral replication. remarkably, cell cycle disruption at g /m phase inhibited viral replication for cv-a , ev-a and ev-d viruses [ , ] . wang and colleagues demonstrated for the first time that cv-a disrupts cell division cycle at g /g phase for viral replication through its nonstructural protein rna-dependent rna polymerase d and c protease proteins [ ] . viruses depends on host cell proteins and processes for efficient genome replication. exploiting the cell cycle process, a highly regulated process enables viruses to have unfettered access to the cell cycle factors for efficient viral replication. future work should look at the cell cycle stage where other enteroviruses disrupt cell division cycle. this will enable better antiviral therapy design and development targeting different viruses associated with hfmd as well as other forms of enteroviral infections. the process of autophagy has been linked to the formation of the double membranous structures which acts a replication site for enteroviruses including poliovirus (pv). the formation of these membranous structures is dependent on the exploitation of the autophagy process by the enteroviruses (pv, cv-b, cv-b among other enteroviruses) where a and bc viral proteins are involved [ ] [ ] [ ] [ ] [ ] [ ] . recent studies have linked autophagy regulators to the formation of the autophagosome/ the replication organelle during coxsackievirus b (cv-b); thus showing that enteroviruses not only target the autophagy process but also its regulators for efficient replication of their genomes [ , ] . wong and colleagues showed that coxsackievirus b (cv-b ) induces autophagosome formation without lysosome degradation of proteins [ ] , clearly highlighting the role of autophagosome in the formation of the replication organelles during enteroviral infections. follow-up studies by zhai and colleagues observed formation of autophagosomes both in cv-b infected fibroblasts and in balb/c mice thus, linking autophagy to the pathogenesis of myocarditis infections [ ] . the shedding of cv-b virus from infected cells was linked by robinson and colleagues to the extracellular microvesicles with autophagosome markers. the role of the autophagomes in the release of cv-b virus from infected cells was later validated by sin and coworkers [ ] . the study by sin and colleagues demonstrated ability of cv-b to egress from cells and infect other cells via a dynamin related protein (drp ) initiated mitochondrial fragmentation; a process vital for the mitochondrial based autophagy elimination/mitophagy [ ] . from this study, cv-b is believed to localize in the mitochondria where it initiates virus induced mitophagy and eventual escape from cells through autophagosomebound-mitochondrion-virus complex [ ] . the role of mitophagosome in the release of cv-b virus, explains possible alternative process used by picornaviruses to release from infected cells and infect other cells thus ensuring the infection cycle is sustained. the disruption of the mitochondrial dynamics through virus induced stimulation of drip to block virus induced apoptosis and eventual persistence of viral infection has also been observed in hcv [ ] . this points to the fact that different single stranded rna viruses may be using the same process to disrupt mitochondrial traffic and eventual apoptosis for viral replication maintenance of viral infection cycle. enterovirus a (ev-a ) induced autophagy had been reported both in vivo and in vitro with ev-a -vp and c proteins localizing with microtubule-associated protein light chain (lc ) and mannose- -phosphate receptor (mpr) resulting in the formation of the amphisome thereby increasing viral replication [ , ] . ev-a bc non-structural protein was recently shown to trigger the formation of autolysosomes in human rhabdomyosarcoma cells thus enhancing ev-a replication [ ] . this study also showed that the bc protein interacts with n-ethylmaleimide-sensitive factor attachment receptor (snare) protein, syntaxin- (stx ), synaptosome associated protein (snap ) and microtubule-associated protein light chain b (lc b) major players in the formation of the autolysosome [ ] . the results of this study are consistent with earlier findings linking enterovirus bc non-structural proteins to the exploitation of autophagy process to support enterovirus viral replication. corona and colleagues showed that enterovirus d (ev-d ) is able to disrupt autophagy processes downstream to promote viral replication and eventual egress from the cells thus promoting viral infection within the cells [ ] . this phenomena linking viral proteins to interact with various regulators of autophagy processes for efficient viral replication and transmission has been reviewed [ , ] . another pending issue has been if the enteroviruses are able to replicate inside the acidic autophagosomes and how do they evade degradation and exit the cells intact. however, this has so far been linked to the enteroviruses' ability to divert cargo traffic away from degradation [ , , ] . cv-b c protease has been illustrated to target cleavage of snare and plekhm proteins which are key in regulation of autophagosome fusion and eventually impairing establishment of snare complexes [ ] . the role of autophagy regulators in enterovirus infections have also been studied. for example, a study by delorme-axford showed that an autophagy regulator; bactericidal/permeability-increasing protein (bpi) foldcontaining family b, member (bpifb ) acts as a host limiting factor during coxsackievirus b virus infection [ ] . this study reported that bpifb may be playing a role in downregulating the key steps involved in the autophagy process proposed to be helping in the formation of the membranes needed for enteroviruses replication [ ] . a study by morosky and colleagues linked bpifb , another protein in the family of bpifb to be a positive regulator of cv-b suggesting that bpifb family of proteins may be having diverse effects in regulating viral infections [ ] . a recent study by delorme-axford and coworkers identified exoribonuclease xrn as a negative post-transcriptional regulator of autophagy [ ] . the same study also showed that xrn maintains the process of autophagy at basal levels thus limiting replication of poliovirus and coxsackievirus b [ ] . a recent study by velazquez and colleagues demonstrated that poliovirus can generate autophagosomes through a downstream of ulk signaling pathway; cleaving the cargo traffickers which may negatively interfere with cargo loading [ ] . this points out to the ability of the picornaviruses to fine-tune the interaction with the autophagy machinery for effective survival within the cells. targeting of autophagy key players and auxiliary factors have been reported for number of picornaviruses. cv-b through its viral aprotease was has been shown to cleave sequestosome /p (sqstm / p ) [ ] ; a known intermediary of selective autophagy degradation of ubiquitinated proteins [ ] [ ] [ ] . this study further showed that cleavage of sqstm resulted in the impairment of nf-kb signaling and eventual disruption of the selective autophagy in infected cells; emerging as an pro-viral strategy to establish an efficient infection during cv-b infection [ ] . a subsequent study by mohamud and colleagues demonstrated that sqstm and another host factor calcium binding and coiled-coil domain-containing protein /nuclear dot protein (calcoco ) regulate cv-b virus infection by targeting autophagy receptors; via their interaction with viral protein [ ] . this study also showed that calcoco targets mitochondrial antiviral signaling protein for degradation thereby blocking the establishment of antiviral state within the infected cells for efficient establishment of cv-b infection [ ] . different strategies used by viruses to trigger and hijack the process of autophagy have been recently reviewed in details by zhang and coworkers [ ] . autophagy is key in controlling various cellular processes including enhancing innate immune signaling during viral infections through a process known as virophagy. the ability of virus infected mitophagosomes to be released out of the infected cells provides an important mechanism of virus egress from the infected cells. enteroviruses have been shown to have the ability of interacting with cellular autophagic process that is conventionally known to degrade the mitochondrial traffic upon fusion with the lysosomes. enteroviruses has evolved ways to evade this process through degradation of various autophagy initiating factors as well as its regulators. this host cellular process has been linked to the non-lytic exit of various enterovirus infections including poliovirus, echovirus , eva and cv-b viruses. however, blocking initiation of mitophagy as a way of controlling viral infections may not be feasible given that observations from different studies have shown only disruption of extracellular micro-vesicles (emv) release and not the replication ability of cv-b virus. thus, this process does not provide an ideal antiviral target. an overview of the human host cell/process: npev viral protein interactions are highlighted in table below. much has not been achieved in the development of antivirals against npev infections. the major challenge for development of the antivirals has always been the mutations on the viral genomes. several compounds have been tested for possible use as antivirals against enteroviruses as shown in table below but no much success has been achieved. most of the drug screening have been done in vitro with little success in vivo and in clinical trials. screening fda approved drugs and repurposing of existing drugs based on known viral-human protein interactions are some of the strategies that has been adopted by scientists to identify antivirals against npevs. for example, li and colleagues evaluated the effects of ribavirin a known antiviral against other rna viruses on ev-a for possible repurposing of the drug [ ] . their study showed decreased ev-a virus yield in vitro and reduced disease status, death and adverse effects associated with its infection in vivo; highlighting the possible role as an antiviral compound against ev-a [ ] . plant metabolites have also been targeted as possible antiviral compounds against enteroviruses. for example, quercetin; a well distributed plant flavonoid has been shown recently to inhibit ev-a infection by inhibiting virus attachment, adsorption and by targeting viral c protease [ ] . the antiviral efficacy of pyrazolo [ , -d] pyrimidines have also been evaluated against enteroviruses; cv-b and ev-a virus infections where they inhibited their infections but the exact mechanism was not established [ ] . more recently, andrographolide has been reported to suppress ev-d replication targeting the viral maturation within the acidified endosomes [ ] . world health organization (who) recommended combination therapy has also been evaluated for possible antiviral development against enteroviruses [ ] . screening of fda approved drugs recognized pirlindole as a strong inhibitor of cv-b [ ] . natural products have recently gained much interest in drug development studies. of these; plant secondary metabolites; flavonoids have been of interest in drug therapy screens against viral infections given that they are freely available and form better part of human dietary. screening of plant metabolites for possible use as antiviral therapy has been reported as reviewed by zakaryan and colleagues [ ] and their biological activity as well as chemistry have also been extensively reviewed [ ] . some flavonoids with antiviral abilities in vitro against viral infections include; isoquercitrin against zika virus infections [ ] , chikungunya infections [ ] , apigenin antiviral effects on a number of viruses such as african swine fever virus (asfv), hepatitis c virus [ , ] . apigenin have also shown antiviral activity against ev-a virus by inhibiting viral ires dependent translation [ ] [ ] [ ] . a recent screen of flavonoid library identified st and st as lead antiviral compounds against ev-a , cv-a and cv-a enteroviruses [ ] . all these concerted efforts towards identifying antivirals against enteroviruses and other viral infections needs a follow-up and validation in animal models. the good news is that most of the already identified compounds have shown no cytotoxicity in cells; thus, may not have toxic effects in animal models. the efficacy of most of the identified compounds have only been elucidated in vitro thus there is need for further studies to identify their effects in vitro. little success has been achieved in terms of antiviral therapy against enteroviruses. given that drug discovery process is an expensive and time-consuming venture, most researchers have relied on the fda approved drugs or drugs that are already in use for possible repurposing. not much success on drug therapy has been recorded in viral infections due to the high mutation rates observed during viral replication. combination therapy of the drugs with different mode of actions ev-a -vp and c proteins induce autophagy through localization with lc and mpr enhanced ev-a replication through formation of amphisome [ , ] ev-a bc protein interacts with snare, stx , snap and lc b proteins leading to formation of autolysosome in rd cells enhanced viral replication [ ] ev-d can disrupt autophagy process downstream promotes viral replication and egress from infected cells; promoting viral infection within the cells [ ] cv-b viral protein c targets cleavage of snare and plekhm proteins impairs establishment of snare complexes thus providing conducive environment for viral replication [ ] cv-b viral a protease cleaves sqstm /p a known intermediary of selective autophagy degradation of ubiquitinated proteins impairs nf-kb signaling and disrupts selective autophagy in infected cells to establish an efficient viral replication/infection [ ] cv-b interacts with calcoco and sqstm targets autophagy receptors; targets mitochondrial antiviral signaling protein for degradation thus blocking establishment of antiviral state in the infected cells [ ] targeting different stages of viral infections would be an alternative in targeting different stages of the enteroviral infection cycle. this will only be achieved with a complete map out of the human host factors hijacked by these viruses during infections. thus, there is need for continued elucidation of molecular mechanisms of the already postulated viral targets as well as identifying other underlying factors and process. vaccines have shown much success against viral infections and the success story of vaccination against poliovirus infection in the world which is a picornavirus; points for the need of continued studies towards identifying vaccine candidates against the enteroviral infections. with outbreaks of enteroviruses being recorded in different parts of the world, if not checked they might have a potential threat to the global health; just soon after near-eradication of poliovirus infection. the emergence of outbreaks of enteroviral infections in different parts of the world point to the need of mapping all the host factors involved in the infection paradigm. given that viruses need host factors in every step of their infection from attachment, entry, replication, virion assembly and eventual entry, there is need to elucidate all the host factors involved for an improved understanding of the molecular dynamics of enteroviral infections. this will be a big boost towards the long overdue antiviral and vaccine development against these epidemiologically important viruses. there is much to be elucidated on the formation of npev replication complex formation as the existing mechanisms do not wholly explain the processes and steps involved in this important process during viral replication. the nuclear host factors involved in the enteroviral replication also needs to be fully described as this is a vital step in maintaining viral replication and eventual life cycle. viral entry studies need to be carried out as the known receptors and viral entry requirements do not fully explain the myriad of disease features observed during viral infections. the role of cellular processes such as autophagy, apoptosis, necroptosis, pyroptosis as well as posttranslational modifications in enteroviral infections also needs to be fully elucidated. this will be specifically important in explaining the little-known stages of viral infections such as non-lytic egress for continuous viral cycle within the host. the paucity of information on the infection dynamics of these viruses calls for concerted efforts to elucidate the viral-human cell interactions. there is still a lot to be investigated to fill the gaps that exist on the life cycle of non-polio enteroviruses. with new cases emerging in different parts of the world, it is just a matter of time before we have a global outbreak of non-poliovirus enteroviral infections in different parts of the world. there is also an urgent need for further studies especially in the field of vaccine developments as well as antiviral therapy against enteroviruses. we would like to thank the authors' whose work has been reviewed and cited in this paper as well the funding organizations that funded the primary research. authors' contributions coo conducted literature research and wrote the draft manuscript, jc provided editorial input and revision for the final version. both authors read and approved the final manuscript. agency for science, technology and research-singapore international graduate award (a*star-singa). some of the work reviewed and cited in this paper was funded by different funding agencies. availability of data and materials not applicable. dtrip ev-a blocks viral replication by targeting the rna dependent rna polymerase. [ ] ribavirin ev-a general reduction of the viral yield [ ] quercetin ev-a inhibits virus attachment, adsorption and the viral c protease [ ] rupintrivir ev-a , cv-a , hrv binds and inhibits viral c protease activity [ ] [ ] [ ] fluoxetine cv-b reduces synthesis of viral rna and protein [ ] dibucaine cv-b , ev-d , rna replication stage; may be targeting cv-b viral c protease [ ] formoterol cv-b , ev-d , ev-a , rv-a , rv-b likely inhibit rv- by reducing icam- levels or acidic endosomes. [ , ] pirlindole ev-d and cv-b rna replication stage; may be targeting cv-b viral c protease budesonide rv- [ ] zuclopenthixol ev-d and cv-b rna replication stage; may be targeting cv-b viral c protease [ ] apigenin ev-a targeting viral ires thereby inhibiting viral translation process [ ] [ ] [ ] ictv virus taxonomy profile: picornaviridae enteroviruses as agents of emerging infectious diseases an apparently new enterovirus isolated from patients with disease of the central nervous system fatal enterovirus encephalomyelitis bulbar poliomyelitis: mr findings with pathologic correlation neurologic complications in children with enterovirus infection mr imaging findings of enteroviral encephaloymelitis: an outbreak in taiwan fatal rhabdomyolysis and renal failure associated with hand, foot and mouth disease nephrotic syndrome in hand, foot and mouth disease caused by coxsackievirus a : a case report acute kidney injury secondary to severe hand, foot and mouth disease caused by 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of zika virus infection in human cells antiviral activity of selected flavonoids against chikungunya virus the flavonoid apigenin inhibits hepatitis c virus replication by decreasing mature microrna levels apigenin inhibits african swine fever virus infection in vitro apigenin inhibits enterovirus- infection by disrupting viral rna association with transacting factors apigenin inhibits enterovirus replication through suppressing viral ires activity and modulating cellular jnk pathway apigenin restricts fmdv infection and inhibits viral ires driven translational activity a flavonoid compound library screen revealed potent antiviral activity of plant-derived flavonoids on human enterovirus a replication novel antiviral agent dtrip- targets rna-dependent rna polymerase of enterovirus enterovirus and coxsackievirus a c proteases: binding to rupintrivir and their substrates and anti-hand, foot, and mouth disease virus drug design in vitro antiviral activity of ag , a potent inhibitor of human rhinovirus c protease multiple classes of antiviral agents exhibit in vitro activity against human rhinovirus type c fluoxetine is a potent inhibitor of coxsackievirus replication formoterol and budesonide inhibit rhinovirus infection and cytokine production in primary cultures of human tracheal epithelial cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no competing interests.received: april accepted: june key: cord- -mxyxwkhx authors: sallie, richard title: replicative homeostasis ii: influence of polymerase fidelity on rna virus quasispecies biology: implications for immune recognition, viral autoimmunity and other "virus receptor" diseases date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: mxyxwkhx much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. rna polymerases (rna(pol)) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. thus, rna(pol )causes more morbidity and premature mortality than any other molecule. the extraordinary genetic heterogeneity defining viral quasispecies results from rna(pol )infidelity causing rapid cumulative genomic rna mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking rnapol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral rna is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. this mechanism – "viral receptor disease (vrd)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type ii diabetes mellitus, and some forms of obesity. viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations. many of the world's population suffer from acute and chronic viral infection. the two common types of chronic viral hepatitis (cvh), hepatitis b (hbv) and c (hcv) are major causes of death and morbidity; conservative esti-mates suggest million people are persistently infected with hbv, while hcv may infect a further million. annually, in excess of two million people will die from cirrhosis or liver cancer caused by cvh, and many more suffer chronic ill health as result. during the years since the human immunodeficiency virus (hiv) was identified, perhaps million people have become infected worldwide and each year about a million die from resulting immunodeficiency and consequent opportunistic infections, particularly tuberculosis, and other complications. poor countries bear a disproportionate burden of disease caused by these viruses that further exacerbate poverty through pervasive economic disruption and diversion of limited resources to healthcare and disease control. emerging viral pathogens including west nile virus (wnv), the sars coronavirus, endemic viruses like murray valley, japanese, and other encephalitis viruses, dengue and yellow fever, and seasonal influenza, hepatitis a (hav) and e (hev) cause enormous further morbidity and mortality, while pandemic outbreaks of virulent influenza strains remain a constant threat. together, these viruses probably kill more people every ten days than the boxing day tsunami. rna viral infections, including foot and mouth, bovine viral diarrhea virus (bvdv) and hog cholera virus (hchv), cause similar devastation of animal populations with enormous economic consequences. rna polymerases generate massive genetic variability of rna viruses and retroviruses that circulate within infected hosts as vast populations of closely related, but genetically distinct, molecules known as quasispecies. after translation, this genetic variability causes near-infinite antigenic heterogeneity, facilitating viral evasion of host defences. tuberculosis, malaria and other cellular pathogens also express broad cell-surface antigenic heterogeneity, generated by dna-dependent rna pol . thus, rna polymerases probably cause more morbidity and premature mortality in man, and other animals, and greater economic loss, than any other molecule. despite a depressing global epidemiology that strongly suggests otherwise, the immune system is thought to "control" viruses. what practical meaning does "immune control" have for the individual? there is no argument for hbv, and other viruses, high affinity antibody, generated by prior vaccination or other exposures and directed against neutralizing epitopes, will prevent hbv infection (excepting vaccine escape mutations [ , ] ), in part by blocking viral ligand interaction with cell receptors, or that most patients exposed to hbv develop neutralizing antibodies (hbsab), clear hbsag from serum, and will normalize liver function long term. however, even patients who develop robust immune responses to hbv, defined by high-affinity antihbsab and specific antiviral cytotoxic t cell (ctl) responses, will have both "traces of hbv [ ] ... many years after recovery from acute hepatitis" [ ] and transcriptionally active hbv demonstrable in peripheral blood mononuclear cells (pbmcs) [ ] . furthermore, occult hbv is detected in liver tissue of patients with isolated antihbc (i.e. hbsag/hbsab negative) [ ] and in patients with hbsag-negative hepatocellular carcinoma [ ] suggesting, at least some patients, hbv in may persist irrespective of any immune responses, implying long term latency and low level basal replication may be a survival/reproductive strategy for hbv. for most patients, acute hcv or hiv infection results in life-long viral persistence. although many patients develop immunological responses, including specific antibody and ctl reactivity to various viral antigens, these responses have little discernible impact on either hcv or hiv replication that occurs essentially unchecked at rates estimated between and virions per day [ , ] , indefinitely, while progressive destruction of liver or immune cells proceeds, commonly resulting in cirrhosis or liver cancer (for hcv) or death from immune deficiency (for hiv). evidence that prior hcv infection confers no protective immunity against heterologous hcv infection in humans [ ] or chimpanzees [ ] or against either homotypic [ ] or heterotypic [ ] human reinfection, confirmation that active hcv infection persists long after either apparent spontaneous [ ] or treatmentinduced [ ] viral clearance, or that vaccines causing specific antiviral b and t cell responses fail to protect against infection in animals [ ] , and that antibodies to hcv envelope protein e are only detected in animals with persistent infection [ , ] , further undermines the potency of "immune control" and suggests, at least for patients with hcv, the definition of "control" may need to broadened significantly. based on observations that stronger specific cd /cd immune responses with t-helper (th ) cytokine profiles are found more frequently in patients with self limiting viral infections than those who develop chronic viral carriage [ , ] it is thought ability to mount robust adaptive immune responses predicts viral clearance while failure to do so results in chronic viral carriage [ ] . however, detailed and very painstaking studies, albeit in small numbers of chimpanzees [ ] and patients following antiviral therapy [ ] , have failed to demonstrate any relationship between t cell responses and viral clearance. although development of th and other immune responses are certainly temporally and, probably, causally related to reduced viral replication and viral clearance the assumed direction of causality (immune response -> reduced viral replication), is not proved by the fact those responses develop, post hoc ergo propter hoc, as comforting a conclusion as it may be to reach. the first part of this paper explores the impact of rna pol fidelity on quasispecies behaviour, specifically in mediating immune avoidance during acute hcv infection. we suggest the primary event causing reduction in viral replication is inhibition of rna pol processivity by variant viral proteins, specifically envelope and envelope-related proteins. we also suggest that immune responses to viruses are thwarted initially by broad antigenic diversity generated by low rna pol fidelity but develop, when they do, after viral replication falls (because of reduced rna pol processivity) and polymerase fidelity increases -linked events that occur because of replicative homeostasisthus restricting antigenic diversity sufficiently to permit focused immune recognition. we further suggest immune responses strategically exploit replicative homeostasis to force viruses to reveal critical dominant antigenic epitopes, facilitating progressively more focused immune responses. the second part explores the ineluctable consequence of viral rna quasispecies: that is, translation of rnas into protein quasispecies with a spectrum of phenotypes and unpredictable properties, among which may be disruption of the cell surface receptors that viruses co-opt for cell entry. this innate property of viral quasispecies may explain a wide variety of diseases apart from viral autoimmunity. acute hcv and hbv infection have characteristic kinetics of viral replication, adaptive immune responses, and cause predictable tissue injury, reflected in elevated serum aminotransferases. these kinetic and transaminase responses are summarized schematically for patients with persistent infection (figure ) [ ]. initial hcv replication is very rapid and viral load increases exponentially until about week , at which point viraemia increases more slowly, and asymptotically, towards ~ genome equivalents (geq)/ml by weeks - (these kinetics alone suggesting competitive inhibition of rna pol ). this exponential increase of viral rna in serum reflects explosive dissemination of virus in tissues, detectable by in-situ hybridisation throughout hepatocytes, including the nuclei, within days of infection [ ] . viral replication declines rapidly from weeks - to weeks - falling by - geq/ml but lower level (~ geq/ml) fluctuating replication persists, generally indefinitely, thereafter. by contrast, neither hbv dna nor hbv antigens are detectable in either viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection figure viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection. data summated from figure [ ] and modified to represent typical patients with chronic viral persistence. note: a) high level hcv replication for - weeks prior to any immune responses, b) onset of humoral immune response well after down-regulation of viral replication [ ] , and c) transaminase peaks occurs ~ weeks later. both hbv and hcv are non-cytolytic and viral clearance from hepatocytes, as well as hepatocyte injury, thought to be immune mediated. however, for both hbv and hcv the brisk fall in viral replication following acute infection paradoxical hcv replication kinetics figure paradoxical hcv replication kinetics. if host immune clearance forces (i c , black arrows) reduce viral replication acutely (point a), then they must exceed viral expansive forces (v e , grey arrows) at that point. at equilibrium (e.g. points b through d), viral concentrations (-) and, therefore, viral forces, have fallen by - hence, immune forces i c must fall by > - from a to b for equilibrium to develop. there is no evidence this happens. http://www.virologyj.com/content/ / / precedes the peak of transaminase rise by at least two weeks (figure ). if falling viral replication is due to adaptive immune responses causing hepatocyte lysis the transaminase peak should either precede or be coincident with falling replication. this temporal relationship is also inconsistent with the belief immune factors cause the falling replication seen during acute hcv or hbv, and is analagous to non-cytolytic reductions of viral replication observed for both hbv and lymphocytic choriomeningitis virus (lcv) experimentally, that suggested either [unspecified] antiviral mechanisms are operative [ , ], or that auto-inhibition of rna pol by viral mechanisms (replicative homeostasis) occurs [ ] . however, if other non-cytopathic host anti-viral mechanism(s) are responsible, the kinetic paradox implies their potency falls significantly between points a and b. hepatitis c replication kinetics and their relationship to immune responses are well documented [ , ] but reveal an unexplained paradox. despite high level viral replication, adaptive cellular immune responses to hcv are completely undetectable for at least - weeks [ ] after infection, while humoral responses are rarely detected before - weeks [ ] , and in some patients [ ] , and some chimpanzees [ ] , are never detected at all. an exhaustive and very careful review of the clinical and experimental data relating adaptive immune response and hcv replication kinetics has been published recently [ ] . seeking to rationalize the enigma posed by a complete lack of immune responses to hcv replication of ~ - geq/ml at week but [variable] immune responses to replication at ~ geq/ml after week , the authors conclude "..[the data]...appear[s] to be consistent with the interpretation that hbv and hcv are ignored by the adaptive immune system for about months after primary infection" and "[in hcv].. the adaptive response seems to really ignore for several weeks a substantial quantity of virus (at least copies/ml)..". this is certainly an accurate synthesis of an extensive and highly complex literature but does it make any sense? if adaptive immune responses really ignore high level hcv replication for two months, as suggested, then the following mechanism(s) are implied: a) an accurate mechanism for prompt detection of infection; b) a timing mechanism; c) a trigger mechanism for immune responses independent of any viral factor (given levels of virus are greater before immune recognition than afterwards the trigger for immune response must be either non-viral or falling (!) viraemia); and, as cytomegalovirus (cmv)-specific cd (+) t cell responses arise within days of cmv infection [ ] ; d) a mechanism allowing the immune system to differentiate hcv from cmv and other viruses (and reasons to do so). while possible, this seems unusually inelegant and pointlessly counterproductive, especially as events soon after infection probably determine whether virus is cleared or chronic infection develops. it is much more likely that adaptive cellular or humoral immune responses do not develop in the first - weeks of hcv infection simply because the virus isn't "seen". why should hcv replicating at - geq/ml at week be invisible to the immune system but visible when replicating at geq/ml long term? dissection of this problem requires explicit analysis of what is being measured and how. assay of hcv rna and detection of hcv by immune responses measure two quite different things. quantitation of hcv is typically performed by branch-chain cdna assay (bdna) or quantitative pcr (qpcr) using probes or primers complementary to conserved 'untranslated ( 'utr) hcv rna sequences. immune responses to hcv typically "measures" envelope proteins translated from envelope-encoding rna (eerna) sequences and are directed at specific antigenic amino acid sequences and polypeptide conformations, not total viral envelope protein concentrations. while concentrations of 'utr rna will be proportional to eerna concentrations in any given sample, they may not be identical for two reasons; i) rna transcription may prematurely terminate making 'utr rnas relatively more prevalent than eernas and ii) hcv 'utr is highly conserved, while eerna s are less constrained, making hybridization efficiencies of pcr primers or bdna probes greater for 'utr rnas than for the population of eernas, causing relative under-estimation of true envelope rna concentration . nonetheless, as 'utr hcv rna concentrations will be proportional to eerna concentration, the question remains; why should envelope proteins translated from eerna sequences present at concentrations corresponding to ~ 'utr geq/ml at weeks be visible immunologically, but envelope proteins derived from eerna sequences corresponding to ~ - 'utr geq/ml at - weeks remain unseen? quasispecies biology, specifically variable rna pol fidelity, replicative homeostasis, and sequence-specific requirements for both genetic and immunological detection suggest an answer. rna viruses replicate by copying antigenomic templates, a process catalysed by rna pol , an enzyme lacking fidelity or proof reading function [ ] [ ] [ ] [ ] . theoretically, an rna viral genome like hcv (about bases) could assume any of (about . × ) possible sequence combinations exceeding, by some margin, population estimates of protons in the known universe (about ), meaning the potential complexity of rna viral quasispecies is infinite, for all practical purposes. an rna pol fidelity rate of - errors per base copied predicts at least one and as many as (estimated for hiv) [ ] genomic mutations will be introduced during each cycle of replication. furthermore, as hcv replication results in synthesis of ~ virions per person per day [ ] , on average, mutations will develop at each genomic locus ~ times/day, while the probability any two genomes synthesized consecutively will be identical is about - . the sum effect is inexorable accumulation of genomic mutationsthat, by itself, should threaten replicative fitness because of muller's ratchet [ ] -and progressive dilution of wildtype genomes (figure ), processes that make long-term stability of rna virus quasispecies highly paradoxical [ ] . as argued previously, a combination of selective genomic replication and variable rna pol fidelity, both mediated by replicative homeostasis, act together to prevent rna quasispecies extinction [ ] . the phenotypic consequences of viral quasispecies biology may be more important. progressive divergence of genomic rna sequences away from wild-type sequences caused by rna pol infidelity generates a massive population of closely related, but genetically distinct, rna molecules (figure ), an effect operative at all scales from each open reading frame (orf) to whole virus species. a quasispecies of orf rnas has but one inevitable outcome; translation of a quasispecies of viral proteins with a vast and highly variable spectrum of phenotypes, some subtly nuanced, others grossly defective. furthermore, mutations simplified, two dimensional clade diagram of hyperdimensional viral rna and protein sequence-space figure simplified, two dimensional clade diagram of hyperdimensional viral rna protein sequence-space. because of rna pol (p) infidelity and müller's ratchet, mutations ( ) are introduced into each rna template synthesized, and progressively accumulate, resulting in an rna quasispecies with sequence progressively divergent from consensus sequence. translation results in a spectrum of proteins ( , , , etc.) with properties that also vary progressively from wild-type sequence ( ) to highly variant proteins ( , , etc.). some rnas will be so abnormal that translation or replication fails or is truncated ( ), while others will code for grossly defective proteins ( , etc.). that create new, or obliterate pre-existing, start or stop codons in a significant proportion of rnas, will cause translation of highly unusual and heterogeneous proteins, particularly during high-level viral replication, a phenomenon that may explain hbeag. viral quasispecies cannot, and will not, produce homogeneous proteins with predictable and consistent phenotypic and antigenic properties. while rna pol infidelity will cause progressive divergence of copied sequences away from wild-type or consensus sequences, the probability of any particular sequence arising will fall dramatically with increasing genetic distance from that consensus sequence (figure ), allowing conceptual representation of the resulting genomic (and consequent phenotypic) diversity as a frequency distribution curve, with increasingly variant sequences surrounding a 'centre of gravity of replication', formed by wild-type sequences. viral quasispecies occupy hyperdimensional sequence-spaces, hence any physical representation is necessarily simplified, but because mutation away from wildtype sequences is equally probable in all directions, variant rna and protein frequencies will be normally distributed and the standard deviation (sd, σ) -insofar as 'normal' or 'standard' can be applied to a hyperdimensional space -of that distribution will be a function of rna pol fidelity; if rna pol is completely faithful, the rnas and proteins will be monoclonal and σ = ; if rna pol has no fidelity, rna will be synthesised randomly, and all rna and consequent protein sequences will arise with equally probability, therefore σ = ∞. while viral rna and related protein sequences are theoretically unconstrained (at least before any consideration of functionality), the sequence specificities of any reagents used in their detection (bdna probes, pcr primers, mabs etc) are not, by definition, and their specificity and the efficiency with which they detect variant molecules will fall progressively the further those variant sequences are from the consensus sequence. a zone of 'reagent specificity' may therefore be defined probably encompassing wild type and some variant sequences, but there will exist some rna sequences and corresponding proteins of any quasispecies that are undetectable with these sequence-specific reagents. a threshold of detection of any assay (including immune detection) may similarly be defined; rna or protein sequences present at concentrations below this conceptual level being undetectable by that particular assay. the hcv "early replication" paradox now partially resolves; the 'utr sequences are both highly conserved and common to virtually all rnas in the quasispecies, therefore, the 'utr concentration -that is, the common measure of hcv viraemia -corresponds to the area under the frequency distribution curve. by contrast, envelope rna sequences (and related envelope proteins) are not so constrained and their relevant concentrations (i.e. whether or not that rna or protein sequence is detectable) corresponds to the frequency of that specific sequence in the quasispecies and that, in turn, depends on rna pol fidelity; if rna pol fidelity is low, the frequency or concentration of any particular rna or protein sequence will also be low and may be below the detection threshold, while increasing rna pol fidelity may increase sequence frequency [i.e. the concentration of specific proteins] above detection threshold. but why should specific eerna sequence frequencies -in other words, hcv rna pol fidelity -increase after week , facilitating adaptive immune responses? viral autoregulation, specifically replicative homeostasis, provides an answer. interactions among species, whether between humming birds and flowering plants, primitive viroids and prokaryotic cells or hcv and man, results in an unremitting process of adaptation and responsive counter-adaptation -in effect, a molecular arms race -for each species just to maintain ecological parity. the price of survival for a species is continual evolution. survival, for viruses, requires cell entry, a precondition long antedating necessity to evade more complex host defenses, including interferons and other cytokines and adaptive immune responses, while for cells, and complex cellular organisms, cell wall defenses, including receptor polymorphisms, form a principal barrier against viral invasion. viral survival -effectively meaning rna pol survival -on an evolutionary timescale, as argued previously [ , ] , requires control of mutation and replication rates in a manner adaptively responsive to constantly changing biota and this implies dynamic linkage of rna pol fidelity and processivity with quasispecies phenotypic and antigenic diversity, meaning an autoregulatory linkage -replicative homeostasis -between rna pol fidelity and processivity and envelope proteins, as argued previously [ ] . by definition, evolutionary co-adaptation occurs in response to adaptations in locally prevalent interacting species. natural selection for beak variation(s) in darwin's finches occurs as a consequence of concrete survival benefits these variations -mediating, for example, enhanced food harvesting interactions with other variable plant or animal species -confer to individual galapagos island birds, rather than any inexorable hypothetical 'improvement' in beak function for finches in general. if a species is widely distributed in space, but population mixing is slow or incomplete, locally prevalent interactions with other species will vary and regional genetic variations will arise and be maintained, hence progressive divergence from the original genotype (speciation) may result. for viruses, and their hosts, genetic variations -reflected in viral genotype and cell surface polymorphisms and resulting disease susceptibilities -would be predicted, and are observed [ ] [ ] [ ] [ ] [ ] [ ] , to have frequencies that vary geographically. consider the following; an enzyme (e) functioning in a closed system synthesizes either product a or b that both interact with e to influence output such that a:e interactions cause production of b, while b:e interactions pro-duce a. irrespective of starting conditions (excluding substrate exhaustion and product inhibition), an equilibrium will eventually develop ( figure ) with the relative concentrations of a:b determined by the relative association constants (k) of a:e (k a:e ) and b:e (k a:b ) and the velocity (ν) of production of a from b:e (ν a ) and b from a:e (ν b ). removal or addition of either a or b will alter equilibrium conditions but not the fact equilibrium is reached; if a is removed, for example, the increased two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent spe-cificity (red shading) and threshold of detection (tod) of any assay figure two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent specificity (red shading) and threshold of detection (tod) of any assay. as mutations ( , ) accumulate and rna sequence progressively diverges from consensus sequence ( ) the probability of that rna sequence and corresponding protein (e.g. envelope, env.) arising falls rapidly. standard deviation (σ) of frequency distribution is proportional to rna pol fidelity. frequency genetic distance threshhold of detection (tod) reagent specificity ♦ ♦ frequency of b:e interactions will cause compensatory increased a synthesis; in this sense enzymatic autoregulation occurs. intuitive analysis suggests that enzymes acting in a milieu of increasing concentrations of inhibitory molecules become progressively less processive until reduced enzyme output is insufficient to further inhibit enzyme activity, and an equilibrium state is reached. considering viral replication, if alteration of rna pol fidelity causes synthesis of either wild-type or variant rna sequences (simplified, as a continuum between these two must exist) that are subsequently translated into either wild-type or variant polypeptides that then interact with rna pol such that wild-type: rna pol are high affinity interactions that induce rapid, low fidelity rna pol replication while variant protein: rna pol interactions are low affinity and cause high fidelity rna pol replication at low rate then an equilibrium will eventually develop. hence, as relative concentrations of wild-type and variant viral proteins vary, alteration of both processivity and fidelity of rna pol results, permitting viruses to adaptively respond to environmental changes, including immune recognition and reaction to evolving cell receptors. stable, highly reactive equilibria not only develop as a result of rna pol /envelope interactions and viral autoregulation, there is no option but for this to occur. generation and maintenance of polymorphisms, that is, replacement of existing genes -that, by operational dar-winian definition, have proved their functionality and evolutionary fitness by surviving to reproduce -with variant genes (polymorphisms) of uncertain functionality, fitness or overall compatibility within an organism, is an evolutionary strategy that will only be sustained on a geological timescale if new polymorphisms confer survival benefits to organisms that exceeds the risks and metabolic costs of generating and sustaining those polymorphisms. for primitive cells, lacking functional humoral, cellular or cytokine defense mechanisms, development of cell-surface protein polymorphisms is an obvious adaptive strategy to thwart invasion by primitive viruses. like other adaptive strategies, cell-surface polymorphisms are strongly selected for, and have been highly conserved over deep time, and are found in all organisms from primitive prokaryotic cells [ ] and thermophilic bacteria [ ] through to plants [ ] as well as mammalian cells, strongly suggesting a critical evolutionary function. the lock and key hypothesis, for which there is very considerable evidence [ ] [ ] [ ] [ ] , first proposed by jbs haldane [ ] , contends polymorphisms arise, and are maintained, as protection against cellular parasitism, particularly by viruses . while dna-encoded protein polymorphisms form necessary defenses against viral access, they may not be sufficient; a quasispecies of cells (e.g. the liver) expressing similar and static receptor variations renders those cells vulnerable to sustained attack from any virus that successfully invades any one cell, and further dynamic modification of cell receptors, triggered by viral infection and mediated at the transcriptional level by modulation of dna dependent rna polymerase fidelity in nearby uninfected cells, by a mechanism similar to replicative homeostasis would seem possible. a clear, unambiguous band at the "c" position on a sequencing gel, causes "cytosine" to be assigned to that genetic locus. but does this certitude reflect reality, at least for viral rna quasispecies? direct pcr sequencing is an "averaging" procedure revealing the most frequent nucleotide at any particular locus. however, nucleic acids and proteins cannot express 'an average', and discrete quanta of specific nucleotides or amino acids are present at every locus. a typical clinical serum sample, containing × geq/ml hcv and mutating at - substitutions/base, will contain examples of each possible nucleotide at every locus, but most variations will remain undetected during sequencing or any other method of quasispecies analysis. analysis of cloned dna gives cleaner data than pcr sequencing but if clones (and multiple hcv quasispecies clones are highly unlikely to be identical) provides definitive sequence, would we process the st to reveal different and, potentially, critical sequence variations? and if we did, how would we recognise its importance? is important sequence likely to be present at frequencies of http://www.virologyj.com/content/ / / < %? infectious virions containing, presumably, fulllength functional genome and corresponding wild-type proteins, are often outnumbered by ~ × : in serum by defective and non-infectious particles [ ] that presumably do not, suggesting that important genetic sequence and associated phenotype may occasionally be extremely rare. how the immune system recognizes uncommon, nondescript, but important protein sequences in a featureless background of similar molecules is a non-trivial problem for which replicative homeostasis may suggest a solution. replicative homeostasis, described in detail elsewhere [ , ] , is an epicyclic mechanism of viral autoregulation that results when viral proteins, notably envelope (env), influence rna pol fidelity and processivity. the predicted consequences of replicative homeostasis for rates of intracellular viral replication and mutation, cellular expression of viral proteins and immunological responses occurring because of replicative homeostasis is represented schematically (figures , ). during early viral replication in a naive cell devoid of inhibitory molecules (panel a, a), high affinity wild-type envelope:polymerase interactions predominate, causing rapid low-fidelity polymerase activity resulting in rapid synthesis of variant viral rnas and subsequently proteins, hence causing a broad spectrum of viral proteins to be expressed on the cell surface, each at concentrations below the threshold of immune detection (tod). rna pol infidelity ensures synthesis of variant viral rnas and proteins predominates early, hence variant protein molecules progressively accumulate within cells relative to wild-type viral molecules (panels b-d) and increasing the probability of variant viral envelope:rna pol interactions. variant viral envelope:rna pol interactions causing progressive inhibition of rna polymerase processivity and increasing rna pol fidelity, reducing diversity of viral rnas synthesized and progressively restricting dynamic progression of rna pol functional properties, processivity () and fidelity () predicted by replicative homeostasis figure dynamic progression of rna pol functional properties, processivity ( ) and fidelity ( ) predicted by replicative homeostasis. initial state (a, corresponding to panel a, figure ): in a newly infected cell, high-affinity wild-type:rna pol interactions will predominate resulting in high rna pol processivity but low fidelity causing high-level viraemia with broad virus phenotypic spectrum, maximizing cell tropism. intracellular accumulation of variant viral proteins (b, c.f. panel b, figure ) reduces rna pol processivity but increases fidelity reducing viral rna synthesis and consequently, viraemia before a dynamic, fluctuating equilibrium (c, c.f. panel c or d, figure ) develops in which inhibition of rna pol by variant viral proteins is balanced by increases in rna pol fidelity (with consequent synthesis of wild-type viral products tending to cause high rna pol processivity). conceptual progression of intracellular viral replication events, including variable rna pol fidelity and processivity, restriction of antigenic diversity and immune recognition under influence of replicative homeostasis env viral protein diversity expressed on the cell surface (panels b to d), increasing cell-surface concentrations of individual viral proteins above the threshold of detection (panels c, c) at which point a polyclonal immune response develops. development of low-affinity polyclonal blocking antibodies, restricting cellular egress of viral proteins, further increasing intracellular concentrations of variant envelope proteins, still further increasing the probability of variant viral envelope:rna pol interactions and inexorably further restricting antigenic diversity increasing relative expression of wild-type proteins thus further exposing these epitopes to immune surveillance and facilitating specific high-affinity immune responses, including cytotoxic t cell responses, (d,d) to wild-type proteins. thus, the immune responses can strategically utilize replicative homeostasis to force viruses to reveal important and dominant wild-type epitopes, but those responses develop initially as a consequence of restriction of rna pol fidelity that occur because of replicative homeostasis. high-affinity responses further deplete intracellular concentrations of wild-type proteins, progressively reducing wild-type envelope:rna pol interactions, greatly reducing rna pol processivity to the point of viral latency (e,e), caused by variant viral envelope:rna pol interactions. the hepatitis c "early replication" paradox now resolves completely when considered in the context of replicative homeostasis; initial high level hcv replication (due to high rna pol processivity) remains immunologically undetectable for - weeks, or more, because of low rna pol fidelity causing a broad spectrum of hcv envelope proteins each expressed on cell surfaces at concentrations below the threshold of detection even while viraemia, reflected in concentrations of 'utr rna common to each rna species, are present at - geq/ml. as replication progresses, intracellular accumulation of variant viral proteins increase rna pol fidelity but decrease processivity (replicative homeostasis), downregulating hcv replication and reducing viraemia but restricting antigenic diversity and increasing expression of hcv envelope proteins to beyond the threshold of immune detection. furthermore, the temporal tissue injury (aminotransferase) paradox also resolves in this light: focussed immune recognition (including cytotoxic t cell responses) doesn't develop until after viral antigenic diversity is restricted by replictive homeostasis the transaminase peak would not be expected until after viral replication falls due to autoinhibition of rna pol processivity. varying expression of viral proteins by modulating rna pol fidelity to facilitate immune escape would seem a useful evolutionary adaptation that might be retained by more complex organisms, including cellular pathogens like tuberculosis and malaria, to optimize their stability within hosts. this mechanism of immune avoidance might also explain maternal-foetal tolerance. the human foetus maintains a stable parasitic existence during gestation (and, i expect, to university age and beyond) that is tolerated despite normal maternal immune responsiveness in general and lack of specific tolerance to paternal antigens in particular, a situation made more problematic as expressed foetal antigens are predominantly of paternal origin [ ] . while immunological isolation of foetal tissue by the placental trophoblastic layer [ ] , and placental display of hla-g [ ] , probably contribute to foetal stability in the face of a potentially robust immune attack, neither mechanism would explain persistence of viable foetal nucleated red blood cells within the maternal circulation [ ] in quantities sufficient to permit clinical prenatal diagnosis [ ] . is it possible foetal tolerance is mediated by regulating the fidelity of foetal dna dependent rna transcriptases to ensure any cell-surface antigens are expressed heterogeneously and at levels below the threshold of maternal immune responsiveness? for many classical autoimmune disorders, including primary biliary cirrhosis [ ] , multiple sclerosis, and rheumatoid arthritis, convincing epidemiological evidence [ ] , including cases clustering [ , ] , strongly suggests these diseases are triggered by infectious agents in genetically predisposed individuals. in others, such as diabetes mellitus, tantalizing epidemiological [ ] , clinical [ ] and laboratory [ ] evidence has implicated enteroviruses, but has suggested viral-triggered autoimmune processes, rather than cytolytic destruction of pancreatic betacells [ ] . similar circumstantial evidence exists for myocarditis, demyelinating diseases, myositis and other post infectious inflammatory disorders. when macfarlane burnet wrote autoimmunity arises from "inability to distinguish self from non-self" hbv, hcv, hiv and other viruses, now established to cause diseases with clear autoimmune features were unknown. viral infections, particularly hepatitis c -and its treatment with interferon -are associated with many varied autoimmune phenomena [ ] , and thyroid disease [ ] [ ] [ ] , diabetes mellitus [ , ] , membranous, membranoproliferative and cryoglobulinemic glomerulonephritis, vasculitis and peripheral neuropathy [ ] , and autoimmune gastritis [ ] are all very well documented, although the mechanism(s) are unknown and causality is certain. classical serological markers of autoimmunity, including rheumatoid factor, antinuclear antibodies (ana), anticardiolipin, antithyroid, anti-liver/kidney/microsomal antibodies (anti-lkm), as well as hcv/anti-hcv immune complex formation and mixed essential cryoglobulinemia are common accompaniments of chronic hcv infection [ ] , raising the obvious question of whether all "autoimmunity" has a viral basis. indeed, zinkernagel's pragmatic and subtly anticipatory; "if we know the infection, we call the disease immunopathologically mediated; if we do not recognize or know it, we call the disease autoimmune [ ] " fully reflects recent explosive growth of information and the deeper questions this information poses. rna virus quasispecies biology, specifically the generation of rna quasispecies by rna pol , and translation of these immensely variable rnas into protein quasispecies, suggests an immediate solution to the problem of viral autoimmunity and, by extension, to autoimmunity in general, as well as suggesting a unifying hypothesis to explain other diseases known to have multi-factorial aetiologies that include inflammatory components -such as coronary artery disease -in addition to other diseasesincluding schizophrenia and some forms of depressionthat currently lack rational and coherent pathogenic explanations. viruses are known to co-opt cell surface molecules, including lectins, hormone receptors and cell signaling molecules, to access cells. receptors, and other cell surface molecules, identified as "viral receptors"or to specifically interact with viral proteins include prostaglandins, catecholamines and acetylcholine receptors [ ] , serotonergic neurotransmitters ( ht) [ ] , endothelial cell glycoproteins [ ] , insulin-like growth factor (igf-ir) and its major signaling molecules insulin receptor substrates irs- [ ] and irs- [ ] , epidermal growth factor (egf) [ ] , neurotrophin receptor [ ] , thyroid hormone receptor tralpha [ ] , an immunoglobulin protein superfamily [ ] , low density lipoprotein (ldl) receptors [ , ] , transferrin receptor (tfr) [ ] , asialoglycoprotein receptor (asgp-r) [ , ] , and angiotensin-converting enzyme [ ] , to cite biologically diverse examples. of necessity, some receptor affinity studies have used cloned viral protein ligands, an artificial situation that cannot approach the phenotypic complexity of rna viral protein quasispecies. nonetheless, variable virus receptor affinities [ , ] , evolutionary adaptation of receptor affinity [ ] , emergence of escape variants with altered receptor affinities [ ] , temporal alteration of receptor usage [ ] and capacity to exploit alternative entry pathways [ ] have all been confirmed, suggesting viruses are capable of generating highly plastic ligands with very broad receptor affinities. if a virus co-opts a receptor for cell entry, then wild-type envelope (consensus sequence) epitopes, coded for by wild-type rna sequences, will probably form the common viral ligand. however, any viral rna quasispecies also contain a vast spectrum of rnas derived from, and similar to, envelope open reading frame (orf) consensus sequence, but variant from it. as the envelope orf quasis-pecies sequences progressively diverge from wild-type, the quasispecies of envelope proteins translated from these variant orfs will also, and inexorably, diverge in sequence, structure and biological function from wildtype envelope sequence proteins. some of these envelope proteins will be functionally identical, but others, and probably the vast majority, will range from subtly different to grossly abnormal, either due to major differences of sequence and/or chemical or steric amino acid incompatibility, or because of premature introduction of stop codons. even minor amino acid differences, as sickle cell anaemia illustrates, and has been confirmed specifically for viral receptor usage [ , ] , may catastrophically alter a proteins' function with respect to co-opted viral receptors, with some having no binding affinity, while others will bind strongly and act as agonists, antagonists or competitive inhibitors of normal receptor function. variant and defective viruses, and their polypeptides, will be in vast molar excess compared to wild-type [ ] but will exhibit similarly high antigenic variability, permitting escape from immune and other scavenger mechanisms. as many variant viral polypeptides will bind tightly to "self" receptors, but contain immunogenic non-self motifs, a polymorphic (because variant viral proteins will themselves be highly polymorphic due to the quasispecies process) immune response, apparently directed against "self" antigens, but actually targeting virus protein-receptor complexes virtually indistinguishable from normal cell receptors, will result causing apparent 'autoimmune' tissue damage. this mechanism suggests an explanation for common autoimmune phenomena. if a virus enters cells because wild-type envelope motifs interact with insulin, insulin receptor substrate [ , ] , tsh or related molecules [ ] , or acetylcholine [ ] receptors, many variant envelope polypeptides, generated by envelope orf quasispecies rnas, would have similar receptor binding affinity, but may effectively disrupt receptor function, predictably causing impaired glucose tolerance or diabetes mellitus, thyroid dysfunction, or myasthenia gravis with secondary resistance to, and elevation of, the normal hormone ligand (insulin, tsh etc.). the expected consequences disruption of receptor function by variant viral proteins might explain many common biochemical pathologies; for example, what effect would chronic blockade of parathyroid (pth) receptors by viral proteins have on pth levels, the parathyroid glands, or bone? leptin is a kda protein hormone secreted by adipocytes and carried across the blood-brain barrier by a rate-limiting transporter to act on hypothalamic receptors [ ] where, among other functions, it regulates thyrotropinreleasing hormone (trh) genes and upregulates alphamelanocyte-stimulating hormone and other anorexigenic neuropeptides [ ] important to appetite-regulation and energy balance [ ] . leptin also regulates a broad spectrum of other processes and behaviours including thermogenesis, blood pressure and immune function. s=serum leptin concentrations and leptin resistance, are independent markers of obesity, weight gain, systemic hypertension [ ] , diabetes mellitus [ ] , obstructive sleep apnoea [ ] and myocardial infarction [ ] , while polymorphisms of the leptin gene are associated with insulin resistance [ ] and long-term risk of developing diabetes mellitus [ ] . predictably, variant envelope proteins generated by envelope orf rna quasispecies from viruses utilizing leptin receptors for cell access would have similar receptor affinity, but exhibit non-physiological leptin antagonist or agonist properties, thus disrupting leptin receptor function, altering energy regulation, and causing either excess caloric intake unrestrained by satiety responses, or inappropriate satiety signals with pathologically reduced caloric intake. as clear evidence exists for viral disruption of leptin function [ ] and virus-associated weight gain in humans [ ] and monkeys [ ] , is it possible the global epidemics of type ii diabetes mellitus, insulin resistance, hyperlipidaemia and obesity now prevalent [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , are just that; epidemics fundamentally caused by viruses that co-opt insulin or leptin or other associated receptors for cell access and generate protein quasispecies that disrupt receptor function? could it also be that ethnically based epidemics of obesity, diabetes mellitus, hypertension and reno-vascular disease (the 'metabolic syndrome'), as seen in pima indians, nauruans and australian aborigines [ ] have developed not primarily because of exposure to "western" foods and lifestyles -that, after all, are all-pervasive without necessarily having so dramatic an effect on other groups -but because of chronic or recurrent exposure to viruses, or genotypes of viruses to which their particular repertoire of receptor polymorphisms confer no protection? or that anorexia nervosa develops, in some patients, when variant viral proteins with aberrant leptin-agonist function arise during the course of viral infection, as the temporal relationship between infection and disease onset, very clearly documented in one study [ ] , suggests. cardiovascular disease, the leading cause of premature death and disability in most western countries, has a wellestablished multi-factorial basis involving a complex interplay between genetic predisposition, environmental and personal risk factors -including systemic hypertension, diabetes mellitus, hyperlipidaemia, obesity and cigarette smoking -and more recently recognized mechanisms, including endothelial dysfunction [ ] , vascular inflammation [ ] and leptin levels [ ] . systemic hypertension, diabetes mellitus and hyperlipidaemia have long-established, but complex, patterns of inheritance, a situation further compounded by evidence receptor polymorphisms -including those of angiotensin ii type receptor [ ] , irs- gene [ ] and low density lipoprotein receptor (ldlr) [ ] -both confer disease susceptibility and have regionally variable prevalences [ , ] . the flaviviradae -including hcv -as a family, and the rous sarcoma virus, utilize low density lipoprotein receptors to enter cells [ , ] , while angiotensin ii [ ] , insulin receptor substrates (irs and irs ) [ ] , and endothelial cell glycoproteins [ ] and other receptors widely distributed in vascular tissues are known to be permissive for virus cell entry establishing, in principle and in fact, viral-protein receptor affinity relevant to cardiovascular diseases. viruses accessing cells through these receptors will generate a quasispecies of variant proteins capable of disrupting receptor function potentially causing hyperlipidaemia, hypertension, hyperglycaemia and endothelial dysfunction, as well as immune-mediated endothelial cell damage, thus establishing the necessary and sufficient conditions and a chain of events that potentially link viruses and vascular diseases, including myocardial infarction. this hypothesis exists at the confluence of established risk factors for coronary artery disease, including genetic susceptibility, polymorphisms predisposing to hypertension [ ] [ ] [ ] , diabetes [ ] and hypercholesterolaemia and substantial new data implicating vascular inflammation [ , ] , endothelial dysfunction [ , ] , leptin dysregulation [ ] and viral infection [ , ] in the pathogenesis of vascular disease. furthermore, this final common pathway can account for that small, but significant, group of patients with vascular diseases but no clinically identifiable risk factors, as well as the non-random co-incidence of depression and coronary artery disease [ ] (as discussed below) in addition to the anti-inflammatory action of hmg-coa reductase inhibitors (statins) [ ] , and their effect in lowering cardiovascular mortality independent of cholesterol reduction [ ] ; if statins compete with variant viral proteins for hmg-coa reductase receptor binding, and displace immunologically attractive molecules, inflammatory responses directed at viral product, but involving endothelial cell receptors, will be ameliorated (figure ). human immunodeficiency virus hiv-associated dementia (hivd) occurs in % of hiv-infected adult patients, and as a major cause of dementia in the young represents "proof of principle" of virus-caused dementia, raising the possibility other forms of virus related dementia exist. although highly active antiretroviral therapy (haart) has reduced the incidence of hiv-d by - % [ ] , it remains a major cause of morbidity and the pathogenesis poorly understood. direct cytopathic effects of hiv or other viruses are unlikely, while active replication of virus, high-level viral protein expression [ ] , and increased viral envelope sequence-diversity in blood and brain [ ] are all important, clearly indicating viral proteins are pathogenically important. the clinical features of hivd, including psychomotor slowing, apathy, and altered gait and posture, strongly suggest a subcortical dementia with involvement of the basal ganglia and striatal dopamine receptor pathways. schizophrenia, depression and bipolar affective disorder, and anorexia nervosa are highly prevalent, chronic conditions of unknown aetiology that cause enormous morbidity and generate significant health care costs. each of these disorders have well documented, albeit regionally variable, associations with receptor -including dopamine -polymorphisms [ , [ ] [ ] [ ] [ ] [ ] , as well as epidemiological evidence that viral infections are aetiologically important, either directly or as precipitating events [ , [ ] [ ] [ ] [ ] , although other sero-epidemiological studies [ ] and work directly seeking viral nucleic acids in patients with schizophrenia have proved negative [ ] . if a virus, or viruses, use dopamine, acetylcholine [ ] , neurotrophin [ ] , serotonergic ( -ht) [ ] , or other neuro-transmitter receptors to access cells (and, given rna virus quasispecies biology, it would be surprising if some didn't), then the rna quasispecies will generate a quasispecies of variant polypeptides potentially reactive to these receptors. while it is difficult to imagine what effect perfusing a functional human brain with a solution of antigenic, inflammatory polypeptides that bind to, and are variably disruptive of, critical neurotransmitter receptor function, might have on cognition, perception, behaviour, attention span, abstract thought, fine motor or emotional control, it is unlikely to be beneficial. in this context, the welldocumented cognitive abnormalities -unrelated to depression -found in patients with early hcv and hiv infection [ ] [ ] [ ] are unsurprising. virus receptor disease (vrd) is quite distinct from either immune complex deposition disease due to deposition of macromolecules in tight vascular arcades, or from disease related to altered cell tropisms and is also completely independent of the primary site of viral replication; both non-inflammatory receptor blockade and immune-mediated inflammation directed at viral protein-receptor complexes could cause pathology of tissues non-permissive for and remote from the primary site(s) of viral replication with "autoimmune" damage to the liver, pancreas, brain, skin or lungs arising, for example, from chronic small intestinal virus infection. viral quasispecies biology predicts vrd will have other characteristics. first, due to replicative homeostasis, the ratio of wild type to variant viral proteins of the quasispecies will both fluctuate with time and will alter dramatically after initial infection; if wildtype proteins are dominantly agonist in function with respect to their receptor, variant proteins, most likely, will predominantly exhibit antagonist function (and vice versa). furthermore, the net effect of viral proteins (because of viral autoregulation) will fluctuate initially between receptor agonist and antagonist function, before becoming predominantly antagonistic, thus providing a possible explanation for transient thyrotoxicosis during early thyroiditis (before hypothyroidism supervenes), for hypoglycaemia seen during early insulin-receptor antibody-mediated insulin resistance [ ] , and for the contradictory functions ascribed to hiv nef [ ] . a corollary of fluctuating phenotypic dominance of viral protein quasispecies is that receptor affinity of these proteins will also fluctuate, and any resulting inflammation may vary in both intensity and anatomical distribution over time. second, because viruses utilize alternate receptors for cell access, apparently homogeneous disease processes could result from multiple different viruses. similarly, because cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship ( ; unbound, ; activated), receptor permis-sive for virus cell entry ( ) or blocked by polymorphism (rp, ) figure cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship ( ; unbound, ; activated), receptor permissive for virus cell entry ( ) or blocked by polymorphism (rp, ). receptor blockade by variant viral envelope proteins (green e, ), blockade by antigenic envelope proteins stimulating "autoimmune"response apparently directed against self receptors (e, ), competitive displacement of antigenic proteins by drug (d, e.g. statin, aspirin) abrogating immune response ( ). virus quasispecies produce a broad spectrum of protein phenotypes, and the receptor polymorphisms permissive for cell entry for specific viruses will be variably distributed in host populations, pathology of widely variable tissues in different individuals could result from the same virus. third, as evolutionary co-adaptation results in progressive genetic co-divergence of interacting species, the receptor polymorphisms predisposing to (or protecting against) infection by any particular virus, and resulting vrd, and the common viruses causing them, would be predicted to vary geographically, an expectation multiply confirmed for disease associated polymorphisms. as a corollary this suggests individuals migrating from regions where hosts and virus strains are stably co-adaptated to other areas, where different viruses are prevalent, might experience increased rates of vrd -beaks optimally adapted for finch survival on the galapagos may be a liability elsewhere -a prediction again amply confirmed [ ] [ ] [ ] ;. finally, if immune mechanisms are unable to clear rna viruses like hcv and do not cause the reduced viral replication seen during acute infection, are they any more likely to be effective against other rna viruses? is it possible that self-limiting infections like influenza and sars also autoregulate their replication, and, like hcv or hbv, become partially dormant, yet remain transcriptionally active, in the face of an active and powerful immune response? pcr amplification of influenza rna from convalescent samples makes this readily testable, while the documented relationship of influenza to myocardial infarction [ ] and juvenile rheumatoid arthritis [ ] makes the question important. if confirmed, the well-documented seasonality of some depressive illnesses [ ] and schizophrenia, [ ] and increased rates of schizophrenia during influenza epidemics [ ] , and the increased incidence of both depression [ ] and schizophrenia [ , ] following in-utero exposure to influenza may be more rationally explained. if 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coleman, and sons matt and tim, for everything important, my parents dick and janet for extraordinary opportunity, and some great physician-teachers -that most noble vocation -of the university of western australia medical school; professors mike mccall, dick joske, bill reed, bill musk, peter pullan, michael quinlan, dick lefroy and ted haywood. special thanks to karl ruckriegel for turning back-of-envelope sketches into first-class graphics. any remaining lack of clarity is my fault. key: cord- -rwmuucsp authors: dicker, kate; järvelin, aino i.; garcia-moreno, manuel; castello, alfredo title: the importance of virion-incorporated cellular rna-binding proteins in viral particle assembly and infectivity date: - - journal: semin cell dev biol doi: . /j.semcdb. . . sha: doc_id: cord_uid: rwmuucsp rna is a central molecule in rna virus biology due to its dual function as messenger and genome. however, the small number of proteins encoded by viral genomes is insufficient to enable virus infection. hence, viruses hijack cellular rna-binding proteins (rbps) to aid replication and spread. in this review we discuss the ‘knowns’ and ‘unknowns’ regarding the contribution of host rbps to the formation of viral particles and the initial steps of infection in the newly infected cell. through comparison of the virion proteomes of ten different human rna viruses, we confirm that a pool of cellular rbps are typically incorporated into viral particles. we describe here illustrative examples supporting the important functions of these rbps in viral particle formation and infectivity and we propose that the role of host rbps in these steps can be broader than previously anticipated. understanding how cellular rbps regulate virus infection can lead to the discovery of novel therapeutic targets against viruses. viruses are obligate intracellular pathogens that represent a threat to human health and the economy of countries. viral genomes are typically small, encoding only a few proteins. to circumvent this limitation, viruses hijack host resources to complete their infection cycle. rna is a central molecule in rna virus biology since it functions not only as a messenger for protein synthesis (i.e. mrna), but also as a genome. since viruses cannot encode all the proteins necessary for viral (v)rna replication/transcription, translation and packaging, viruses usurp cellular rna-binding proteins (rbps) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . on the other hand, cellular rbps are also central to the antiviral defences [ , ] . the host cell senses viruses through specialised rbps that recognise unusual signatures present in vrnas, known as pathogen-associated molecular patterns (pamps), leading to the stimulation of interferon production and the activation of mechanisms to inhibit protein synthesis [ ] . cellular rbps are thus fundamental components of the virus-host cell battlefield, either sustaining or restricting virus infection. understanding how viruses interact with these cellular proteins can thus be instrumental for the development of novel antiviral therapies. rbps interact with rna forming dynamic complexes known as ribonucleoproteins (rnps), which are critical for mediating gene expression [ ] . recently, the repertoire of human cellular rbps was increased dramatically to over proteins by the development of a proteome-wide approach known as rna interactome capture (ric) [ ] [ ] [ ] . ric employs ultraviolet (uv) crosslinking of 'zero' distance (< Å) protein-rna interactions, followed by cell lysis under denaturing conditions, isolation of rbps crosslinked to poly(a) rna via oligo(dt) capture and quantitative mass spectrometry. additionally, new methods based on organic-aqueous phase separation to detect also proteins bound to non-poly(a) rnas have contributed to greatly extend the number of rbps in the cell [ ] [ ] [ ] . while a substantial proportion of these proteins interact with rna using a defined set of well-characterised rna-binding domains (rbds), such as rna-recognition motifs (rrms), many rbps lack them, suggesting the existence of unconventional modes of rna binding [ ] [ ] [ ] . indeed, dozens of novel rbds were later discovered using proteomic-based methods. these domains included enzymatic cores, protein-protein interaction surfaces and dna-binding domains [ , ] , as well as intrinsically disordered regions that emerged as a prominent mode of rna binding [ , ] . importantly, both classical and unconventional rbps have been linked to virus infection and immunity, as reviewed before [ ] . moreover, analysis by comparative ric of the 'rna-binding proteome' of cells infected with the rna virus sindbis (sinv), revealed that the complement of cellular rbps is pervasively remodelled upon infection [ ] . strikingly, many rbps activated by sinv either sustain or repress infection, highlighting the critical roles of cellular rbps as regulators of virus infection. because sinv rna is polyadenylated, ric isolates both host and viral rna. indeed, many of the proteins enhanced by sinv infection were shown by orthogonal approaches to accumulate within the viral replication factories, suggesting a direct interplay with vrna. however, ric will also discover rbps with differential rna-binding activity that interact with cellular rna instead of vrna. several approaches have been developed to elucidate the composition of viral ribonucleoproteins (vrnps). initially, studies on influenza a virus (iav) and vesicular stomatitis virus (vsv) focused on isolating viral rna polymerase complexes, as these should reflect the composition of replicating vrnps to some extent [ ] [ ] [ ] . while useful, these datasets were biased towards direct protein-protein interactions. more recently, different groups have employed diverse approaches to capture vrna together with its interacting proteins, which are then detected by mass spectrometry. all these methods start by 'freezing' protein-vrna complexes. this can be achieved by uv protein-rna crosslinking exploiting the excitability of natural bases upon irradiation with uv light at nm [ ] . alternatively, photoactivatable nucleotides, such as thiouridine ( su), can be incorporated into nascent vrna, and protein-rna crosslinking is achieved by irradiation with nm uv light [ , ] . uv crosslinking only promotes covalent bonds between proteins and rnas placed at 'zero' distances, thus displaying high specificity with the cost of low efficiency. another approach typically used to immobilise protein-rna interactions is formaldehyde crosslinking [ , , ] . while more efficient than uv, formaldehyde also induces covalent bonds between proteins, which promotes the isolation of indirect binders through protein-protein bridges. once protein-rna complexes are 'frozen', the second step is to isolate the vrna together with its covalently bound proteins. typically, vrna is captured with antisense dna probe(s). this approach substantially enriches for vrna [ , , ] , although can still co-purify rnas in a non-specific fashion through the formation of partial hybrids with non-target sequences. if vrna is labelled with su (see above), it can alternatively be isolated by biotinylation of the sulfhydryl group in the su base, coupled with streptavidin pull down [ ] . since exposed cysteines on protein surfaces can also be biotinylated, it is not surprising that this approach identifies a large proportion of cellular proteins lacking rna-binding activity. collectively, these methods have been applied to several virus models including dengue virus (denv) [ , , ] , zika virus (zikv) [ ] , sinv [ ] , poliovirus [ ] and human immunodeficiency virus (hiv- ) [ ] , representing important advances towards understanding vrnp composition. the interactomes between host proteins and incoming viral particles have been recently unravelled using a novel technique named vir-clasp [ ] . vir-clasp uses su to label the genomic rna in infected cells, which is later incorporated into viral particles. viruses released to the supernatant harbouring su-labelled genomes are then used in very high multiplicity to infect new cells. infection is followed by crosslinking with uv light at nm, solid-phase capture of protein-rna complexes and protein identification by mass spectrometry. vir-clasp has been successfully applied to chikungunya virus or iav and have revealed important regulators of the initial steps of the infection cycles of these viruses [ ] . despite their strengths and limitations, all these studies agree that both classical and unconventional rbps engage with vrna and play critical roles in infection. in general, infection cycle of rna viruses consist of three main phases ( fig. ) : ( ) attachment of the viral particle to the cell and entry; ( ) viral genome replication and expression; and ( ) assembly and egress of the virus progeny [ ] . the viral cycle begins when a virus binds to specific surface receptors and enters a host cell. once inside the cell, the genomic rna of positive-stranded viruses engages directly with the host protein synthesis machinery to produce the viral proteins required for replication. conversely, negative-stranded rna viruses typically carry the transcription machinery with them into the newly infected cell. retroviral rna must be reverse transcribed into dna, which is then imported into the nucleus and integrated into the host chromosome to be transcribed by the cellular rna polymerase ii. the next stage involves the synthesis of the components (i.e. viral structural proteins and genomes) required to assemble the progeny viral particles that leave the producer cell to infect a new one. virtually all these stages can be regulated by cellular rbps (fig. ) , although most work so far has focused on viral replication and protein synthesis [ ] [ ] [ ] [ ] [ ] [ ] . however, new discoveries have highlighted the importance of cellular rbps in the assembly of viral particles as well as in the very initial steps of infection, when the rna genome is delivered into a newly infected cell. these steps are the focus of the present review. one of the main open questions is how vrna is recognised specifically by viral proteins with limited, if not non-existent, specificity for vrna during the assembly of viral particles [ , ] , and whether highspecificity cellular rbps may cooperate with viral proteins to recognise the vrna. moreover, vrna is generally highly structured [ , ] . thus, it is plausible that cellular rbps with helicase and chaperone activities facilitate the binding of capsid/nucleocapsid proteins across the vrna to enable rna packaging into virions. immediately upon entry into the cell, vrna must either be translated (positive-stranded rna viruses), transcribed and replicated (negative-stranded rna viruses) or reverse transcribed (retroviruses) without alerting the host immune defences, especially in the initial steps of infection where the virus is more vulnerable. how viruses achieve this remains largely unknown; however, recent evidence supports the idea that in certain cases such as hiv- , some of the lifecycle of the vrna takes place inside the 'protective walls' of the capsid shell [ ] [ ] [ ] . another interesting idea is whether vrnas carry the necessary cellular components from the producer cell to maximise the efficiency of the subsequent initial steps of infection. different proteomic studies have identified hundreds of cellular factors within the particles of several rna viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , many of which are rbps. here, we discuss the 'knowns' and 'unknowns' of the roles that virion-incorporated cellular rbps could play in the assembly of viral particles and the early steps of infection in the new host cell. in the last two decades, the composition of the particles of several rna viruses has been elucidated by proteomics. however, no work has currently focused on identifying the scope of cellular rbps that are present in virions. to gain insights into this question, we compiled the proteomes of particles from ten different human rna viruses (fig. ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . upon building the superset of host proteins present in virions (table s a) , we cross-referenced it to the complement of experimentally determined human rbps [ ] as well as gene ontology (go) terms and protein domains related to rna binding. the resulting in virion rbps (ivrbps) are listed in table s b , including the dataset in which each protein was reported. we discover that virions contain many rbps (fig. s a ), % of which harbour known rbds, while the other % were just recently classified as rbps by ric studies and their modes of rna binding remain unknown [ ] . analysis of go biological process terms (fig. s b ) and proteinprotein interaction networks (fig. s ) indicate that ivrbps can potentially participate in a wide variety of processes, e.g. transport, exocytosis, response to stress, immune system pathways, cytoskeleton organization, translation, rna processing, rna stability or protein folding. however, the main limitation of the cited proteomic analyses is that budding of the viral particles results in the uptake of a portion of the cytoplasm and plasma membrane. hence, it is challenging to discriminate between abundant proteins randomly incorporated into virions and with no function in virus biology, from those with an active regulatory role. we reasoned that host proteins with a true function in infection are likely to be identified in multiple datasets and across different virus species and cell lines (fig. s ) . strikingly, we noticed that ivrbps are commonly detected in the particles of different viruses (at least viruses out of , table s b ). for example, cofilin (cfl ) was identified in of the viruses studied here. cfl is a regulator of actin cytoskeleton reorganisation and has been shown to play a crucial role in measles virus (mev) rnp formation for vrna synthesis [ ] , iav assembly and budding [ ] and the production of infectious respiratory syncytial virus (rsv) [ ] . however, whether the rna-binding activity of cfl plays a role in infection requires further investigation. moreover, the importance of cfl incorporation into particles should also be further tested functionally, since it could also be due to passive uptake with actin, which is often present in viral particles. many ivrbps such as annexins, heat shock family proteins (hsp), peptidylprolyl isomerase a (ppia -also cyclophilin a), eukaryotic translation elongation factors (eef), heterogeneous nuclear ribonucleoproteins (hnrnp) or poly(rc) binding protein (pcbp ), have been linked to infection in multiple ways (fig. s ) , and here we show that they are incorporated in the particles of several viruses (table s b) . whether these proteins are incorporated passively due to high abundance or through convergent, virus-driven active mechanisms such as specific interactions with viral proteins or rna, requires further investigation. however, ppia illustrates well why the second option should not be underestimated, as it interacts specifically with the capsid of hiv and this interaction is critical for capsid core stability [ , ] . whether ppia rna-binding activity plays a role on this recruitment remains unknown. many ribosomal proteins are also present in the superset of ivrbps, together with rna helicases that aid the unwinding of rna (fig. s ). interestingly, several ivrbps are restricted to the virions of a certain superclass of viruses. for example, heat shock protein alpha (hsp aa ), transgelin (tagln ) and hnrnpd are found exclusively in negative-stranded rna viruses. nevertheless, it is important to note that all these virion proteomes have been generated using different i) virion isolation methods, ii) proteomic approaches and iii) data analyses. these divergences are noticeable when comparing datasets generated in closely related viruses or even the same virus (fig. s ). for example, each of the six hiv- virion proteomes that we examined identified a distinct number of proteins ( fig. ), reflecting differences in virion isolation stringency, proteomic depth and/or statistical thresholds. the analysis of virion proteomes is complicated by potential contamination with cellular proteins, either within extracellular vesicles that copurify with viral particles or via nonspecific interactions with the virus exterior. most of the viruses used in our metanalysis were purified on sucrose cushions or density gradients, and some lack additional purification steps [ , , , , , ] . few of the latter studies showed that the vast majority of their samples were intact viral particles using transmission electron microscopy. interestingly, several studies incorporated further purification strategies: ( ) treatment of viral particles with proteases such as subtilisin [ , ] or proteinase k [ , ] to remove non-specific binders and vesicles due to an alteration in their density; ( ) cd immunoaffinity depletion as cd is present on microvesicles but not in hiv particles [ ] ; ( ) solubilisation of lipid bilayers for isolation of hiv cores by including a detergent layer on the sucrose density gradient [ ] ; ( ) haemadsorption to and elution from red blood cells to select for iav particles with receptor binding and cleavage activity, provided by haemagglutinin and neuraminidase [ ] ; ( ) sequential affinity purification with a heparin column to select for hcv particles containing the viral e protein, followed by antibody-or tag-mediated capture of envelopecontaining particles [ ] . by focusing on proteins identified in multiple datasets, we expect to minimise technical differences between datasets, as well as biological factors, such as the incidence of randomly incorporated proteins (see above). however, passive incorporation due to abundance cannot be excluded a priori, and we recommend interested readers to test the abundance of their candidates in proteomic analyses of whole cell lysates, if available. table s highlights many of these robustly identified ivrbps and we discuss below several ivrbps with well-known roles in infection, and others that remain poorly characterised. k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx . do cellular rbps participate in viral particle assembly? the cytoplasm of the cell is a hostile environment for vrna due to the presence of antiviral sensors. these cytoplasmic immune surveillance factors are mostly specialised rbps that detect viral doublestranded (ds)rna, under-methylated cap structures or triphosphate ′ ends, which are common signatures present in vrna [ ] [ ] [ ] [ ] . the recognition of vrna as 'foreign' nucleic acid can trigger the antiviral response and direct the rna decay machinery towards the vrna [ , ] . hence, it is crucial for vrnas to effectively engage with cellular rbps to increase their stability and translatability [ ] . upon accumulation of viral rna and proteins, these components must travel to the virus assembly areas and, in some instances, vrna trafficking has been proposed to be mediated by the cytoskeleton [ ] [ ] [ ] [ ] [ ] [ ] . the role of protein chaperones in the trafficking of vrnps is becoming increasingly evident. many cellular chaperones and peptidylprolyl cis/trans isomerases (immunophilins) are hijacked by viruses to enable viral replication and translation by folding viral proteins [ , ] . interestingly, new proteome-wide approaches have revealed that both chaperones and immunophilins interact with rna in vivo and exert critical functions in rna metabolism, including regulatory roles within the spliceosome, control of transcription and mrna stability and ribonucleoprotein remodeling [ ] [ ] [ ] . moreover, their rna-binding surfaces have recently been revealed, suggesting that their ability to act on proteins and bind to rna is interconnected [ ] . ric analysis of sinv-infected cells revealed that the rna-binding activity of several protein chaperones and immunophilins is enhanced upon infection and that their functional perturbation severely impairs viral fitness [ ] . the importance of host chaperones in infection is well illustrated by hiv- rnps. viral genomic rna interacts with staufen double-stranded rnabinding protein (stau ) in the cytoplasm forming hiv- -dependent stau -rnps [ ] [ ] [ ] . compositional characterisation of these rnp complexes revealed the presence of the chaperones hspd (hsp ), hspa (hsp ) and hspa (hsc ) ]. it has been proposed that the stress-inducible chaperone hsp and its constitutive form, hsc , interact with nascent hiv- gag-vrna complexes and hold them in an assembly-competent conformation during their transport towards the plasma membrane [ ] . strikingly, these proteins are also present within hiv- virions (fig. s c) , suggesting that they associate with vrna at different stages of the infection cycle ]. the participation of hsc in viral particle assembly is also illustrated by hepatitis c virus (hcv) infection. hsc interacts with hcv rna and colocalises with the hcv capsid and e proteins at assembly sites [ ] . knockdown of hsc [ ] or abrogation of protein activity with allosteric inhibitors [ ] decreases viral particle production, but has no effect on viral replication. whether direct binding of hsc to the hcv rna genome contributes to the formation of infective virions is still unclear. the roles of cellular rna-binding chaperones, therefore, may span replication, translation, trafficking and virion assembly (fig. s ) . cellular rbps are also thought to contribute to capture the vrna, amongst all the cellular rnas in the cytoplasm, to be packaged into the virion. several viruses, such as the bacteriophage ms , encode capsid proteins with high specificity and affinity for stem loops present within the vrna [ ] . however, other viruses, such as hiv- , do not encode for any protein with high specificity and affinity for the vrna. hence, how these viruses select and package their vrna into virions against cellular counterparts remains poorly understood. for example, it was believed that the hiv- nucleocapsid subunit of the viral polyprotein gag recognises vrna from the pool of cellular rnas by a unique binding event at a cis-acting packaging element in the ′ leader of the vrna [ ] . however, a recent study using clip-seq (crosslinking and immunoprecipitation followed by sequencing) discovered that, in addition to the binding site in the ′ leader, gag interacts in the cytoplasm with other discrete sites across the hiv- rna [ ] , placed at the rev recognition element (rre) and ′ utr. however, when the vrna reaches the plasma membrane, this binding pattern changes dramatically, and gag covers virtually the whole vrna. how gag switches from selective to non-selective binding is not understood. since many cellular rbps interact with hiv- rna and gag [ , , ] , it is tempting to speculate that they might cooperate with hiv- nucleocapsid to recognise hiv- genome in the cytoplasm of the infected cell. in agreement, it has been proposed that stau assists gag interaction with hiv- rna [ ] . the resulting rnp is transported through the cytoplasm to the plasma membrane where stau facilitates the multimerization of gag on the vrna [ ] . this multimerization step enables the formation of immature virus particles, where the hiv- genomic rna acts as a nucleation site for assembly [ , ] . interestingly, stau only modulates gag assembly once the tail of gag has anchored into the plasma membrane suggesting that stau may play a role in ensuring the rnp is delivered to the membrane [ ] . stau has also been shown to promote the activity of hiv- rev protein in exporting vrna from the nucleus, along with dexh-box helicase (dhx ) and arfgap with fg repeats (agfg ) [ ] [ ] [ ] . together, these studies suggest that staufen proteins play crucial roles in the transport of hiv- rnps from the nucleus to the plasma membrane for viral particle formation. additional studies should be carried out to determine if other cellular rbps cooperate with viral capsids and nucleocapsids to recognise the vrna and to enable vrnp trafficking. the cellular endosomal sorting complex required for transport (escrt) is a large multi-component machinery comprised of five complexes, escrt- , escrt-i, escrt-ii, escrt-iii and vps , which assemble sequentially in a multi-step manner following ubiquitination of escrt- [ ] . the escrt machinery is hijacked for viral particle release across most types of viruses including retroviruses, filoviruses, arenaviruses, paramyxoviruses, rhabdoviruses, flaviviruses, reoviruses and picornaviruses [ ] . viruses can either hijack escrt for assembly and budding at the plasma membrane or for budding at endosomal membranes into the cytoplasm. escrt is typically involved in the budding of vesicles from the endosomal membrane into the endosomal compartment. however, escrt proteins can also perform a "reverse topology" membrane fission. escrt is the only cellular machinery with such ability and this is perhaps why so many viruses have evolved to exploit it [ ] . escrt-i/ii essentially function to recruit the escrt-iii protein chmp (charged multivesicular body protein ) to membranes where the formation of escrt-iii filaments is then promoted. the formation of escrt-iii filaments drives the budding of the membrane, but it is not understood exactly how this is achieved. programmed cell death interacting protein (pdcd ip or alix) recruits chmp and the escrt-iii complex to the plasma membrane. several studies have shown that many viral structural proteins initiate escrt driven budding from the membrane by recruiting alix. examples include gag of hiv- [ ] , the accessory c protein of human parainfluenza type [ ] , ebola virus vp [ ] , px of hepatitis a virus [ ] and the ns protein of denv [ ] and yellow fever virus [ ] . although alix has largely been studied from a proteo-centric perspective, ric studies have revealed that it is endowed with rnabinding activity [ ] . in addition, alix has recently been implicated in the recruitment of cellular rnas to extracellular vesicles [ ] . importantly, the interaction of hiv- gag with alix relies on the presence of vrna. the n-terminal bro domain and the central v-shaped domains of alix interact with hiv- nucleocapsid and p domains of gag respectively [ , ] . interestingly, the interaction between alix bro domain and hiv- nucleocapsid is disrupted by rnase treatment, suggesting that the vrna molecule forms a bridge between the two proteins. both alix bro and nucleocapsid are highly positively charged and are believed to establish electrostatic interactions with the phosphate backbone of the vrna. the recruitment of alix by vrna raises the question of whether alix is recruited to the hiv- rnps in the cytoplasm or in the plasma membrane as the virus assembles ]. additionally, proteomic-based compositional analysis of the zikv and denv rnps revealed that alix also interacts with these vrnas [ ] , although the exact role of these interactions remain unexplored. another class of unconventional rbps implicated in virus release is the annexin protein family. annexins are calcium-dependent membrane binding proteins that carry out a diverse range of functions. they can reversibly associate with components of the cytoskeleton or with regulatory proteins and rnas that mediate stress-induced intra-and inter-cellular signalling [ ] . annexin a (anxa ) is a multifunctional, ubiquitously expressed protein with roles in membrane domain organisation, membrane fusion, vesicle aggregation and exocytosis [ ] . it has been shown to play a role in cell attachment and entry or replication of enterovirus, rsv, hcv and iav, amongst other viruses [ ] . however, we still lack a molecular understanding of the relationship between anxa rna-binding activity and its regulatory roles in infection. anxa was shown to be involved in the formation of the hcv replication complex and can bind both hcv rna, in a sequence-specific manner, and non-structural protein ns b forming a ternary complex [ ] . the silencing of anxa has no effect on vrna levels suggesting that it does not influence replication. instead it significantly reduces the number of produced viral particles indicating that anxa plays a role in hcv virion formation or release, although the mechanism by which this is achieved remains unknown [ ] . anxa has also been shown to be involved in virion assembly of mev through recruitment of the viral matrix protein to the plasma membrane [ ] . anxa and other members of the annexin family have been identified within the viral particles of ebola and marburg virus [ ] , hiv- [ ] , iav [ ] , vsv [ ] , rift valley fever virus [ ] and rsv [ ] (table s b ). together, these data suggest that anxa proteins may be involved in the formation and release of particles of a broad range of viruses. however, the mechanisms of action of anxa proteins and their rna-binding activity in infection remains largely uncharacterised. the compositional analysis of different virions has also revealed the presence of some members of the adp ribosylation factor (arf) gtpase family, suggesting a role in viral particle formation and/or release [ ] . this is a ras-related subfamily fundamental for the regulation of vesicle formation, trafficking and docking at target membranes, and has been implicated in the infection cycles of many pathogens [ ] . arfs have been involved in the recruitment of hiv- rnps to the plasma membrane and the release of hiv- particles [ ] , and arf regulates hiv- trafficking to the virological synapse [ ] . additionally, arf is necessary for hcv rna replication and production of infectious particles [ ] . the possibility that the rna-binding activity of arf facilitates the localisation of vrnps at the sites of viral particle assembly and egress calls further investigation. after entry into the host cell, most positive-stranded rna viruses release the genomic mrna into the cytoplasm to be immediately translated into the viral replicase complex by the cellular translation machinery (fig. ) . this is the case for sinv, a representative member of the alphavirus genus [ ] . it was recently described that sinv infection of mammalian cells produce two subpopulations of infectious viral particles. one of them, known as 'heavy' viral particles, exhibits an enhanced translation of the vrna once inside the newly infected cell [ ] . only one homogeneous population of virions is released from the other natural host of sinv, mosquito, with an infectivity that matches that of 'heavy' viral particles. authors showed that virion-incorporated host-derived factors, including the ribosomal components rps , rps and s ribosomal rna, and the cellular rna binding motif protein (rbm ), are responsible for vrna superior translation. rbm has been shown to promote translation in different contexts [ ] and its incorporation into sinv particles was recently confirmed [ ] , together with other ribosomal proteins and many cellular rbps (table s c) . interestingly, while sinv particles with increased infectivity can be produced in either mammalian or mosquito cells, the enhanced vrna translation only occurs in a newly infected mammalian cell, but not in a mosquito cell [ ] . this striking phenomenon is recapitulated in animal models and in other alphaviruses [ ] . this suggests that rbps pre-loaded in the viral particle may interact specifically with mammalian proteins controlling translation initiation. the identity of these mammalian proteins remains unknown and uncovering them will be a challenge. however, recent work has shown that the mammalian ribosome establishes interactions with hundreds of cellular rbps, which represent potential regulators of translation [ ] . as many of these ribosome interactors are indeed found inside virions (table s ), we anticipate that they may accompany the vrna into the newly infected cell. once the vrna is released into the cytoplasm, these proteins may facilitate translation initiation by interacting with the ribosome. to what extent virion composition determines translation of incoming viral genomes awaits mechanistic characterisation. moreover, whether this phenomenon can be extended to other rna viruses beyond sinv remains to be explored. particles of negative-stranded rna viruses contain a viral rna-dependent rna polymerase and accessory proteins to transcribe the vrna into mrna upon entry into the host cell (fig. ) . later, this 'transcriptase' complex is modified to form a 'replicase' complex that synthesizes an intermediary positive-stranded rna that serves as a template for producing new copies of the negative vrna genome [ ] . thus, viral transcription and replication are two separate processes controlled by distinct vrnp complexes. a representative example is vsv that forms two complexes composed of the viral rna polymerase (l) and different viral and host proteins [ ] . the vsv transcriptase complex contains the viral proteins l and p and the host proteins rna guanylyltransferase and ′phosphatase (rngtt) capping enzyme, eef a and hsp [ , ] . the last two proteins are important for the rna polymerase activity of l and can be found within virions (table s b ). in addition, ppia is also bound to the vrnp complex inside purified vsv particles and is used for post-entry primary transcription [ ] . interestingly, the mentioned host rbps are absent in the vsv replicase complex, which contains n, l and p viral proteins and synthesizes the plus strand 'antigenome' vrna used as template for copying the viral genome [ ] . this suggests that differential association of the rna polymerase complex with host proteins may regulate the switching from transcription to replication. the cellular rbp hnrnpu interacts with the vsv leader rna that is required for vrna replication and inhibition of cellular transcription [ ] . this protein is also packaged into vsv particles, but the potential association of virion-incorporated hnrnpu with the transcriptase or replicase complexes and its activity in the early steps of vsv infection is not well understood. in iav, the viral polymerase consists of three subunits: pb , pb and pa. the cellular chaperone hsp is involved in the transport of pb and pb to the nucleus and modulates the interaction of pa with pb [ ] . after binding of pa to pb -pb , hsp dissociates from the complex. in this way, authors suggested that hsp regulates the assembly of the mature trimeric polymerase complex. on the contrary, hsp was found to be associated with the trimeric polymerase complex in a different study [ ] . hsp relocates to the nucleus after iav infection [ , ] , and this could reflect its interaction with newly synthesized polymerase subunits. interestingly, hsp has been found inside purified iav particles (as well as in other viruses, table s b), but k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx the possibility that virion-incorporated hsp participates in transport of the incoming vrnp to the nucleus and/or the initial viral transcription has not been explored so far. in vitro, hsp stimulates iav rna synthesis by interacting with pb ] , and binding of hsp to pb is increased during rna synthesis [ ] . authors suggested that hsp may participate in the early steps of transcription elongation by dissociating the polymerase from the vrnp and stabilizing the different subunits during their transfer between rna templates [ ] . however, further investigation in the context of iav-infected cells is required to elucidate the role of hsp and its recently described rna-binding activity [ ] in early iav transcription. it was believed that the capsid shell protecting hiv- rna was disassembled upon entry into the host cell, allowing viral rna and proteins to associate with host rbps in the cytoplasm of the newly infected cell to initiate reverse transcription. however, more than a decade of intensive work has challenged this model by discovering that reverse transcription takes place inside the capsid core [ ] [ ] [ ] [ ] [ ] [ ] , protected from the hostile intracellular environment, and uncoating occurs at the nuclear pore complex [ , ] or even inside the nucleus [ ] (fig. ) . recently, it was shown that the presence of pores in the capsid hexamers allows deoxynucleotides to traverse the capsid shell to feed reverse transcription [ ] . however, at only Å wide, these pores are too small to allow proteins to pass through. hence, host rbps participating in reverse transcription must be present inside the capsid core prior to cell entry, likely being incorporated in the producer cell during the assembly of viral particles. the idea of bringing up key proteins from the producer cell is well characterised for negative stranded rna viruses, which incorporate viral polymerase complexes including host factors within the viral particles to enable replication upon entry in the host cell (see above). similarly, hiv- particles contain reverse transcriptase and integrase, two viral enzymes that are critical in both reverse transcription and proviral dna integration into the host chromosome. recently, integrase was shown to bind not only dna but also specific structures in hiv- rna within virions, and these interactions are critical for the correct localization of the vrnp inside mature virions [ ] . several studies have revealed that specific cellular ivrbps either promote (e.g. upf rna helicase and atpase [upf ] [ ] , y-box binding protein [ybx ] [ ] , dhx [ ] , eef a [ , ] , ppia [ ] and aminoacyl-trna synthetases [ ] ) or inhibit (e.g. mov risc complex rna helicase [mov ] [ ] , pyruvate kinase m / [pkm] [ ] , enolase [eno ] [ ] ) reverse transcription (table s e) . nonetheless, the scope of cellular rbps hijacked into hiv- particles and whether they play critical roles in these initial steps of infection inside the virion is not well defined yet. as example, upf is a cytosolic rna helicase that plays crucial roles in hiv- infection and is incorporated into virions (table s e) . strikingly, reverse transcription fails if virions are generated in cells depleted of upf or expressing an atpase-defective upf mutant [ ] . authors suggested that upf could mediate the remodelling the vrnp to facilitate reverse transcription or, alternatively, promote the annealing of trna lys primer to the viral genome, as shown in other helicases such as dhx [ , ] . conversely, recent evidence suggested that dhx participates in the elongation phase of reverse transcription but not in the annealing of trna lys to the vrna [ ] . further research is required to elucidate the exact mechanism of action of upf , dhx and other cellular rbps during hiv- reverse transcription. interestingly, cellular rbps have also been found in hepatitis b virus (hbv) nucleocapsids. hbv is a 'gapped' dna virus but initially a singlestranded rna pre-genome (pgrna) is packaged into viral particles. the pgrna is reverse transcribed to a circular dsdna within the viral capsid before the virus matures and is secreted from the cell [ ] . the pgrna is packaged through recognition of epsilon (ε) stem-loop structure in the ′ terminus by hbv polymerase (pol). it was shown that eukaryotic translation initiation factor (eif) e enables this recognition by forming an eif e-pol-ε rnp complex that is incorporated into the nucleocapsid [ ] . reverse transcription of the pgrna then requires hsp [ ] , which forms a rnp together with other cellular rbps including hsc and dnaja (hsp ) [ , ] to enable chaperone-mediated specific binding of hbv reverse transcriptase to pgrna. how the rna-binding activity of these proteins contribute to viral capsid assembly and/or reverse transcription remains poorly understood. finally, other cellular ivrbps such as dead-box helicase x-linked (ddx x) [ ] , apolipoprotein b mrna editing enzyme catalytic subunit g (apobec g) [ , ] and mov [ ] negatively regulate the very early steps of hbv reverse transcription. ddx x interacts with hbv polymerase and requires atpase but not helicase activity for minus-strand dna synthesis inhibition [ ] ; apobec g acts via an unknown deaminationindependent mechanism [ ] that may involve direct binding to reverse transcriptase [ ] , while mov binds directly to hbv rna but not the viral polymerase [ ] . however, the precise mechanisms by which these proteins affect hbv dna synthesis still remain unclear. it is an evocative idea that cellular antiviral factors may be also packaged with the vrna into virions in order to interfere with the initial steps of infection. well-known antiviral rbps such as zinc finger ccch-type containing antiviral (zc hav ), apobec c and apobec f are detected in the proteome of purified sinv particles (table s c) . however, studies exploring their role in the context of the viral particle or the early phase of sinv infection are non-existent to our knowledge. in addition, antiviral rbps might be under-represented in the different virion proteomes analysed here (table s ). this is most likely due to ) the existence of sophisticated mechanisms to avoid the presence of restriction proteins inside virions, or ) because the viral particles used in these proteomic studies were often produced in highly permissive cell lines with damaged interferon pathway. the first possibility has been widely studied in hiv- , which excludes antiviral proteins from virions by different mechanisms. for example, serine incorporator (serinc ) and serinc are relocalized from cell surface to endosomes by the accessory viral protein nef [ , ] ; yth n -methyladenosine rna binding protein (ythdf ) is cleaved by hiv- protease inside the viral particle [ ] , and apobec proteins are targeted for degradation by the viral protein vif [ ] . ythdf has a strong affinity for n -methyladenosine (m a), a posttranscriptional rna modification that has recently emerged as a key regulator of vrna fate [ ] . m a can be found in the rna of iav, denv, zikv, hcv, yellow fever virus, west nile virus, enterovirus and hiv- . in the case of hiv- , m a modification can influence different steps of the virus infection cycle including reverse transcription. recently, ythdf was found to be encapsidated into hiv- particles by interacting with nucleocapsid and negatively affected reverse transcription once the capsid core is delivered into the newly infected cell [ ] . interestingly, hiv- protease cleaves ythdf into smaller fragments inside the virion, thus counteracting its antiviral activity. other 'readers' of m a rna can be detected inside the viral particles of different viruses, including eif , insulin like growth factor mrna binding protein (igf bp ) and hnrnpa b (table s b) . further work is required to discover the potential role of m a-modified vrna and ivrbps in the early steps of infection. one of the best studied cases of antiviral rbp acting inside virions is the apobec proteins [ ] . these are cellular cytidine deaminases that catalyse the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine. to exert their antiviral effects in hiv- , these proteins are recruited into viral particles through binding to the vrna and nucleocapsid. apobec f, g and h have preference for g-rich and a-rich sequences which resemble the rna-binding pattern of the hiv- nucleocapsid domain in gag polypeptide, as determined by clip-seq in cells and purified virions [ , ] . apobec proteins bind to a large proportion of cellular mrnas in infected cells due to their low specificity. however, the g/a-rich sequence bias ensures that a proportion of apobec molecules interact with hiv- rna [ ] as well as with other retroviruses [ ] . once inside the viral particle, apobec proteins induce cytidine deamination of the reverse-transcribed dna strand. changes from c to u cause complementary g to a conversion during second strand synthesis and this hypermutation inhibits hiv- replication [ ] . in addition, apobec proteins also restrict hiv- by a deamination-independent mechanism that consist of altering reverse transcription template switching frequency [ ] . apobec g has also been shown to hinder replication of mev, mumps virus and rsv [ ] . however, it is unclear whether the deaminase activity is responsible for the inhibition of replication of these viruses. apobec proteins, including apobec c, f and h, also restrict replication of the human coronavirus nl via two mechanisms: cytidine deamination and binding to nucleocapsid protein [ ] . apo-bec c restricts zikv infection in a non-editing-dependent manner and is partially counteracted by a small subgenomic flavivirus rna that sequesters it [ ] . while apobec s suppress the infection of a wide range of viruses by different, poorly understood mechanisms, it remains unknown if they are incorporated into these viral particles and exert their antiviral activity in the early steps of infection. this possibility deserves further investigation. in summary, many cellular rbps are hijacked by viruses to sustain infection and are involved in virtually all stages of their infection cycle. we have highlighted here many previously known and novel rbps that are found inside virions from different human rna viruses and depicted a clear gap in our understanding of their function in infection. cellular ivrbps may facilitate trafficking and selection of vrna into assembling viral particles, protect the vrna from the hostile cellular environment, or streamline the initial processes of vrna metabolism upon entrance in a new host cell. alternatively, some of these ivrbps may function as part of the immune surveillance system of the cell and are incorporated into viral particles to interfere with infectivity. whilst we have discussed several roles for ivrbps in this review, much of these remain poorly characterised and mechanistic data is still missing. future research must focus on deciphering the roles of ivrbps and their newly described rna-binding activities in virus infection. by broadening our understanding on these proteins, it will be possible to identify new targets with potential for host-based antiviral therapies. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.semcdb. . . . thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification identification of proteins bound to dengue viral rna in vivo reveals new host proteins important for virus replication identification of rna binding proteins associated with dengue virus rna in infected cells reveals temporally distinct host factor requirements elucidating the in vivo interactome of hiv- rna by hybridization capture and mass 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replication on co-opted host factors viral subversion of the host protein synthesis machinery host factors in the replication of positive-strand rna viruses tinkering with translation: protein synthesis in virus-infected cells regulation mechanisms of viral ires-driven translation a protein ballet around the viral genome orchestrated by hiv- reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed rna to vpr-bridged dna global changes in the rna binding specificity of hiv- gag regulate virion genesis physical and functional analysis of viral rna genomes by shape exploring the architecture of viral rna genomes hiv- capsid uncoating initiates after the first strand transfer of reverse transcription hiv- uses dynamic capsid pores to import nucleotides and fuel encapsidated dna synthesis reverse transcription mechanically initiates hiv- capsid disassembly actin-binding cellular proteins inside human immunodeficiency virus type proteomic profiling of cellular proteins interacting with the hepatitis c virus core protein proteomic and biochemical analysis of purified human immunodeficiency virus type produced from infected monocyte-derived macrophages proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins cellular proteins in influenza virus particles analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach quantitative proteomic analysis of lentiviral vectors using -de protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein in virus particle assembly identification of essential filovirion-associated host factors by serial proteomic analysis and rnai screen virus-producing cells determine the host protein profiles of hiv- virion cores comparative proteomic analysis of hiv- particles reveals a role for ezrin and ehd in the nef-dependent increase of virus infectivity conserved and host-specific features of influenza virion architecture multi-faceted proteomic characterization of host protein complement of rift valley fever virus virions and identification of specific heat shock proteins, including hsp , as important viral host factors proteomics of hcv virions reveals an essential role for the nucleoporin nup in virus morphogenesis comparative characterization of the sindbis virus proteome from mammalian and invertebrate hosts identifies nsp as a component of the virion and sorting nexin as a significant host factor for alphavirus replication host proteins identified in extracellular viral particles as targets for broad-spectrum antiviral inhibitors actin-modulating protein cofilin is involved in the formation of measles virus ribonucleoprotein complex at the perinuclear region cofilin- is involved in regulation of actin reorganization during influenza a virus assembly and budding new host factors important for respiratory syncytial virus (rsv) replication revealed by a novel microfluidics screen for interactors of matrix (m) protein the host proteins transportin sr /tnpo and cyclophilin a exert opposing effects on hiv- uncoating cyclophilin a stabilizes the hiv- capsid through a novel non-canonical binding site the dsrna protein kinase pkr: virus and cell control rig-i detects viral genomic rna during negative-strand rna virus infection ifits: emerging roles as key antiviral proteins cytosolic innate immune sensing and signaling upon infection rna degradation in antiviral immunity and autoimmunity attacked from all sides: rna decay in antiviral defense strategies for viral rna stability: live long and prosper the taking of the cytoskeleton one two three: how viruses utilize the cytoskeleton during egress hiv trafficking in host cells: motors wanted! host cytoskeleton in respiratory syncytial virus assembly and budding microtubule regulation and function during virus infection friend or foe: the role of the cytoskeleton in influenza a virus assembly the role of host cytoskeleton in flavivirus infection role of rna chaperones in virus replication emerging picture of host chaperone and cyclophilin roles in rna virus replication identification of staufen in the human immunodeficiency virus type gag ribonucleoprotein complex and a role in generating infectious viral particles novel staufen ribonucleoproteins prevent formation of stress granules but favour encapsidation of hiv- genomic rna characterization of staufen ribonucleoproteins by mass spectrometry and biochemical analyses reveal the presence of diverse host proteins associated with human immunodeficiency virus type specific incorporation of heat shock protein family members into primate lentiviral virions chaperones in hepatitis c virus infection allosteric heat shock protein inhibitors block hepatitis c virus assembly bacteriophage ms genomic rna encodes an assembly instruction manual for its capsid life of psi: how full-length hiv- rnas become packaged genomes in the viral particles proteome analysis of the hiv- gag interactome the double-stranded rna-binding protein staufen is incorporated in human immunodeficiency virus type : evidence for a role in genomic rna encapsidation the host protein staufen participates in human immunodeficiency virus type assembly in live cells by influencing pr gag multimerization dimeric rna recognition regulates hiv- genome packaging hiv- rna genome dimerizes on the plasma membrane in the presence of gag protein hrip, a cellular cofactor for rev function, promotes release of hiv rnas from the perinuclear region the cellular hiv- rev cofactor hrip is required for viral replication human protein staufen- promotes hiv- proliferation by positively regulating rna export activity of viral protein rev teis, escrt and membrane protein ubiquitination virus budding and the escrt pathway escrt-ii's involvement in hiv- genomic rna trafficking and assembly alix serves as an adaptor that allows human parainfluenza virus type to interact with the host cell escrt system alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp : a role for alix in ebola virus egress hepatitis a virus structural protein px interacts with alix and promotes the secretion of virions and foreign proteins through exosome-like vesicles assembly and release of dengue virus: role of alix protein interaction between the yellow fever virus nonstructural protein ns and the host protein alix contributes to the release of infectious particles role of alix in mirna packaging during extracellular vesicle biogenesis human immunodeficiency virus type gag engages the bro domain of alix/aip through the nucleocapsid identification of the hiv- nc binding interface in alix bro reveals a role for rna functional association between regulatory rnas and the annexins annexin a in virus infection characterization of interactions between hepatitis c virus ns b polymerase, annexin a and rnaeffects on ns b catalysis and allosteric inhibition role of annexin a in the production of infectious hepatitis c virus particles annexin a mediates the localization of measles virus matrix protein at the plasma membrane arfs at a glance gga and arf proteins modulate retrovirus assembly and release identification of host trafficking genes required for hiv- virological synapse formation in dendritic cells role for adp ribosylation factor in the regulation of hepatitis c virus replication the regulation of translation in alphavirus-infected cells encapsidation of host-derived factors correlates with enhanced infectivity of sindbis virus cold-inducible proteins cirp and rbm , a unique couple with activities far beyond the cold encapsidated host factors in alphavirus particles influence midgut infection of aedes aegypti the mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity the rna synthesis machinery of negative-stranded rna viruses rna polymerase of vesicular stomatitis virus specifically associates with translation elongation factor- alphabetagamma for its activity requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype specific interaction of heterogeneous nuclear ribonucleoprotein particle u with the leader rna sequence of vesicular stomatitis virus involvement of hsp in assembly and nuclear import of influenza virus rna polymerase subunits identification of hsp as a stimulatory host factor involved in influenza virus rna synthesis hiv- dna flap formation promotes uncoating of the pre-integration complex at the nuclear pore isolated hiv- core is active for reverse transcription ip is an hiv pocket factor that prevents capsid collapse and promotes dna synthesis hiv- capsid undergoes coupled binding and isomerization by the nuclear pore protein nup single hiv- imaging reveals progression of infection through ca-dependent steps of docking at the nuclear pore, uncoating, and nuclear transport hiv- uncoats in the nucleus near sites of integration hiv- integrase binds the viral rna genome and is essential during virion morphogenesis upf is crucial for the infectivity of human immunodeficiency virus type progeny virions y-box-binding protein supports the early and late steps of hiv replication virion-associated, host-derived dhx /rna helicase a enhances the processivity of hiv- reverse transcriptase on genomic rna hiv- uncoating and reverse transcription require eef a binding to surface-exposed acidic residues of the reverse transcriptase thumb domain specific interaction between eef a and hiv rt is critical for hiv- reverse transcription and a potential anti-hiv target cyclophilin a promotes hiv- reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cdna role of host trnas and aminoacyl-trna synthetases in retroviral replication p body-associated protein mov inhibits hiv- replication at multiple stages virion-packaged pyruvate kinase muscle type affects reverse transcription efficiency of human immunodeficiency virus type by blocking virion recruitment of trna(lys ) virion-incorporated alpha-enolase suppresses the early stage of hiv- reverse transcription coordinate roles of gag and rna helicase a in promoting the annealing of formula to hiv- rna hepatitis b viruses: reverse transcription a different way incorporation of eukaryotic translation initiation k. dicker, et al. seminars in cell and developmental biology xxx (xxxx) xxx-xxx factor eif e into viral nucleocapsids via interaction with hepatitis b virus polymerase hsp is required for the activity of a hepatitis b virus reverse transcriptase hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids ddx dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids reverse transcriptase-and rna packaging signal-dependent incorporation of apobec g into hepatitis b virus nucleocapsids deamination-independent inhibition of hepatitis b virus reverse transcription by apobec g the mov helicase restricts hepatitis b virus replication by inhibiting viral reverse transcription hiv- nef promotes infection by excluding serinc from virion incorporation serinc and serinc restrict hiv- infectivity and are counteracted by nef hiv protease cleaves the antiviral m a reader protein ythdf in the viral particle apobecs and virus restriction n( )-methyladenosine and viral infection the rna binding specificity of human apobec proteins resembles that of hiv- nucleocapsid promiscuous rna binding ensures effective encapsidation of apobec proteins by hiv- broad antiretroviral defence by human apobec g through lethal editing of nascent reverse transcripts apobec host restriction factors of hiv- can change the template switching frequency of reverse transcriptase the innate antiviral factor apobec g targets replication of measles, mumps and respiratory syncytial viruses apobec -mediated restriction of rna virus replication zika virus noncoding sfrnas sequester multiple host-derived rna-binding proteins and modulate mrna decay and splicing during infection key: cord- - tdtia w authors: li, iris w.; hung, ivan f.; to, kelvin k.; chan, kwok-hung; wong, samson s.y.; chan, jasper f.; cheng, vincent c.; tsang, owen t.; lai, sik-to; lau, yu-lung; yuen, kwok-yung title: the natural viral load profile of patients with pandemic influenza a(h n ) and the effect of oseltamivir treatment date: - - journal: chest doi: . /chest. - sha: doc_id: cord_uid: tdtia w background the natural history of viral shedding from the upper respiratory tract of the new pandemic influenza a(h n ) and the effect of oseltamivir treatment were uncertain. methods a retrospective cohort study involving consecutive patients with specimens positive by reverse transcriptase-polymerase chain reaction for the matrix and new h genes was conducted. results the nontreated and oseltamivir-treated patients were comparable in their viral load at presentation, demography, and the presenting symptoms. no correlation was observed between viral load with age and number of symptoms. viral load of nasopharyngeal aspirate (npa) was significantly lower in treated than in nontreated patients at day after symptom onset. when oseltamivir was initiated ≤ days after symptom onset, a greater rate of viral load reduction in npa of treated patients than that of nontreated patients was observed (− . [ % ci, − . to − . ] vs − . [ % ci, − . to − . ] log copies/ml/d post-symptom onset), and the viral load was undetectable at day after oseltamivir initiation, which was day earlier than that of those whose treatment was initiated > days of symptom onset. the viral load was inversely correlated with concomitant absolute lymphocyte count in nontreated patients (pearson correlation coefficient [r] = − . , p = . ) and treated patients (pearson r = − . , p < . ). resolution of fever was . days later in nontreated than treated patients (p = . ) conclusions the natural viral load profile was described. oral oseltamivir suppresses viral load more effectively when given early in mild cases of pandemic influenza a(h n ) infections. has disseminated globally from mexico and the united states since april . because of the sustained human-to-human transmission at all continents, the world health organization raised the pandemic alert level from phase to phase on june , . because most of the global population aged , years has undetectable neutralizing antibody, , the disease is expected to spread throughout the next few years with a more severe second wave in winter / . the initial animal studies suggested that pandemic a(h n ) also replicates in the lower respiratory tract and elicits more infl ammatory damage than seasonal infl uenza a/h n viruses. [ ] [ ] [ ] although the case-fatality rate outside mexico was comparable to that of seasonal infl uenza, it was much higher in mexico, and most occurred in those aged , years. , because an effective pandemic a(h n ) vaccine was not available during the fi rst months of this pandemic, oral oseltamivir was recommended for chemoprophylaxis and treatment as containment and mitigation measures. , although cases of oseltamivir-resistant isolates in relation to the neuraminidase histidine -to-tyrosine mutation (h y) were reported, most of the viral isolates are still susceptible to oseltamivir on in vitro testing. , oseltamivir treatment had been shown to be safe and reduce disease duration by up to . days and incidence of secondary swab (nts) and sent in viral transport medium at presentation and days , , , and after hospitalization. total nucleic acid extraction using nuclisens easymag instrument (biomerieux; durham, nc) and rt-pcr for infl uenza a virus matrix, the h of pandemic a(h n ), and seasonal h and h genes was performed as previously described. [ ] [ ] [ ] [ ] positive controls with a swine h virus (a/sw/hk/ / ), h n (a/california/ / ), and human seasonal h n and h n viruses were included. for quantitative assay, rt-pcr targeting matrix gene was performed with forward primer [ -cttctaaccgag-gtcgaaacg- ] and reverse primer [ -ggcattttgga-caaakcgtcta- ] . the cdna was amplifi ed in a lightcycler instrument with a faststart dna master sybr green i mix reagent kit, and a reference standard was prepared using pcrii-topo vector (invitrogen; san diego, ca). nontreated pandemic a(h n )-infected patients and those treated with oseltamivir were compared regarding their demographics, underlying comorbidities, initial presenting symptoms, laboratory parameters, and viral load of npa at different days post symptom onset. viral load of npa of nontreated and those on oseltamivir initiated Յ and . days post symptom onset were compared. viral load of nontreated and treated patients at each interval post symptom onset were compared. x test or fisher exact test were used for categorical variables and independent t test for continuous variables between two groups. one-way analysis of variance was used to compare viral loads of npa, nps, and nts at each interval post symptom onset within nontreated and treated patients. linear regression was used to determine the rate of viral load reduction. pearson correlation was used to test the correlations between the age, number of presenting symptoms, concomitant total wbc count, absolute lymphocyte (lym) count, hemoglobin (hb) level, and platelet (plt) count with viral load, respectively. spss . for windows (spss inc.; chicago, il) was used for statistical computation. a twotailed p value , . was considered signifi cant. one hundred forty-five patients diagnosed with pandemic a(h n ) infection from the period since the beginning of the epidemic in hong kong were included. sixty-one ( %) were male patients. seventy-six patients ( . %) were aged , years but none were aged , year. twenty-seven patients ( . %) who refused treatment were enrolled as cases and ( . %) patients who received oseltamivir were controls. refusals were related to fear of possible side effects of oseltamivir despite counseling by attending clinicians. none had diabetes mellitus or chronic obstructive airway disease. only two ( . %) patients had mild pneumonic changes over right middle and lower lobes on chest radiograph, respectively. none had evidence of rhabdomyolysis, myocarditis, or encephalitis. the nontreated and treated patients were comparable in demographics, comorbidity, presenting symptoms, and initial laboratory parameters ( table ) . oseltamivir was initiated on the same day ( / , . %), day ( / , . %), days ( / , . %), days complications, such as pneumonia or otitis media, when initiated within h of symptom onset in seasonal infl uenza a. , it can be easily administered orally and is the only safe drug in patients with asthma. the evidence of its effectiveness for the treatment of pandemic a(h n ) remains uncertain. the current recommendations were based on the assumption that pandemic a(h n ) will have similar biologic characteristics as seasonal infl uenza a/h n viruses. we compared the viral load profi le of the fi rst group of pandemic a(h n )-infected patients with or without oseltamivir treatment in hong kong. during the containment phase of the pandemic a(h n ) outbreak in hong kong from april , , to june , , all patients with laboratory-confi rmed pandemic a(h n ) infection, under the local prevention and control of disease ordinance, were compulsorily isolated. , only those isolated in the three hospitals of hong kong were included in this study. the study was approved by our institutional review board. in addition to drawing blood for routine hematologic and biochemical tests, a chest radiograph was taken if clinically indicated. if nasopharyngeal specimens tested positive by reverse transcriptase-polymerase chain reaction (rt-pcr) for the infl uenza a matrix gene and the pandemic a(h n ) hemagglutinin gene, but negative for h and seasonal h , days of oseltamivir was recommended at a dosage adjusted for age and renal function. those who refused oseltamivir treatment were regarded as cases for our study of natural viral load profi le from upper respiratory specimens, whereas those treated were controls. demographic, clinical, laboratory, and radiographic data were retrospectively retrieved from the computerized clinical management system for entry on a standard form for analysis as previously described. the number of classic initial clinical symptoms as predictors of infl uenza infections were used to correlate with viral load in respiratory specimens. , these included the presence of fever (temperature Ն . °c), cough, sore throat, nasal symptoms, myalgia, and headache. , ( / , . %), and Ն days ( / , . %) post symptom onset. the mean (range) interval between symptom onset and oseltamivir initiation was . ( - ) days. six patients had nonspecifi c complaints of nausea, vomiting, or loose stool on the initial days of oseltamivir therapy but it was diffi cult to differentiate from clinical symptoms of pandemic a(h n ) infection. none reported major side effects from oseltamivir therapy. the viral loads of npa, nps, or nts at different intervals post symptom onset in nontreated patients were comparable ( fig a , table ), whereas significant differences were observed at days to to days to post symptom onset in treated patients ( fig b , table ). in treated patients, viral loads of npa were higher than those of nps and nts, and signifi cant differences were observed at day to post oseltamivir initiation but not at day before to day after oseltamivir initiation ( fig , table ). similar fi ndings were observed in days and post oseltamivir initiation in those who received oseltamivir Յ days post symptom onset, and day post oseltamivir initiation in those who received oseltamivir . days post symptom onset. when only npa specimens are analyzed, viral load of npa was signifi cantly lower in treated than nontreated patients at day post symptom onset irrespective of the relation between oseltamivir initiation and the day post symptom onset ( fig a , table ). when oseltamivir was initiated Յ days post symptom onset, a greater rate of viral load reduction in npa of treated patients ( . [ % ci, . to . ] vs . [ % ci, . to . ) log copies/ml/d post symptom onset) than that of nontreated patients was observed. similar rate of viral load reduction in npa was observed in those who received oseltamivir Յ and . days of symptom onset ( . [ % ci, . to . ] vs . [ % ci, . to . ] log copies/ml/d post oseltamivir initiation). at day post oseltamivir initiation, . % of these patients had undetectable viral load level in respiratory specimens ( fig b ) and their viral load level of npa was undetectable, which was day earlier than those received oseltamivir . days post symptom onset. moreover, the viral load of npa in those who received oseltamivir Յ days post symptom onset was consistently lower than that of nontreated patients, and signifi cant differences were observed at days to and days to post symptom onset ( fig , table ). among the viral load tests performed, out of samples from nontreated patients and out of samples from treated controls had concomitant peripheral blood taken for hematologic test. the viral load level was inversely correlated with concomitant lym count (pearson r . , p , . ), hb level (pearson r . , p . ), and plt count (pearson r . , p . ) in treated patients. antiviral therapy for infl uenza is hampered by the early peaking of the viral load, which leaves a narrow window of opportunity for antiviral treatment. , in both seasonal and pandemic a(h n ) infl uenza a infection, the viral loads peaked at around to days post symptom onset in natural infections of healthy adults with or without oseltamivir treatment. , as no data has yet been reported on the treatment effectiveness of oseltamivir on pandemic a(h n ) infections, it is reassuring to observe that treated patients similar fi ndings were obtained with lym count (pearson r . , p . ) and wbc counts (pearson r . , p . ), but not for hb level and plt count of nontreated patients. no signifi cant correlation was observed between the viral load level with patient's age and the number of classic presenting symptoms. resolution of fever (temperature Յ . °c) was . days earlier in treated than in nontreated patients ( . vs . days, p . ). none required ventilatory or critical care and no death was reported. the recent few years. oseltamivir resistance, which is largely associated with h y mutations in neuraminidase gene of influenza a(h n ) and h n viruses, - developed in . % to . % of patients following oseltamivir treatment. , [ ] [ ] [ ] [ ] in , h y mutants of infl uenza a(h n ) brisbane-like variant, which were fi rst detected in norway, have spread globally and become the dominant seasonal h n virus. initial bioinformatics analysis and phenotypic antiviral susceptibility tests showed that the h n is susceptible to oseltamivir and zanamivir but resistant to amantadine or rimantadine because of a serine -to-asparagine mutation. with the dramatic increase in oseltamivir use for treating pandemic a(h n ) infection, resistance may continue to increase worldwide, including hong kong. , even with the availability of a safe and effective pandemic a(h n ) vaccine, antiviral stockpiling remains an important armamentarium for the epidemiologic control of future pandemic. the early control of viral load in patients not treated with oseltamivir may be explained by the actions of innate immunity followed by early anamnestic adaptive immune response, possibly cytotoxic t lymphocyte response against cross-reacting epitopes of internal proteins or nonneutralizing surface proteins common to all infl uenza a viruses. failure to mount this early immune response because of innate humoral or cellular immunodefi ciencies, concomitant use of immunosuppressive drugs, or transient immunoparesis due to severe concomitant coinfection may predispose some patients to develop severe clinical progression. they are often those who presented late with hospitalization at a median of days with respiratory failure and death at a median of days post symptom onset. viral load refl ects the dynamic interaction between viral replication and clearance by body defense mechanisms. monitoring viral load throughout the disease course has been used as an objective means of checking the clinical progress or response to antiviral therapy. we showed the inverse correlation between the absolute lymphocyte count and concomitant viral load level in had a greater rate of viral suppression in respiratory specimens throughout the disease course, with significantly lower viral load at day post symptom onset. moreover, when oseltamivir was initiated Յ days of symptom onset, viral load was not detectable at day post oseltamivir, which was day after treatment completion, and was day earlier than that of those initiated . days of symptom onset. the effect of oseltamivir as early treatment in suppressing pandemic a(h n ) was similar to that of seasonal infl uenza, although its effectiveness for pandemic a(h n ) chemoprophylaxis was not yet established. fever subsided . days earlier in treated patients, which is consistent with previous fi ndings that oseltamivir shortens fever duration and reduces the quantity and duration of viral shedding in adults with seasonal infl uenza. , , although oseltamivir was shown to hasten recovery and reduce viral load in this study, its long-term effectiveness for pandemic a(h n ) remains uncertain. primary resistance to the neuraminidase inhibitors among wild strains of human infl uenza viruses a(h n ), a(h n ), and b has been uncommon until table values given are mean sd viral load in log copies/ml (no. of specimens). the detection limit of the quantitative rt-pcr was . log copies/ml. see table for expansion of abbreviations. values given are mean sd viral load in log copies/ml (no. of specimens). the detection limit of the quantitative rt-pcr was . log copies/ml. fnps fl ocked nasopharyngeal swab; npa nasopharyngeal aspirate; nps nasopharyngeal swab; nts naso-throat swab; snps standard nasopharyngeal swab; rt-pcr reverse transcriptasepolymerase chain reaction. aspirate is more likely to get a good sample of infected cells than swabbing the throat, anterior nares, or even the nasopharynx. as the viral load is decreased by oseltamivir, the difference would be accentuated in the suboptimal sampling methods by nps and nts. but these factors are unlikely to affect the overall conclusion of this study because the results were similar when only nasopharyngeal aspirates were analyzed. similar to previous studies, oseltamivir is associated with few side effects. the most frequent side effects are gastrointestinal symptoms, which occurred in up to % of school children who received oseltamivir prophylaxis for the infl uenza a(h n ) virus. previous reports suggesting serious neuropsychiatric manifestations after oseltamivir were not demonstrated in later studies and only % of school children taking oseltamivir prophylaxis had reported mild symptoms. [ ] [ ] [ ] nontreated and treated patients irrespective of the days post symptom onset at the time when the specimens were sampled, as in the case of sars coronavirus and infl uenza a(h n ) virus infections. , , [ ] [ ] [ ] lymphopenia was noted in pandemic a(h n )infected patients of greater severity, especially those who were pregnant or morbidly obese. , , previous study in immunocompromised patients showed that lymphocyte recovery was associated with viral clearance, independent of oseltamivir. there was no difference in viral load with different sampling methods in nontreated patients, whereas the differences seen with npa, nps, and nts in treated patients could be attributed to increased statistical power with the higher number of patient specimens in this group. as the dominant site of viral replication in these mild cases is the nasopharynx rather than the lower respiratory tract, the suction by nasopharyngeal values given are mean sd viral load in log copies/ml (no. of specimens). the detection limit of the quantitative rt-pcr was . log copies/ml. see table values given are mean sd viral load in log copies/ml (no. of specimens). the detection limit of the quantitative rt-pcr was . log copies/ml. see table for expansion of abbreviations. despite the apparent effi cacy of oseltamivir in mild cases, its effi cacy in stopping further disease progression of late cases remains uncertain. a randomized control treatment trial is not possible at the beginning of the epidemic in our locality because of the uncertainties of its disease severity and the international recommendations on oseltamivir treatment. moreover, patients presented to us on different days post symptom onset and some have refused further nasopharyngeal sampling once their symptoms improved. despite these limitations, the nontreated cases are still comparable to the treated patients in demographics, symptoms, and laboratory fi ndings. a previous viral load study of pandemic a(h n ) infection focused on oseltamivir-treated patients; this is the fi rst study, to our knowledge, of natural viral load profi le of these patients without such treatment. neuraminidase inhibitor flu treatment investigator group . effi cacy and safety of oseltamivir in treatment of acute infl uenza: a randomised controlled trial antiviral therapy for respiratory tract infections neuraminidase inhibitors for preventing and treating infl uenza in healthy adults: systematic review and meta-analysis the government of the hong kong sar. legislative council panel on health services. prevention and control of human swine infl uenza infection in hong kong strategy and management of human swine infl uenza (hsi) interim guidance on antiviral treatment, chemoprophylaxis and pneumococcal vaccination for human swine infl uenza (hsi) / infl uenza a (h n ) infection viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory syndrome predicting infl uenza infections during epidemics with use of a clinical case defi nition diagnosis of infl uenza in the community: relationship of clinical diagnosis to confi rmed virological, serologic, or molecular detection of influenza comparison of the nuclisens easymag and qiagen biorobot nucleic acid extraction systems for detection of rna and dna respiratory viruses in nasopharyngeal aspirate samples viral load in patients infected with pandemic h n infl uenza a virus medical treatment of viral pneumonia including sars in immunocompetent adult cdc realtime rt-pcr (rrtpcr) protocol for detection and characterization of influenza (version differential susceptibility of different cell lines to swine-origin infl uenza a h n , seasonal human infl uenza a h n , and avian infl uenza a h n viruses use of the oral neuraminidase inhibitor oseltamivir in experimental human infl uenza: randomized controlled trials for prevention and treatment viral shedding in children with infl uenza virus infections treated with neuraminidase inhibitors european infl uenza surveillance scheme . oseltamivir-resistant infl uenza virus a (h n ), europe, - season chan: contributed to performing the laboratory tests, interpreting the data, and performing statistical analysis human infection with new infl uenza a (h n ) virus: clinical observations from mexico and other affected countries statement to the press by who director-general dr margaret chan update: novel infl uenza a (h n ) virus infection-mexico cross-reactive antibody responses to the pandemic h n infl uenza virus transmission and pathogenesis of swine-origin a(h n ) infl uenza viruses in ferrets and mice pathogenesis and transmission of swine-origin a(h n ) infl uenza virus in ferrets emergence and pandemic potential of swine-origin h n influenza virus iner working group on infl uenza . pneumonia and respiratory failure from swine-origin infl uenza a (h n ) in mexico who guidelines for pharmacological management of pandemic (h n ) infl uenza and other infl uenza virus interim guidance on antiviral treatment, chemoprophylaxis and pneumococcal vaccination for human swine infl uenza (hsi)/ infl uenza a (h n ) infection centers for disease control and prevention . cdc health alert network (han) info service message: three reports of oseltamivir-resistant novel infl uenza a (h n ) viruses avian infl uenza virus infections in humans oseltamivir resistance during treatment of infl uenza a (h n ) infection clinical features and rapid viral diagnosis of human disease associated with avian infl uenza a h n virus resistant infl uenza a viruses in children treated with oseltamivir: descriptive study oseltamivir-resistant infl uenza viruses a (h n ), norway update: drug susceptibility of swine-origin infl uenza a (h n ) viruses clinical effectiveness of oseltamivir and zanamivir for treatment of infl uenza a virus subtype h n with the h y mutation: a japanese, multicenter study of the oseltamivir-resistant infl uenza a pandemic (h n ) virus infl uenza a vaccine based on the extracellular domain of m : weak protection mediated via antibody-dependent nk cell activity fatal co-infection with swine origin infl uenza virus a/h n and community-acquired methicillinresistant staphylococcus aureus the severe acute respiratory syndrome (sars) viral loads in clinical specimens and sars manifestations functional tumor necrosis factor-related apoptosis-inducing ligand production by avian influenza virus-infected macrophages clinical fi ndings and demographic factors associated with intensive care unit admission in utah due to novel infl uenza a(h n ) infection hospitalized patients with novel influenza a (h n ) virus infection -california prolonged infl uenza virus infection during lymphocytopenia and frequent detection of drug-resistant viruses comparison of fl ocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients oseltamivir adherence and side effects among children in three london schools affected by infl uenza a(h n )v assessment of neuropsychiatric adverse events in infl uenza patients treated with oseltamivir: a comprehensive review infl uenza associated central nervous system dysfunction in taiwanese children: clinical characteristics and outcomes with and without administration of oseltamivir dr cheng: contributed to collecting the clinical data and samples. dr tsang: contributed to collecting the clinical data and samples. dr lai: contributed to collecting the clinical data and samples. dr lau: contributed to collecting the clinical data and samples. dr yuen: contributed to designing, executing, and supervising the study. financial/nonfi nancial disclosures: the authors have reported to chest that no potential confl icts of interest exist with any companies/organizations whose products or services may be discussed in this article. other contributions: we thank dr york y. n. chow, secretary for food and health; dr ping-yan lam, director of health; and dr rodney allen lee and dr alan wl wu, department of pathology of pamela youde eastern hospital, the hong kong sar, for their kind support. we also thank dr herman tse for helpful discussions, and the clinical and laboratory staff of queen mary hospital for their contribution in patient management and laboratory service support. key: cord- -bu pzbnv authors: miller, craig s. title: pleiotropic mechanisms of virus survival and persistence date: - - journal: oral surg oral med oral pathol oral radiol endod doi: . /j.tripleo. . . sha: doc_id: cord_uid: bu pzbnv viruses are enormously efficient infectious agents that have been implicated in causing human disease for centuries. transmission of these pathogens continues to be from one life form to another in the form of isolated cases, epidemics, and pandemics. each infection requires entry into a susceptible host, replication, and evasion of the immune system. viruses are successful pathogens because they target specific cells for their attack, exploit the cellular machinery, and are efficient in circumventing and/or inhibiting key cellular events required of survival. this article reviews some of the advances that have taken place in human virology in the past years, emphasizing mechanisms that contribute to, and are involved with, virus survival and persistence. the field of virology was barely half a century old in when dr thomas francis wrote articles, ''viruses as agents of disease'' and ''the prevention of virus disease,'' that were published in this journal. early leadership by mayer, ivanovsky, loeffler, frosch, walter reed and others, allowed progress from ''contagium vivum fluidum'' and a simple understanding of the existence and predatory nature of viruses to the characterization of viruses with regard to size, resistance to chemical and physical agents, host and tissue selectivity, and pathogenic and immunologic effects. these investigations made it clear that viruses were a very diverse group of pathogens. however, our knowledge of viral-cell interactions and the effect of viruses on the immune system was rudimentary. we held the understanding that a recovered individual is not susceptible to reinfection with the same virus, and that serum contained components that when mixed with virus and injected into a susceptible animal that animal was protected. these basic concepts served as the basis for classic studies of active and passive immunization. but several important advances that occurred between and jump-started the field of modern virology. these included the development of cultures of single animal cells, , watson and crick's identification of dna and the genetic code, the development of optimal medium for growing cells, and the development of the viral plaque assay. by the early s, max theiler and jonas salk (killed virus) had developed vaccines for yellow fever and polio, respectively, and through the benefits of growing the viruses in cell culture, shortly thereafter sabin developed the oral (live attenuated virus) vaccine. introduction of these vaccines into the human masses remains to this day one of the greatest accomplishments of preventive medicine. in the s, the transition from basic virology to molecular biology began. viruses and components of viral infections were analyzed using gel electrophoresis, protein-antibody interactions, and biochemical assays to answer basic biologic questions. as a result, greater knowledge of virus replication, viral and cellular receptors, and immunologic interactions was achieved. specifically, research during this time led to an understanding of the regulation of gene expression including transcription factors, enhancer elements, promoters, aspects of rna polymerase, and reverse transcriptase, as well as the discovery of proto-oncogenes and tumor suppressor proteins. scientists tagged viruses to identify intracellular locations of viral proteins, understand nuclear and cytoplasmic shuttling, and map neural circuitry. complete genomic sequences of viruses have been recorded and entered into public databases. the benefits of databases such as fasta and blast have led to searches in homology between motifs characteristic for specific gene products and the identification of novel viral genes and their functions. our knowledge also has advanced from the use of positive and negative selection procedures. positive selection being the method whereby genomic fragments or single candidate genes are expressed in a suitable cell system and tested for functionality (ie, infection phenotype). in contrast, negative selection is based on the construction of viral mutants that lack specific genes and the implementation of studies that identify a change in phenotype when the viral mutant infects a particular this work was supported in part by nih grant de . cell or animal. use of these techniques has led to the identification of virulence factors, novel mechanisms of regulation of cell surface receptors, and signal transduction pathways. within the last decade, the emerging fields of genomics and proteomics have allowed for the functional analysis of a large number of transcripts and protein sequences that are expressed during viral infection that provide new clues as to the regulation of acute, chronic, and persistent viral infections, as well as reactivation and malignant transformation. excitingly, the last decade has demonstrated that scientists have the knowledge and skill to harness unique features of viruses (eg, adenoviruses, retroviruses, herpesviruses) in implementing gene therapy and targeting processes important in chronic disease and cancer. however, mastery of this field remains to be seen. despite the exponential growth in virology during the last years, mankind still suffers from transmission and disease when humans serve as hosts to viruses. moreover, the outcomes are often severe when humans serve as novel hosts to emerging virus infection (eg, avian flu virus, ebola virus, equine hemorrhagic fever viruses, hanta virus, human immunodeficiency virus (hiv), hendra virus, nipah virus, sudden acute respiratory syndrome (sars) coronavirus, and west nile virus). of great importance is the fact that the oral cavity continues to be the source of transmission of many viruses, the site of replication and asymptomatic shedding of viruses, and a site where persistent viral infections exist, the latter being a prerequisite for virally induced malignant transformation. clearly, the field of virology has grown to the extent that a ''state-of-the-art'' paper would be exhaustive in length. accordingly, this review focuses on specific viral cell interactions that allow the virus to survive the cellular attack and evade the immune system, establish persistent infections, and cause chronic disease. additional topics will be covered in future reviews. viruses have developed numerous strategies for subverting the host defenses that are launched during infection. the first innate defense encountered is cellular selectivity during the entry process. viral attachment proteins bind to specific cell receptors (proteins, carbohydrates, or glycolipids) and coreceptors. the absence of a specific receptor shields the cell from attack. if this level of defense is foiled, upon binding receptors can sense microbial infection and trigger a multitude of antimicrobial and inflammatory responses. the toll-like receptor (tlr) family which consists of to members are well characterized in their ability to detect bacterial components (ie, lipoproteins and lipoteichoic acids, flagellin) as well as unmethylated cpg motif dna of bacteria and viruses (detected by tlr ), double-stranded rna (detected by tlr ) and single-stranded viral rna (detected by tlr ). in particular, tlrs , , , and specialize in viral detection and recognition of nucleic acids within the intracellular compartments which results in defensive signaling. after receptor binding, entry is modulated by either direct fusion with the plasma membrane or clathrin-or nonclathrin-mediated endocytosis. viruses that gain entry uncoat and deliver their genetic material and undergo a permissive or nonpermissive infection. a permissive cell permits virus replication and ultimate lysis of the host cell. in contrast, a nonpermissive cell downregulates virus replication and lytic gene expression resulting in little to no viral progeny. nonpermissive infections can be abortive or persistent, and persistent infections can be active or latent. latent infections are characterized by silencing of gene transcription, intermittent reactivation, or rarely oncogenic transformation. at the onset of infection, for a virus to survive within a cell, the virus must balance its own growth with death of the host and circumvention of the immune response. strategies for survival involve regulating apoptosis, inhibiting interferon production, modulating the major histocompatibility complex (mhc) class i function that ultimately affects the cytotoxic lymphocyte (ctl) and natural killer (nk) response, and limiting cytokine and chemokine production/function. long-term survival (ie, latency) requires downregulation of lytic gene expression, inhibition of apoptosis, and minimizing the inflammatory response. apoptosis, or programmed cell death, is a highly regulated and conserved series of sequential cellular events that results from receptor-or mitochondrialmediated pathways in response to a variety of stimuli, including viral infection and the appearance of doublestranded rna. the process is regulated (fig ) by a family of aspartate-specific cysteinyl proteases, or caspases, that converge at a number of downstream points resulting in proteolytic cleavage and enzyme activation. caspases are segregated into distinct subfamilies. the ''apoptotic'' caspases ( , , , , , , and ) are involved in the cascade that results in protease production, chromatin condensation, and cellular degradation. the ''inflammatory'' caspases ( , , and ) provide a second round of defense against viral infection. the inflammatory caspases are involved in the proteolytic maturation of key cytokines (ie, interleukin (il)- b and il- ). cytokine il- , also known as interferon (ifn)-inducing factor, directs the production of ifn-c. in turn, ifn-c induces expression of proteolytic active subunits that lead to proteolysis and antigenic processing by tap proteins. tap proteins are critical for displaying viral antigens on the cell surface (see cellular immunity, below). clearly, apoptosis is an important target of virus defense, because early destruction of an infected cell could greatly reduce replication and the number of viral progeny produced. interestingly, viruses have evolved several methods for suppressing or delaying apoptosis as well as encoding proteins that function as inducers of apoptosis. this apparent yin-yang relationship with apoptosis is important to prolong the life of the cell yet facilitate the release and spread of viral progeny at the appropriate time. , viruses regulate apoptosis by several mechanisms including the targeting of the tumor suppressor gene product p , the fas death receptor, and by producing caspase inhibitors and viral bcl- homologs. adenovirus, for example, encodes several gene products that influence apoptosis. the e a gene product stabilizes p and induces p -dependent apoptosis. , in contrast, the adenovirus e gene product promotes degradation of fas, and the adenovirus e b proteins antagonize p function. viral homologs of bcl- , an apoptosis suppressor that binds with bax, are produced by adenovirus, epstein-barr virus (ebv, bhrf protein), and other viruses. , there are several classes of caspase inhibitors encoded by viruses. these include the serine proteinase inhibitors (serpins: crma/spi- ), viral inhibitors of apoptosis (viaps), p , and inhibitors of procaspase protease (also known as flice). crma and p block caspase , previously termed il- beconverting enzyme (ice). caspase functions primarily as an activator of proinflammatory cytokines, but also has apoptosis-inducing ability in select mammalian cells, such as neurons. the viaps appear to inhibit bax-mediated apoptosis in human cells rather than directly inhibiting caspases. several human herpesviruses encode fliceinhibitory proteins (flips) that block trail-mediated cell death by interfering with procaspase protease (flice) activation. for example, the b-herpesviruses (cytomegalovirus (cmv)) encode a viral inhibitor of caspase activation (vica) which inhibits caspase (flice) activation, and c-herpesviruses encode vflips (eg, k ) which inhibit activation of caspases by molecular mimicry. cmv also encodes a viral mitochondrial inhibitor of apoptosis (vmia) (encoded by the u l gene) which inhibits activation of mitochondrial pores in a manner similar to members of the antiapoptotic bcl family. , the alpha herpesvirus hsv- encodes several antiapoptotic gene products (ie, icp , icp , c . , u s , gj) [ ] [ ] [ ] [ ] [ ] that modulate apoptosis at several levels, including antagonism of double-stranded rna-activated protein kinase (pkr), a downstream induction molecule of the interferon signaling pathway , of note, all c-herpesviruses express viral homologues of cellular antiapoptotic genes, including or bcl- homologues. interferon, discovered in the late s when scientists observed that virus-infected cells secrete a factor that mediates the transfer of a viral-resistant state, is a family of regulatory glycoprotein cytokines that modulate both innate and adaptive antimicrobial immunity. they are products of an infected cell genome and one of the key factors in the host response against viral infection. ifns serve as an early defense system that precedes the onset of the immune response and are triggered by envelope glycoproteins, cpg dna, or double-stranded rna. in recombinant formulations, they have been used in medicine and dentistry to combat various viral infections. , human ifns are classified based on the sequence of amino acids into main groups -a, b, and c -and that are less extensively studied (x, j, and s, not discussed further in this review). ifn-a and -b are produced rapidly when viral factors interact with cellular patternrecognition receptors such as tlrs and cytosolic receptors. historically, synthesis of ifn-a has been attributed to macrophages and b cells, and ifn-b has been considered to be produced by fibroblasts. more recently, plasmacytoid dendritic cells have been shown to produce ifn-a preferentially to ifn-b. both ifn-a and -b prevent the replication of viruses by inducing formation of secondary messengers which include ifn regulatory factor (irf) , irf- , irf- , c-jun/atf- , and nf-jb. ifn-c is synthesized by activated t lymphocytes and natural killer (nk) cells following receptor-mediated stimulation or in response to cytokines produced by macrophages or antigen-presenting cells (ie, primarily il- , il- , and ifn-a/b) or by stimulation through t cell receptors (tcrs) or nk cell receptors. it is a powerful activator of mononuclear phagocytes, thus enhancing their ability to destroy intracellular microorganisms and tumor cells. ifns mediate their antiviral action through ifn-stimulated genes (isg), which number in hundreds. ifns also regulate the cell cycle and have antiproliferative effects. viral evasion of ifn occurs by several strategies. in the majority of infections, viruses encode products that antagonize either the ifn signal transduction pathway or cellular proteins induced by ifn that are responsible for inhibiting virus replication (fig ) . adenovirus, ebv, papillomavirus, and members of the paramyxovirinae subfamily encode proteins that inhibit the jak-stat (janus kinaseesignal transducer and activator of transcription) signaling pathways that are required for ifn production. specifically, adenovirus encodes the oncoprotein e a which inhibits the activation of isg factor (isgf ). , paramyxovirinae reduce the effectiveness of the ifn response by targeting stat for degradation or by interference with stat phosphorylation or stability. , kaposi's sarcomaeassociated herpesvirus (kshv) encodes the pleiotropic gene product latency-associated nuclear antigen (lana) that acts downstream of isgf and inhibits p . , in an alternate approach, hpv encodes proteins, e and e , that bind to irf- and irf- , respectively, both of which inhibit the transactivation functions of the bound irf. , viruses also encode proteins that mimic cellular components of the ifn signal transduction pathway, including homologs of the ifn receptors, a viral isrelike promoter element, and viral homolog of irf (virf). for example, poxviruses antagonize ifn signals by encoding soluble ifn receptor homologs. , ebv encodes a viral isre and hhv encodes virf from the k orf that functions as a repressor of transcriptional activation induced by ifn-a, -b, and -c. in addition, several viruses have developed strategies to inhibit ifn-inducible, rna-dependent protein kinase (pkr). pkr, when antagonized, leads to phosphorylation of eif- a which results in inhibition of the ifn-induced antiviral response of the host. adenovirus, herpesviruses, influenza, and sv antagonize pkr by different mechanisms involving degradation of pkr, prevention of pkr activation, and resistance to downstream kinase activation. [ ] [ ] [ ] the mhc class ierestricted t cell response can result in a lethal hit before virus replication. thus, many viruses have developed strategies for interfering with antigen presentation to mhc class i molecules and intracellular trafficking of mhc molecules. viruses target the mhc-i at almost all steps of its trafficking: in the endoplasmic reticulum (er), in the cytoplasm on its way to the surface, and after the mhc reaches the cell surface (fig ) . one key target in the viral defense against the cellular arm of the immune system is attack of the transporter protein associated with antigen processing (tap). tap loads short antigenic peptides to the mhc which stabilize the class i complexes and allows their migration to the cell surface. without the peptide cargo, mhc class i molecules are unstable and dissociate. hsv- and hsv- encode infected cell polypeptide (icp)- , an immediate early gene product, that interacts with the tap protein in the cytosol to prevent peptide binding to tap. human cytomegalovirus (hcmv) encodes u s , a eamino acid glycoprotein, that blocks peptide transport by binding to tap in the endoplasmic reticulum. although efficient at both retaining mhc-i molecules and preventing ctl recognition, hcmv also uses additional viral proteins (u s , u s , u s , and u s ) to evade the immune system. a different approach is taken by ebv. this human c-herpesvirus encodes a glycine-alanine repeat (gar) domain on ebv-encoded nuclear antigen (ebna) that inhibits ubiquitin/proteasome-dependent proteolysis of ebv antigens. thus, processing (ie, degradation) of viral proteins into antigenic peptides is restricted. kshv, a lymphotropic c-herpesvirus, interferes with mhc-i antigen presentation by ubiquitinating the cytosolic domain of the mhc-i. herpesviruses also produce proteins that target mhc class i molecules for degradation in lysosomal compartments and downregulate expression of major histocompatibility complex molecules by shutting off host cell protein synthesis by the gene known as virion host shutoff (vhs, u l ). in contrast, hiv with its simple genome encodes fewer proteins but accomplishes similar immune evasion by pluripotent accessory proteins. for example, nef, of the regulatory proteins encoded by hiv, has multiple functions. in addition to enhancing virion infectivity, nef binds to and inhibits the surface expression of the major mhc-i, downregulates the cell surface expression of cd (the main hiv receptor), and facilitates cd receptor endocytosis. through the function of nef in redirecting the trafficking of immune receptors, infected t lymphocytes are able to hide from the immune system allowing for viral spread. hpv utilizes early proteins (e and e ) to persist undetected within epithelial cells. the early gene product e of the oncogenic strains hpv- and - downregulates mhc-i expression at the transcriptional level by inhibiting the promoters of the mhc-i heavy chain, tap- , and lmp- . e decreases mhc-i expression at the transcriptional level and causes retention of mhc-i in the golgi apparatus. within the golgi, hpv e inactivates the atpase proton pump system. as a result, acidification is blocked, local ph rises, and mhc-i trafficking is perturbed. thus, it is clear that viruses have achieved ingenious methods for interference with mhc-i antigen presentation and inhibition of the cellular immune response. viruses persist in cells because they are able to downregulate key processes that if left unattended would result in cell death. originally attributed in part to the immune response, increasing evidence suggests regulation of key genes plays an important role in the process. specifically, regulation of viral transcription and genomic replication allows for long-term viral stability and survival. many viruses, including those that cause persistent infections and chronic disease (ie, hepatitis c virus, hepatitis b virus, hiv, human herpesviruses, hpv, and jc virus), are successful because of their cell tropism and ability to autoregulate their replication efficiently within specific cells. common features of autoregulation include sensors to the external environment, negative feedback loops, transcriptional enhancers specific for cells that host the persistent infection, and transcriptional silencers. in some cases autoregulation results in steady-state levels of virus replication; in other infections, the virus enters latency only to reactivate intermittently. the importance of autoregulation is apparent from both in vivo and in vitro studies. for example, during lentivirus (hiv, simian immunodeficiency virus, and feline immunodeficiency virus) infection, viremia peaks early after infection then declines to a steady-state level. the effect is not altered by the presence of steroidinduced immunosuppression, and clearance of infected white blood cells is not associated with an earlier presence of antibody, cytokine response, or cytotoxic lymphocyte activity. in another common clinical example, successful antiviral therapy results in dramatic drops in viremia, often to undetectable levels. however, replication of virus often rebounds rapidly to pretreatment levels upon drug withdrawal, and in vitro studies indicate that cellular and immune functions are not contributory to the observed outcome. even when antiviral therapy achieves a sustained virologic response (ie, absence of viremia months after the end of treatment), highly sensitive assays (ie, polymerase chain reaction) detect residual viral genomes in most patients, indicating the ability of viruses to persist and autoregulate based on their environment. , herpesviruses are well known to establish latency in a variety of cell types, and this family of viruses has the ability to autoregulate. this is illustrated by hsv- and hsv- , which can undergo dichotomous life cycles: a lytic infection in epithelium and a latent infection in neurons. in fact, when neuronal cells are infected with hsv- or hsv- at low multiplicity of infection in vitro, the majority of cells can survive for many days even in the absence of immune cells and without the addition of antiviral drugs to the culture medium. similarly, if the rare neuronal cells supporting replication are eliminated using acycloguanosine in the above mentioned system, over % of the remaining population harbors a quiescent infection for weeks after the antiviral drug is removed, again in the absence of immune cells. , viruses regulate replication of their genome in a complex manner, but achieve the outcome through use of viral sensors, repressors, and effectors. viral sensors ''sense'' perturbations in the viral equilibrium within the cell and signal change at the appropriate time. with many viruses (ie, hiv, hepatitis c virus, hepatitis b virus), envelope proteins play the role of sensors, because envelope proteins can influence virus replication in both a positive and a negative manner. [ ] [ ] [ ] [ ] for hpv, regulation is through cellular factors that bind to the promoters of e and e . for hsv- , transcription of the immediate-early (ie) genes during the lytic infection is regulated by the binding of a tegument protein (vp ) with the cellular protein host cell factor (hcf) and oct- . thus, vp would seem to be a logical choice for a sensor. however, latency can be established in the presence of vp and reactivation occurs without the transactivating domain of vp . thus, downstream factors of vp (eg, icp , icp , or other unknown factors) serve as sensors of the environment and regulate the balance between latency and reactivation. the ''effectors'' (transactivators and replicative enzymes such as rna polymerase) modulate virus replication and are the targets of the sensors. effectors are tightly regulated (ie, repressed at certain times) but dynamically modifiable, typically by proteins bound to critical regions of the genome. these proteins afford protection by limiting changes in conformation of, or enzymatic action on, the restricted gene. histones are the most notable guardians of the effectors. histones permit access of dna to specific activators or repressors, general transcription factors, and rna polymerase by posttranslational modification (acetylation, methylation and phosphorylation) of their amino terminal tails. , for example, hyperacetylation of histones is associated with an ''open chromatin'' conformation and transcriptional activation, whereas hypoacetylation of the histone complex is associated with condensed (hetero-) chromatin and gene silencing. several human herpesviruses - utilize these mechanisms for regulating latency and reactivation. in addition, the active regions of the latent a-herpesvirus genome appear to be segregated from the repressed gene regions by boundary or insulator elements, similar to that found on cellular chromosomes (d bloom, personal communication). these chromatin insulators appear to be able to protect genes in one region from the regulatory influence of adjacent regions through conserved ctcf motifs. herpesviruses also encode proteins such as lana and latency-associated transcripts (lat) that appear to regulate viral transcription during latency. , integration, and the site of integration, into the host chromatin is another mechanism that can regulate viral gene transcription. for example, viruses that integrate (ie, hiv) preferentially select chromosomal sites where high-level transcription of key transactivators is maintained. this is accomplished by viruses preferentially integrating in chromatin regions characterized by an open structure (a hallmark of actively transcribed genes). the process by which this is regulated is not completely clear, but it has been suggested that cellular proteins may interact with integrase, the viral protein that catalyzes the integration reaction, in a manner that is site specific. viral persistence increases the likelihood of chronic infection and replication, but under certain circumstances also contributes to increased risk of oncogenic transformation. this can occur through chromosomal instability and virus integration, and the ability of several specific viral proteins to bind and inactivate p or less frequently prb (fig ) . p is a checkpoint protein that interacts with cdk/cyclin inhibitors and p , p , and p to arrest the cell cycle in the g phase and can send signals for apoptosis through the regulatory proteins bax, bcl- , and c-myc. the clinical importance of p inactivation is exemplified in that persistent hpv infection is associated with an increased risk of developing cervical cancer in young women, and recent findings suggest that the persistence of hpv dna in treated tissue after cancer therapy is highly predictive of local recurrence. in this brief review, examples of mechanisms that contribute to survival and persistence of viruses within their host were presented. emphasis was placed on mechanisms 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virus latency insulators: many functions, many mechanisms a lat-associated function reduces productive-cycle gene expression during acute infection of murine sensory neurons with herpes simplex virus type integration site selection by retroviruses viral and host factors in human papillomavirus persistence and progression perspectives in studies of human tumor viruses human tumor suppressor p and dna viruses persistence of human papillomavirus infection as a predictor for recurrence in carcinoma of the cervix after radiotherapy key: cord- - xo jn authors: greninger, alexander l. title: a decade of rna virus metagenomics is (not) enough date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: xo jn it is hard to overemphasize the role that metagenomics has had on our recent understanding of rna virus diversity. metagenomics in the st century has brought with it an explosion in the number of rna virus species, genera, and families far exceeding that following the discovery of the microscope in the th century for eukaryotic life or culture media in the th century for bacteriology or the th century for virology. when the definition of success in organism discovery is measured by sequence diversity and evolutionary distance, rna viruses win. this review explores the history of rna virus metagenomics, reasons for the successes so far in rna virus metagenomics, and methodological concerns. in addition, the review briefly covers clinical metagenomics and environmental metagenomics and highlights some of the critical accomplishments that have defined the fast pace of rna virus discoveries in recent years. slightly more than a decade in, the field is exhausted from its discoveries but knows that there is yet even more out there to be found. in , using direct cloning of s rdna genes norman pace presciently noted that the method "seems to have no upper limit as to the complexity of the populations" (pace et al., ) . thirty years later, pace's maxim only seems ever more true. deeper and deeper sequencing technologies have enabled the discovery of thousands of viral species. metagenomic viral discovery can be applied to almost any sample type and novel viruses emerge. growth in virome and metagenomics papers is outpaced only by that of the viruses discovered ( fig. ). this review is meant to cover the field of modern-day rna virus metagenomics, where rna is perhaps best defined as "not dna", mostly due to the use of dna-specific nucleases in library preparations. many excellent reviews on viral metagenomics already exist, but no specific review focuses on rna virus metagenomics (bexfield and kellam, ; delwart, ; edwards and rohwer, ; mokili et al., ; rosario and breitbart, ; tang and chiu, ) . since there is absolutely no way to cover all the samples sequenced or genome organizations found even if one was restricted to a single order or family, the author apologizes in advance if he failed to cite your contribution to the field. where do we begin with rna virus metagenomics? construction and sequencing of cdna libraries share most of the features of rna viral metagenomic protocols and have existed since the late s (sim et al., ; efstratiadis et al., ) . one could argue about the metaor the −genomic nature of the cdna sequencing example, but the method is nearly unchanged since its introduction. a single organism contains multitudes and now one of the more common ways of discovering new viruses is from searching said cdna sequencing data (feng et al., ; shi et al., ; krishnamurthy et al., ) . clearly, the depth of sampling has changed in recent years but sequencing even two clones could be considered metagenomics. rna virus metagenomics began with a dna virus and a contaminant. in a prescient manuscript, allander et al. added dnase to spin-filtered serum prior to extraction to enrich for viral particles (allander et al., ) . nucleic acids were extracted and separately processed for dna and rna libraries and then randomly amplified, enabling the discovery of two bovine parvoviruses from the commercial bovine serum that was used to dilute their viral spike-in controls. surprisingly, the novel dna viruses were present in the rna half of the experimentthe side that was specifically reverse transcribed and amplified in order to test the protocol's ability to detect gb virus c (allander et al., ) . this experiment is worth focusing on as it foreshadowed several themes in the world of rna virus metagenomics. the protocol used in the paper is surprisingly similar to that used today (conceição-neto et al., ) . host or environmental background dna dwarfs most nucleic acids and sequencing of rna species circumvents the problem so well that it may be the best way to find high concentration dna viruses that are being actively transcribed. even today concerns over whether metagenomics is sufficiently sensitive bedevil its use as a gold standard (schlaberg et al., ; wylie et al., ) . finally, in the world of metagenomics, you do not know what is in your sample until you sequence, and the sensitivity of your assay is perhaps best measured by your ability to recover ubiquitous reagent contaminants (bhatt et al., ; naccache et al., ) . environmental rna virus metagenomics arose somewhat later. as recently as , degenerate pcr targeting the rna-dependent rna polymerase from picorna-like viruses had shown the potential for viral discovery in marine virus communities with the detection of four new rna virus families (culley et al., ) . large scale, high-profile efforts such as sailboat-based shotgun sequencing of the sargasso sea came two years later and used . - . μm filters that predominantly enriched for dna from microbial communities (venter et al., ) . three years later, robert edwards and forest rohwer were still calling for the development of methods that would allow for sequencing of rna viruses (edwards and rohwer, ) . allander's original method that detected the bovine parvovirus contaminants gave rise to clinical metagenomics in by discovering a novel human parvovirus (human bocavirus) and detecting a novel human coronavirus (coronavirus hku ) by screening pooled human nasopharyngeal aspirates (allander et al., ; woo et al., ) . reads to influenza a virus, human orthopneumovirus (rsv), metapneumovirus, human adenovirus, and ttv-like virus were also recovered from the reads. that same year , colony sequences from dnase-treated rna extracted from viral concentrates of human feces revealed a preponderance of pepper mild mottle virus and other plant viruses, but no new human viruses (zhang et al., ) . the first non-human environmental-focused rna viral metagenomic survey used dnase-treated extracted nucleic acids from nuclease-treated viral concentrates from coastal communities in british columbia (culley et al., ) . the resultant pure rna was reverse transcribed and double-stranded cdna was synthesized, blunt-end ligated, and cloned to reveal nothing, for there was not enough rna present even in - l of seawater, highlighting the importance of amplification in most any rna virus metagenomic preparation. after amplification, two novel picorna-like viruses and one novel +ssrna virus that remains taxonomically unassigned were discovered at high concentrations among the reads. it is incredible to note that only one year later, researchers were reporting rna viral metagenomic sorcery of honeybees with hundreds of thousands of reads (cox-foster et al., ; koonin, ) . nextgeneration sequencing and rapid bioinformatics gains allowed researchers unbelievable opportunities ( fig. ) (delwart, ) . due to the short read length of solexa sequencing at the time, initial viral metagenomic efforts required sequencing, which allowed for hundreds of thousands of reads a few hundred bases long (rothberg and leamon, ) . the result was an array of human viral discoveries, including merkel cell polyomavirus, human bocavirus , salivirus, lujo arenavirus, dandenong arenavirus, sfts bunyavirus, and a variety of new human astroviruses including mlb and va through va (briese et al., ; feng et al., ; finkbeiner et al., a finkbeiner et al., , b holtz et al., ; kapoor et al., a,b; koonin, ; li et al., ; meyer et al., ; palacios et al., ; yu et al., ) . groups often found the same viruses within days of each other and discrepant names for the same virus were common holtz et al., holtz et al., , kapoor et al., ; li et al., ) . pyrosequencing yielded impressive gains in environmental rna virus sequencing with profiling of a freshwater lake yielding tens of novel ssrna and dsrna viruses (djikeng et al., ). the rna virus metagenomic age was officially underway. . why has rna virus metagenomics been so successful? while early studies of metagenomics lamented the lack of rna viruses discovered compared to dna viruses, rna virus metagenomics publications by year for "rna virus metagenomics" (a) and "virome" (b) over the past decade using the "results by year" graph from pubmed. number of viral species (c), genera (d), and families (e) assigned by the ictv over the past four decades ("international committee on taxonomy of viruses (ictv), " n.d.). changepoint analysis with the "at most one change" statistical test was significant at year for genera and family data and for species data ("changepoint, " n.d.). a.l. greninger virus research ( ) - appeals to the viral discoverer for a number of reasons. the first is that success in viral metagenomics is often measured by the genetic distance of a newly discovered virus from other known viruses. while finding a new virus in the environment is not necessarily a problem in either dna or rna, the low fidelity of rna polymerases and the sequence space they are capable of sampling, along with the possibility of recombination, lend themselves to new species and genera that are the trophies of metagenomic viral discovery. these properties also favor the metagenomic library preparation process. sufficient homology to land pcr primers for amplicon-tiling library preparation approaches may not exist, and the number of oligos needed to cover the sequence space of a given rna virus such as hepatitis c virus can make hybrid capture approaches cost-prohibitive. de novo assembly methods that allow for the rapid reconstruction of near complete genomes have also dramatically improved over the past decade and can handle widely disparate coverage for each contig (grabherr et al., ; bankevich et al., ) . all of these properties often make metagenomics the first method of choice when trying to recover sequence from even a known rna virus (greninger et al., a; ogimi et al., ) . another reason for the provision of rna virus metagenomics is the ease and increased depth and sensitivity associated with removing background dna. indeed, one of the difficulties of rna virus metagenomics is that rna virus genome sizes are generally measured in the kilobases, but the size and quantity host dna background measures in the gigabases. if not partitioning sequence selectively across rna and dna, a single contaminating host cell can be the equivalent of half a million virions. removal of host dna is often required for one to recover complete rna virus genomes. the library preparation process simply requires a -min treatment with dnase i or benzonase to remove all background dna leaving the rna viruses behind. in addition, rna viral capsids are measured in nanometers and can be selectively enriched using . um or . um filtration. even if one is interested in dna viruses, rna metagenomics allows the recovery of mrnas that are associated with actively replicating dna viruses, which may be helpful information when so many dna viruses can be latent or integrated into host genomes (perlejewski et al., ) . recovery of these rna viruses is also made easier by the fact that, once host dna is removed, they can be present at extraordinarily high concentrations in total rna or constitute a surprisingly large proportion of transcripts in an mrna library, up to almost % of non-ribosomal rnas in some invertebrates (li et al., n.d.; shi et al., ) . based on total masses of nucleic acid in virus like particles, it has been estimated that half of marine viruses are rna viruses due to the larger burst size of eukaryotic rna viruses (steward et al., ) . a particularly instructive example was the discovery of the lake sinai virus in honeybees (runckel et al., ) . it was originally discovered and assembled metagenomically at such high titer that when researchers went to perform northern blots, they could see the viral rna staring back at them after rna electrophoresis. the author has also previously taken part in studies where the method of screening for known segmented rna viruses such as rotavirus was to run gel electrophoresis on extracted stool rna without amplification and look for banding (greninger et al., ; yu et al., ) . mining of publicly available transcriptome data has contributed greatly to the discovery of novel rna viruses (basler et al., ; schomacker et al., ) . indeed, genome-transcriptome studies of eurkaryotes sometimes miss the viruses present in their sequence when they map their rna-seq reads to their assembled dna-seq genome (babb et al., ; clarke et al., ) . rna viruses are lurking in the unaligned portion of the reads and often can be readily assembled and aligned to ncbi to find many new viruses in a given host (bekal et al., ) . most recently, six novel rna viruses were discovered in publicly available orb weaver spider transcriptome data (debat, ) . such sequences can be downloaded from the short read archive, assembled, and deposited into ncbi's third party annotation database ("ncbi third party annotation, " n.d.). research parasites of the world can now identify with their obligate intracellular parasitic brethren (longo and drazen, ) . rapid increases in sequencing depth achieved with next-generation sequencing have been matched by the increasing ease of library preparation. original viral sequencing protocols involved the preparation of multiple micrograms of nucleic acid and about a week of work (thurber et al., ; willerth et al., ) . protocols were slowly refined to reduce library preparation to a matter of days (greninger et al., ) . the development of transposon-based library creation and amplification have reduced rna metagenomic library preparation such that most labs perform the process in days, and when coupled to nanopore sequencing the entire sample-to-sequence process can be as little as h (greninger et al., , b . a typical rna virus metagenomic protocol entails filtration, nuclease treatment, double-stranded cdna synthesis, followed by nextera xt tagmentation and pcr amplification (alexander l. hall et al., ) . the benefit of the protocol is the minimal hands-on time, low cost given the dilutability of the transposase, ease of dual-indexing, and direct compatibility with illumina sequencers. the protocol can be performed in as little as - h and is amenable to automation (greninger et al., b (greninger et al., , c . variants include the use of additional pcr cycles or amplification prior to tagmentation. however, in the author's experience these are not necessary given the amplification step present in the tagmentation library preparation protocol and the possibility of extra pcr cycles creating coverage bias (conceição-neto et al., ) . steps to increase the amount of nucleic acid used as input are helpful for the retrieval of longer reads and reducing pcr jackpotting, although often practitioners move forward with the rna present. while no doubt the best way to enrich for viral like particles, and perhaps to help define a sequence as likely viral in origin, the use of ultracentrifugation is not necessarily required for viral metagenomics. rather ultracentrifuge is kept for defined "virome" work, especially where there is a high bacterial background such as feces (kohl et al., ; temmam et al., ; thurber et al., ) . indeed a direct headto-head comparisons of ultracentrifugation and filtration for viral particle enrichment found the main benefit of ultracentrifugation to be the removal of host dna background, which is less of a concern for rna viral metagenomics if extracted nucleic acids are dnase treated (kleiner et al., ) . as evidenced from the early history, discovery of new etiological agents of human disease drove the adoption of viral metagenomics (lysholm et al., ; tang and chiu, ) . clinical metagenomics, or direct shotgun sequencing from clinical samples with minimal sample processing, for diagnostic testing has largely been driven by the diversity of rna viruses that preclude the routine use of single-plex or even multiplex pcr for broad clinical detection. indeed, current rapid diagnostics followed by s or its pcr and sanger sequencing cover most of the actionable and more exotic organisms present in clinical specimens. while metagenomics delivered on the promise of finding novel human viruses, viral discovery in humans has increasingly become a tragic story of patients interacting with the wrong squirrel or tick on the wrong day and most samples sequenced are frankly negative (hoffmann et al., ; mcmullan et al., ) . in part because of the declining number of viral discoveries in humans, clinical metagenomics has taken on the mantle of detecting all known pathogens instead (naccache et al., ; wood and salzberg, ) . metagenomics has been especially successful at finding known rna viruses in unexpected sample types (doan et al., ; naccache et al., ) . provided there is sufficient coverage, it can also provide single nucleotide resolution to define strain relatedness for outbreak epidemiology and, where available, antiviral resistance (barzon et al., ; capobianchi et al., ; greninger et al., b greninger et al., , c . furthermore, the vast majority of clinical samples sent for clinical metagenomic sequencing are entirely negative, yielding a greater potential role for "ruling out" an infectious cause through metagenomic testing prior to treating for other diseases such as autoimmune disorders with immunosuppressant medication. the excitement over using clinical metagenomics for one definitive test for clinical microbiology is tempered by questions around its sensitivity, actionable-ness of the sequence recovered (including crosscontamination), and cost. since clinical labs are highly unlikely to perform ultracentrifugation, metagenomics likely has the best sensitivity in acellular environments such as cerebrospinal fluid rather than respiratory or stool specimens (schlaberg et al., ) . human cells become an interfering substance and at concentrations much lower than those that interfere with other clinical chemistry measurements (ranjitkar et al., ) . detection of viruses at low concentration such as zika virus make shotgun metagenomics problematic for a rule-out result in diagnostic virology (bingham et al., ; landry and st george, ; naccache et al., ) . as with other culture-independent testing, culture will likely be required to provide antimicrobial sensitivity. while early detection can reduce further diagnostic testing, most rna viruses in humans have no treatment and are essentially unactionable. the ease and ubiquity of rna virus metagenomics is perhaps best illustrated by the increasingly diverse samples that are sequenced. when sequence divergence is the metric of success, metagenomicists boldly go where no human has gone before. for the purposes of raw discovery, the use of extraordinary mixed samples allow for one-stop shopping and diversify discovery risk, albeit with the drawback that host species cannot be confidently defined. and the continuing need to fund and sell discovery from the basis of security has focused metagenomic efforts on species with high zoonotic pandemic potential (racaniello, ; temmam et al., ) . here, bat guano routinely ranks high on the rna virus metagenomic list as it checks all three requirements listed above. bats aggregate a wide array of arthropods, plants, and other animals and produce copious amounts of guano and have been associated with multiple zoonoses. cameroonian fruit bats, chinese bats, myanmar bats, hungarian bats, french bats, american bats, big brown bats, tricolored bats, and little brown bats have all been profiled showing a vast array of rna viruses from eukaryotic hosts (dacheux et al., ; donaldson et al., ; ge et al., ; he et al., he et al., , kemenesi et al., ; li et al., ; yinda et al., ) . bats have been used to put an upper limitin the hundreds of thousands − of the total number of eukaryotic viruses that remain to be discovered (anthony et al., ) . other insect and arthropod aggregators such as spiders and birds have also yielded many new rna virus species (debat, ; ducatez and guérin, ; shean et al., ; zhou et al., ) . the greatest paradigm shifter in recent viral metagenomics work has been the sheer number and diversity of novel rna viruses present in arthropods and invertebrates. yong-zhen zhang and team at the chinese cdc have been systematically profiling the transcriptomes of invertebrates to upend our understanding of animal virology. a haul of novel negative-stranded rna viruses from arthropods surpassed the known diversity of mononegavirales (li et al., n.d.) . these included the first circular negative stranded rna viruses ever found, which were part of a larger family of viruses (chuviridae) that may yet become an order given that it is phylogenetically ancient to existing segmented and unsegmented viruses and was arranged in a number of topologies. and yet those were just the negative-stranded viruses. when the team increased the number of hosts profiled to , they found novel rna viruses (shi et al., ) . the genomes found illustrated widespread recombination among rna viruses, viruses with multiple copies of structural genes, loss of structural genes, as well as transfer of genes between virus and host. indeed, these discoveries so dwarf all previous rna virus discoveries, especially in the picorna-like superfamily, that the paper itself would be worth reprinting here in full. three additional novel families of positive-stranded viruses and two novel families of negative stranded viruses were described. rather than detailing what was found, the surprises are the families in which novel rna dependent rna polymerase (rdrp) sequences were not found such as the arenaviridae, picornaviridae, hepeviridae, paramyxoviridae, arteriviridae, and secoviridae. of course, whole undiscovered taxa basal to these taxa were also recovered. perhaps most impressively, zhang's team finished complete genomes with rapid amplification of cdna ends (race) for each of these viral genomes, a feat that not many metagenomic viral discovers undertake when finding more than a few novel viruses. no doubt evolutionary rna virologists will have just begun to comb through this data trove before the next ten thousand whole genomes of novel rna viruses is deposited from peat moss. most importantly for veterinarians, rna virus metagenomics has revealed a number of candidate etiological agents for a variety of animal ailments including avian proventricular dilatation disease, mink shaking syndrome, snake inclusion body disease, python respiratory illness, dairy cow disease, and tilapia die-offs (kistler et al., ; gancz et al., ; blomström et al., ; stenglein et al., stenglein et al., , uccellini et al., ; hoffmann et al., ; bacharach et al., ) . animal feces has proven fruitful hunting for metagenomicists, with canine, feline, porcine, equine, goose, duck, and shrew fecal rna viromes turning up a plethora of mostly novel picorna-like viruses, albeit with an unclear relationship to disease and perhaps some more closely linked to the invertebrates indicated above (fawaz et al., ; greninger and jerome, ; li et al., ; moreno et al., ; nagai et al., ; ng et al., b; phan et al., ; reuter et al., ; sano et al., ; sasaki et al., ; zhang et al., ) . the recovery of ancient northwest territories cripavirus from -year old frozen caribou feces illustrated an incredible stability of encapsidated rna (ng et al., a) . screens for fish viruses are still in their infancy and yet the phylogenetic diversity of fish and perhaps their intense exposure to marine rna viruses may prove them to be the chordate arthropods of viral discovery. novel members of the orthomyxoviridae, picornaviridae, and reoviridae have recently been described in fish (bacharach et al., ; reuter et al., reuter et al., , . our understanding of reptile and amphibian viral diversity is also still in its early days. the discovery of a non-old world, non-new world arenavirus in boid snakes with an envelope protein most closely related to filoviruses proved a tantalizing hint toward new variations on old taxonomies (stenglein et al., ) . for the basic scientists, a number of novel rna viruses have been discovered in model organisms such as c. elegans or d. melanogaster (félix et al., ; webster et al., ) . thousands of new viruses have been found in plants through metagenomics, with most awaiting further characterization or even finished assembly (roossinck et al., ) . plant virus metagenomics is probably one of the more fertile areas of growth for viral discovery in the coming years given their incredible biodiversity (roossinck, ) . rna viral metagenomics has made a number of contributions to our understanding of viral diversity at the far reaches of life. sequencing of a hot, acidic lake and wastewater revealed a novel ssdna virus with an rna virus-like capsid protein, suggesting past recombination between dna and rna viruses (diemer and stedman, ; "rdhv-like virus sf replication-like protein and capsid protein genes, complete cds," ). while there are still no definitive rna viruses of archaea, viral rna metagenomics isolated a . kb contig that contained an capsidlike protein and an rna-dependent rna polymerase that differed from the rdrps of viruses from eukaryotes and bacteria (bolduc et al., ) . the same can be said of the ciliates, who were previously noted among the eukaryota for having no known rna viruses (koonin et al., ) . metagenomic sequencing of wastewater after a rainstorm revealed two rna contigs that were highly suggestive of ciliate rna viruses in a metagenomic background of tetrahymena and several viruses best translated in the ciliate genetic code were found in the invertebrate surveys detailed above (greninger and derisi, a; shi et al., ) . given the incredible growth in bacteriophage diversity from metagenomics, rna phages were a surprisingly late arrival to the metagenomic discovery party. as recently as , genome sequences from rna phages were surprisingly few and far between with only ssrna and dsrna genomes compared to over dna bacteriophage genomes from species (greninger and derisi, b; krishnamurthy et al., ) . mining of metagenomic datasets yielded an additional partial genomes from novel rna phages covering > novel species including the first known rna phage number of a gram-positive bacteria (krishnamurthy et al., ) . these sequences included multiple unique genomic arrangements as well as orfs that had no similarity to known proteins. invertebrate virus sequencing efforts detailed above also found a similar number of levi-like viruses (shi et al., ) . given the many uses of the ms phage coat protein for understanding rna-protein interactions and as a tool for bioengineering rna affinity purification, the expansion of the rna phageome will likely create a number interesting tools for molecular biology that can build on ms . the ridiculous number of virus discoveries in the past several years has put an incredible strain on the whole system of viral taxonomy (fig. c-e) . the inclusion of uncharacterized metagenomic viral sequence in taxonomy has long been of some debate at the international committee on taxonomy of viruses (ictv), the adjudicator of novel families, genera, and species of the viral world. only this past year did the ictv codify its existing policy through issue of a consensus statement on the inclusion of metagenomic data in its consideration of taxonomical placement of viruses . the torrid pace of discoveries has forced the hand of viral discoverers to come up with euphonious names for viruses for which there is almost no biological understanding ("international committee on taxonomy of viruses (ictv), " n.d.). convention previously dictated something like location, host, and number, even if the world health organization now recommends against ruining the tourist economy along the ebola river or in coxsackie, new york (fukuda et al., ) . since sequence similarities govern our understanding of genomic function, there is a temptation to name based on homology and let past discoveries anchor the novel (e.g., picobirnavirus or dicipivirus/cadicivirus/picodicistrovirus) (woo et al., ) . default nomenclature is approaching that of drug manufacturers or therapeutic antibody naming, with alternating consonant-vowel pairs that might have some basis in latin or relation to the location but obscures the actual place (e.g. avisivirus, aquamavirus, hunnivirus, harkavirus) reuter et al., ) . recently, five ancient chinese states formed the basis for naming novel family lineages (shi et al., ) . detection of ancient recombination in rna virus sequences is revealing genomic abominations that only a liger could love. what do you get when you cross a picornavirus and a calicivirus? probably a non-functional, nonreplicating piece of rna, but picalivirus a-d are still a thing (greninger and derisi, c; ng et al., ) . tombusviridae and nodaviridae? tombunodavirus (grasse and spring, ; greninger and derisi, d) . the inventiveness of nomenclature for novel virus discovery in a space of anarchy combats a steady march of rules and reason by the ictv . not even clinically relevant viruses such as human parainfluenza viruses or respiratory syncytial virusesnow human respirovirus, rubulavirus, orthopneumoviruscan resist binomial nomenclature and taxonomical reassignment (adams et al., ) . however, if discoverers get out far enough of the rationalization, their original names can take advantage of the movement to have an outsized influence on viral nomenclature runckel et al., ; woo et al., ) . in a field with seemingly no end to frontiers, it is curious to define even more. no doubt the 'virtuous cycle' of metagenomic sequencing will only increase in breadth and depth in the coming years with a concomitant growth of new bioinformatic algorithms leading to discovery of even more new organisms and further decrease in so-called unalignable viral "dark matter" (fig. ) krishnamurthy and wang, ) . turning the crank on rna virus discovery metagenomics is now the provenance of undergraduate theses shean et al., ; zaaijer et al., ) . other than sampling more broadly and maintaining the exponential growth in genbank, what are orthogonal challenges for future rna virus metagenomic studies? fig. . the conjoined circles of metagenomic success. the increased output of modern-day sequencers has led to increased metagenomic sequencing of samples, both environmental and clinical. this in turn has led to an explosion in the ncbi genbank and wgs databases. new discoveries beget the discovery of more divergent new viruses and organisms as they are now alignable to new references in the database. rather than searching through gapped alignments, the increased coverage of the genbank reference database allows for more exact k-mer searching, which allows for faster, more sensitive alignments of reads. this in turn makes metagenomic sequencing more useful, especially in the clinic, which in turn begets more sequencing. a.l. greninger virus research ( ) - . . easier, faster, better, broader protocols to start in the wet lab, better methods of host depletion and recovery of full viral genomes are absolutely required. too many analogies to "finding a needle in a haystack" in the literature necessitate the use of cellulases (allen et al., ; kowalchuk et al., ; lax and gilbert, ; lecuit and eloit, ; naccache et al., ; soueidan et al., ) . even if rna viral transcripts constitute > % of an arthropod, they rarely do so in mammalian tissue (feng et al., ; shi et al., ) . in both clinical metagenomics and complex mixtures there are always low concentration viral sequences, either due to time of sampling, viral biology, or the long tail of ecological abundance, which deeper sequencing alone may not solve. synthetic biology and programmable nucleases may allow new options for depletion beyond the ribosome although current efficiencies must be improved (gu et al., ; matranga et al., ) . even though sample preparation methods have become considerably easier in the past decade, they still take several hours of hands-on time and have not routinely been ported to automated liquid handlers. with plunging sequencing costs, library preparation costs for rna virus metagenomics have taken a front seat. the time and cost of library preparation hamper adoption of metagenomics in the clinical virology lab. similar to the expanding suite of cas programmable sequence-guided nucleases, an rna-directed transposase for adapter tagging followed by one-step, dual-indexed amplification would speed up library preparation as the bulk of time in current preparations is spent on double-stranded cdna synthesis (gertz et al., ) . such an enzyme might be discovered through the approaches highlighted here, or perhaps through directed evolution of existing dna transposases (adey et al., ) . another solution might be a one-pot mix of enzymes that performed library preparation in a similar fashion as gibson cloning. even in a host-free environment, current amplification methods do not recover full viral genomes from end-to-end. instead, the common paradigm is random priming with amplification followed by a follow-up step of race to recover ends (li et al., n.d.; shi et al., ) . this second step is highly laborious, especially for segmented rna viruses, and difficult to execute on viral sequences at low concentration or in complex mixtures. furthermore, current transposase-based library methods can produce inversions or sequencing artifacts at the end of genome segments or in areas of rna secondary structure. as "unbiased" or "agnostic" as metagenomics can be, we still rely on sequencing by synthesis and a four base read-out. the ultimate immunoevasion strategy may be virally-encoded nucleotides (bryson et al., ; murphy et al., ; weigele et al., ) . host and virallyencoded rna editing already bedevils calling of accurate whole genome sequences in both positive-and negative-strand rna viruses (park et al., ; pelet et al., ; piontkivska et al., ; vidal et al., ) . adenosine-based modifications of viral genomes have shown effects on viral and host biology, but detecting them requires additional modifications to most sequencing protocols (gokhale and horner, ; gonzales-van horn and sarnow, ; kennedy et al., kennedy et al., , . our understanding of rna base modifications in viral genomes is still in its infancy. here, nanopore-based approaches to sequencing may hold the key to new discoveries as they can directly detect modified nucleotides, although current approaches are as dependent on the biology of pore proteins as we are on polymerases (ayub et al., ; carlsen et al., ) . the ultimate answer may be direct mass spectrometry-based detection of viral nucleotides (gooskens et al., ; cobo, ) . improvements in sensitivity of nanopore sequencing in terms of sequencing depth and the host depletion strategies highlighted above are required before nanopore-based metagenomics becomes routine or meaningful . other than the retroviruses, rna viruses do not readily provide host linkage information in metagenomics like dna viruses and phages often do. linking rna virus and host has been imputed based on transcript levels or dinucleotide usage, although the latter has recently been shown to more correlated with viral family than host species (kapoor et al., ; giallonardo et al., ; shi et al., ) . even when sequencing discrete organisms such as a singular honeybee, the co-existence of eukaryotic parasites means that imputation of viral host cannot be exact (runckel et al., ) . host promiscuity seems to be a common theme among newly-described rna viruses (nunes et al., ) . highly-indexed libraries, chemical linkage of rna species, and physical partitioning through single cell transcriptomics currently provide the best potential solution to the host imputation problem in complex mixtures (burton et al., ; chow et al., ; turner et al., ) . to date, most metagenomics has adopted the th century british naturalist approach of cataloging inventory and diversity. this focus of the field has been somewhat punishing for those involved, with burnout of both scientists and reviewers from not fully understanding the why of it all (canuti and van der hoek, ) . this author does not necessarily have a better plan, but perhaps phenotype might be a reasonable start. mass spectrometry was available to previous generations of biochemists, but they did not necessarily stop to catalog the contents of every fraction, focusing instead on function. this is not to understate the critical impact of the technical training that comes from viral metagenomics, nor to minimize the power of the method. but once we know that viruses are diverse, some concern for function is most likely in order. although they are now no doubt contributing, viral metagenomicists have previously borrowed liberally from the biochemical functions assigned to strings of sequence without confirmation. genomics has been the wires that allowed biochemistry to scale ( , ). a number of options for functional characterization of these viruses are available but they unfortunately require work. additional culture models are needed to handle all the new discoveries and genetic engineering methods for more easily establishing cell lines from exotic species are worth pursuing (ettayebi et al., ; finkbeiner et al., ; stenglein et al., ; bell-sakyi and attoui, ; janowski et al., ) . focusing discovery on genetically tractable organisms has allowed for relatively rapid functional studies of the new discovered c. elegans orsay virus jiang et al., ) . a number of culture-independent methods exist now to characterize the novel viruses and their genes. biochemical assays for rdrp, proteases, helicases, capsids, and internal ribosome entry sites all exist and synthetic biology allows for easy cloning from a database to test these new proteins found through metagenomics (ladd effio et al., ; o'donoghue et al., ; peersen, ) . profiling known functions across the new viral species, such as rna binding strength and sequence specificity of novel rna phage proteins related to ms phage or viral protease specificity and kinetics, might be a place to start (o'donoghue et al., ) . affinity purification-mass spectrometry is a powerful method that allows discovery of viral-host protein-protein interactions in the absence of culture (jäger et al., ; greninger et al., ; medina et al., ; greene et al., ) . for vertebrate viruses, serological assays have long been used to confirm roles in disease (o'sullivan et al., ; bao et al., ; coller et al., ) . the ubiquity of these viruses also leads to questions of how we publish in the absence of experiments and what a sequence is worth. the number of novel viruses discovered needed for a high-profile paper has increased by logarithms (krishnamurthy et al., ; shi et al., ) . in the absence of particular phenotypic data or wet-lab viral characterization, many authors have turned to genome announcements, biorxiv, or simply uploading to genbank with extra metadata, figuring that sequencing is the most likely future method of both detection and discovery (debat, ; greninger and derisi, e,f; karamendin et al., ; sharman et al., ; sparks et al., ) . given the glut of new viruses, expansion of sequencing, and the time it takes to publish, scientists may be more likely to align to your novel virus rather than read about it in a journal and decide to screen for it. this method has allowed for rapid sharing of data and de facto global screens of novel rna virus prevalence, tropism, and evolution that then lead to something resembling a story. perhaps the most difficult challenge to spring from sequence gazing at all the novel rna viruses is our understanding of the origins of rna viruses. at what point in host evolution does an rna viral pathogen arrive? and what exactly did the host look like then? rna virus metagenomics still does not provide the answer. results from initial rna virus genomics work suggested picorna-like viruses predated the radiation of the five supergroups of eukaryotic organisms (koonin et al., ) . since then, hints of genetic exchange between hosts and viruses, prominent in dna virus and bacteriophage genomics, are showing up in rna viruses (hughes and stanway, ; sasaki and taniguchi, ; shi et al., ; staring et al., ) . accounting for each of the domains commonly present in rna viruses is a challenge for any origin story, though greater knowledge of domain organization and swapping between different rna viruses may help firm up these dates (koonin et al., ) . biochemical characterization of the incredibly diverse rna viruses discovered through ongoing metagenomic screens will also test theories of the origin of rna viruses and better date the origin of different rna virus clades relative to different epochs of eukaryotic evolution. recent genetic and proteomic screens of eukaryotic rna viruses have indicated key reliance on host lipid modifying enzymes that regulate vesicular transport sasaki et al., ; greninger, ; carette et al., ; marceau et al., ; borawski et al., ; berger et al., ; arita et al., ; nagy and pogany, ; xu and nagy, ; salloum et al., ) . membrane repurposing is a critical determinant of rna virus replication which should be added to the "hallmark genes" of rna viruses, though they currently escape sequence alignment profiling (koonin and dolja, ) . these results have led to a hypothesis in which part of the rna virus origins involve selfish vesicular transport that likely expanded with the rise of intracellular eukaryotic transport (greninger, ; koonin et al., ; kuehn and kesty, ) . the list of rna viruses that have acquired a cellular envelope continues to grow feng et al., ; mcknight et al., ) . new concepts in bacterial vesicular transport might be consistent the close evolutionary link between the eukaryotic rna virus polymerases and bacterial retroelements (jan, ; kuehn and kesty, ) . to put it scientifically, it is nuts that all this and much more have happened 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in febrile and afebrile children enrichment of phosphatidylethanolamine in viral replication compartments via co-opting the endosomal rab small gtpase by a positive-strand rna virus parallel evolution of influenza across multiple spatiotemporal scales highly diverse population of picornaviridae and other members of the picornavirales, in cameroonian fruit bats discovery of a novel polyomavirus in acute diarrheal samples from children using mobile sequencers in an academic classroom rna viral community in human feces: prevalence of plant pathogenic viruses changepoint: an r package for changepoint analysis | killick identification and genome characterization of the first sicinivirus isolate from chickens in mainland china by using viral metagenomics the author would like to thank keith jerome samia naccache, sue greninger, and ryan shean for helpful comments. key: cord- - kg oe g authors: agol, vadim i.; gmyl, anatoly p. title: viral security proteins: counteracting host defences date: - - journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: kg oe g interactions with host defences are key aspects of viral infection. various viral proteins perform counter-defensive functions, but a distinct class, called security proteins, is dedicated specifically to counteracting host defences. here, the properties of the picornavirus security proteins l and a are discussed. these proteins have well-defined positions in the viral polyprotein, flanking the capsid precursor, but they are structurally and biochemically unrelated. here, we consider the impact of these two proteins, as well as that of a third security protein, l(*), on viral reproduction, pathogenicity and evolution. the concept of security proteins could serve as a paradigm for the dedicated counter-defensive proteins of other viruses. supplementary information: the online version of this article (doi: . /nrmicro ) contains supplementary material, which is available to authorized users. all bona fide rna viruses encode at least two proteins, a capsid protein and an rna-dependent rna polymerase (rdrp). in this article, we do not consider 'abnormal' rna viruses such as hepatitis delta virus, which does not possess its own replication machinery and borrows a capsid from another virus , or the capsid-less narnaviruses, which encode only an rdrp ; hepatitis delta virus and narnaviruses are closer to viroids and plasmids, respectively, than to fully fledged rna viruses. rna viruses encoding only a capsid and an rdrp are known to infect protists such as giardia spp. . some rna phages and fungal rna viruses express only three proteins. however, most rna viruses do not rely on such a scant protein repertoire. the proteomes of the majority of rna viruses comprise up to different proteins, with the genomes of some coronaviruses encoding nearly proteins. to infect, viruses must reach an appropriate intracellular environment and, if necessary, adapt this environment to their own requirements. thus, viral infection requires not only replication but also interactions with host defences. to carry out these tasks, rna viruses have only a few proteins at their disposal, with the available protein arsenal being limited by the genome size. as the proteomes of these viruses are strikingly small and specific viral functions generally require more than one protein, most proteins encoded by rna viruses are multi functional. at the same time, however, there is a certain division of labour between viral proteins. we consider some aspects of this problem using, as an example, the picornaviruses, a large family of small animal viruses with a medium-sized, single-stranded, positive-sense rna genome (box ) . although they share a common genome organization, these viruses exhibit sufficient genetic variation to be separated into at least distinct genera (box ) . the family includes important human and animal pathogens that cause a range of disorders, including poliomyelitis, foot-and-mouth disease, the common cold, gastroenteritis, hepatitis, meningitis, myocarditis and uveitis. in addition to acute diseases, these viruses can also cause chronic persistent disorders. of the 'mature' (fully processed) picornaviral proteins (box ) , the most ancient group includes the capsid proteins and a set of conserved proteins considered to be picornavirus signature proteins , comprising the rdrp ( d pol ), the primer for rna synthesis (vpg), a protease ( c pro ) and an atpase ( c atpase ). the capsid and signature proteins are indispensable for viral viability. two less conserved proteins, b and a, are also essential for viability but are less directly involved in viral reproduction. the main functions of these two proteins are to target replicative proteins to the correct destinations and aid in the creation of a suitable replicative niche. finally, two other non-structural proteins that flank the capsid precursor in the polyprotein molecule, the leader (l) and a proteins, constitute a distinct group, although they have no common structural or biochemical features. they are the most variable among the picornavirus proteins, and some viruses even lack l altogether (fig. ; tables , ) . this group also includes the so-called l* protein, which is encoded in a different reading frame of the rna of certain cardioviruses. functionally, these proteins (l, l* and a) have been characterized in some detail for only a few picornaviruses, and in these cases their major function has been shown to be counteracting host defences, thereby ensuring optimal conditions for viral reproduction. in this review we argue that picornaviral l and a proteins that have yet to be characterized are likely to fulfil the same biological role. we have proposed that this group of proteins should be called security proteins . they are sometimes also referred to as virulence factors ; such a designation, although it may be more familiar, is somewhat less preferable to us for the reasons explained below. viruses have no special 'desire' to be virulent, that is, to harm, even less to kill, their hosts. the pathogenic properties of a virus are not a prerequisite for viral fitness. in fact, the most severe harm in a viral infection comes not from viral reproduction but from the (sometimes miscalculated) host defence response. host damaging or even suicidal defensive reactions include rna degradation, inhibition of translation, and the induction of endoplasmic reticulum stress, apoptosis, autophagy and inflammation, and these reactions are aimed at limiting viral reproduction and spread. as a natural response, viruses have evolved tools directed not only at overcoming the specific innate and adaptive immune responses but also at inhibiting the general host metabolic functions on which these specific defences are based -that is, processes such as transcription and translation, cell signalling and intracellular trafficking. inhibiting these processes is the major function of the security proteins. accordingly, the capacity to withstand host defences, rather than virulence (the ability to harm the host) per se, is the property that is selected for during viral evolution. the long-term co-evolution of a virus and its host will probably result in their mutual adaptation accompanied by a decrease in viral virulence. thus, in our opinion, the term 'security protein' is a better reflection of the evolutionary origin of the relevant proteins than the term 'virulence factor' . moreover, the possession of efficient security proteins does not necessarily make a virus particularly virulent. the aim of this review is to consider the properties and biological significance of picornavirus security proteins and, on the basis of this knowledge, to put forward a general view of security proteins as dedicated counterdefensive proteins that evolved primarily to overcome various mechanisms of host resistance. both l and a are extremely heterogeneous with respect to size, sequence and biochemical properties (fig. ; tables , ). the length of l varies from ~ amino acid residues in cardioviruses to ~ residues in some sapeloviruses, and the variation is striking even in a single genus; for example, in sapeloviruses, the length varies from ~ residues (one l protein) to ~ residues (two l proteins). although certain sapeloviruses seem to express two l proteins (supplementary information s (figure)), approximately half the picorna virus genera lack l altogether. the length of a varies ~ -fold (from residues in senecaviruses to ~ residues in avihepatoviruses and some sapeloviruses), and it can comprise one to three separate polypeptides. the l proteins of cardioviruses and klasseviruses are strongly acidic and markedly basic, respectively, whereas the a proteins of these viruses exhibit the reverse ratio of acidity. the overall charge of the proteins will influence the choice of their interaction partners. the primary structures of l and a are, in most cases, totally unrelated when compared across different genera, and even intra-genus conservation among seemingly homologous proteins can be low, as is the case, for example, with the l proteins of kobuviruses and a proteins of cardioviruses and sapeloviruses. with regard to their biochemical activities, only a few security proteins have been characterized in any detail. a of enteroviruses ( a pro ) is a protease with a chymotrypsin-like fold in which the catalytic serine residue has been replaced by cysteine . a pro performs an essential function in viral reproduction through its involvement in the so-called 'primary' co-translational cis-cleavage of the polyprotein between the amino-terminal amino acid of a pro and the carboxy-terminal residue of the capsid protein vp . aphthovirus l is also a protease (l pro ) with a papain-like fold . translation of aphthovirus rna can start at two in-frame aug codons, generating distinct l proteins, lab (~ residues) and lb (~ residues) , with lb predominating in vivo. l pro cleaves the bond at the l pro -vp boundary , . erbovirus l pro exhibits similar picornaviruses possess a single-stranded positive-sense rna genome (see the figure) comprising approximately , - , nucleotides. the rna is packaged into an icosahedral capsid usually composed of four distinct proteins (vp -vp ), but in some viruses vp and vp are fused (forming vp ). after productive contacts with specific cellular receptors, which differ between viruses, the genome is uncoated and enters the cytoplasm, where the main steps of viral reproduction occur. the viral rna, which contains a single orf (with one exception described in the main text), is translated in a cap-independent manner into a , - , amino acid polypeptide, which is eventually processed by limited proteolysis into a dozen 'mature' proteins. these proteins include: capsid proteins; an rna-dependent rna polymerase ( d pol ); a protein (vpg, or b) that serves as a primer for the initiation of rna synthesis; an atpase with a conserved superfamily helicase motif ( c atpase ) and an essential but poorly defined role in viral rna replication; a chymotrypsin-like protease ( c pro ), which, as the mature protein or as a precursor, is a major factor in polyprotein processing; two hydrophobic membrane-binding proteins ( b and a) that participate in the generation of a virus-friendly environment; and, flanking the precursor of the capsid proteins, one or two highly variable proteins (l and a), the structure and functions of which are the subject of this review. the entire coding region of the rna is surrounded with untranslated regions (utrs) harbouring regulatory elements that control viral translation (for example, the internal ribosome entry site (ires)) and replication (the origins oril and orir), and this entire rna sequence is flanked by the covalently linked viral protein vpg and a poly(a) tract at the ′ and ′ termini, respectively. there are four structurally and functionally distinct types of ires and many types of oril and orir. the mode of translation for which initiation is dependent on the presence of the so-called cap structure (m gpppn) at the ′ end of an mrna. specific cap-binding proteins (translation initiation factors) recruit the ribosome to the ′ end of the mrna, and the ribosome scans the template until it encounters the initiation codon. this translation mode is exploited by most mrnas in eukaryotic cells. a protein that binds to the ′ poly(a) tail of eukaryotic mrnas on the one hand and to cap-dependent translation initiation factors on the other hand. this dual binding results in a non-covalent circularization of the mrna template, which is accompanied by a significant increase in translation efficiency. enzymatic activity . owing to the presence of a characteristic motif, the a proteins of some sapeloviruses have also been proposed to be proteases . a of aphthoviruses is a short peptide that promotes co-translational interruption of polyprotein synthesis just downstream of its own coding sequence (and before the b moiety); cleavage at the aphthovirus vp - a boundary is accomplished by c pro . interruption of polyprotein synthesis (also known as ribosome skipping) is proposed to be caused by an interaction between the a peptide and the exit tunnel of the ribosome, preventing the interaction of the ribosome with pro-trna (proline is the amino-terminal residue of aphthovirus b) , . to achieve this, a short peptide harbouring an npg(p) motif (the parentheses mark the interruption site) is sufficient. short a peptides from erboviruses , teschoviruses , senecaviruses and cosaviruses also possess npg(p) motifs and seem to be involved in polyprotein processing at the a- b border. separation of these peptides from the vp capsid protein has been assumed but not yet clearly demonstrated. as these peptides possess no other known activities, their assignment as security proteins is not warranted; we refer to them here as a sp ( a short peptide). the ribosome-skipping npg(p) motif is also present in some 'composite' a proteins, such as those of ljungan virus (a parechovirus), avihepatoviruses and seal picornavirus type (ref. ), in which they seem to ensure self-separation from the downstream a moieties. in cardiovirus a, the npg(p) motif is located at the c terminus and interrupts polyprotein synthesis at the a- b boundary , which corresponds to the polyprotein 'primary' cleavage site in this genus . apart from the npg(p)-containing peptides, a proteins of cardioviruses, avihepatoviruses, ljungan virus and seal picornavirus type contain distinct but poorly characterized sequences. the cardiovirus encephalomyocarditis virus (emcv) encodes a a protein with rnabinding affinity . the bipartite a of ljungan virus, the tripartite a of avihepatoviruses and the npg(p)lacking a proteins of kobuviruses, tremoviruses and some parechoviruses all have a ~ - -residue h-nc domain, which contains a histidine with a downstream asparagine-cysteine dipeptide and a putative transmembrane domain , . the observation that a similar h-nc domain is present in certain cellular tumour suppressors suggests that these viral proteins are also involved in the control of host activities. between the npg(p) and h-nc motifs, a of the avihepatovirus duck hepatitis a virus possesses an additional moiety that contains the so-called aig domain , which is also found in representatives of the ras-like gtpase superfamily. cardiovirus l proteins , which are devoid of any enzymatic activity and exhibit noticeable intra-genus variability (table ) , contain a non-classical but functional zinc finger (cys-his-cys-cys) motif , and a downstream acidic motif that, in some of these l proteins, contains potential phosphorylation sites , . certain strains of theiler's murine encephalomyelitis virus (tmev) are unique among picornaviruses in expressing a functional protein, l*, that is encoded in an alternative translation frame , that starts within the l coding sequence, goes through the vp coding sequences and terminates in the vp coding sequence of the main reading frame. the a and l proteins of other picornaviruses neither contain easily identifiable amino acid motifs nor have known specific biochemical activities. effects on general host metabolism security proteins contribute substantially to the shut-off of host macromolecular synthesis that occurs in response to infection with many picornaviruses. one could perhaps argue that such effects of security proteins contradict the key proposal of this review that these proteins are dedicated to counter-defensive functions. however, as already mentioned, inflicting harm on their hosts does not bring viruses any benefits per se. the only reason (or at least, the main reason) why viruses evolved the ability to damage infected cells is their need to incapacitate the cellular defensive machinery. this machinery includes several specific mechanisms (such as innate and adaptive immunity), the implementation of which requires general cellular functions such as translation, transcription and controllable nucleocytoplasmic trafficking. therefore, virus-induced impairment of these all-purpose metabolic functions can be regarded as a component of the viral counter-defensive strategy. the effect of security proteins on cap-dependent translation of cellular mrna is particularly important. a pro from diverse enteroviruses [ ] [ ] [ ] [ ] and l pro from aphthoviruses , cleave eukaryotic translation initiation factor eif g. interestingly, erbovirus l pro does not seem to cleave eif g and does not trigger translational shutoff . poly(a)-binding protein (pabp ; also known as members of the picornaviridae family are small (~ nm), non-enveloped animal viruses with similar genome organizations and mechanisms of reproduction (box ) . this family contains important human and animal pathogens, studies of which have made crucial contributions to our understanding of the molecular biology of viruses and the mechanisms of virus-cell interactions. the first animal virus (foot-and-mouth disease virus; fmdv) and the first human virus (poliovirus) to be discovered belong to this family. the current official classification of picornaviruses (see the international committee on taxonomy of viruses) includes genera: aphthovirus (for example, fmdv), avihepatovirus (for example, duck hepatitis a virus), cardiovirus (including important model viruses such as encephalomyocarditis virus, mengovirus and theiler's murine encephalomyelitis virus, as well as human and animal pathogenic viruses such as the newly recognized saffold virus), enterovirus (a large genus that includes poliovirus and many other human and animal pathogenic viruses, such as coxsackieviruses, echoviruses and other 'numbered' enteroviruses, as well as rhinoviruses, the causative agents of the common cold), erbovirus (equine rhinitis b virus), hepatovirus (hepatitis a virus), kobuvirus (for example, aichi virus, which induces gastroenteritis in humans, as well as some animal pathogens), parechovirus (the causative agents of various human diseases, particularly in children; also isolated from rodents), sapelovirus (including an avian, a porcine and a simian pathogen), senecavirus (seneca valley virus, a candidate oncolytic agent), teschovirus (a porcine pathogen) and tremovirus (avian encephalomyelitis virus). in addition, there are two candidate genera, not yet officially approved, that are associated with human diarrhoea: cosavirus and klassevirus. finally, seal picornavirus type is a distinct virus not assigned to any of the above genera. several new picornavirus genera have been identified recently, and it seems plausible that this number will grow, particularly with the breakthroughs in sequencing techniques and the achievements of metagenomics. cytoplasmic pabp), a host protein that is also involved in translational control, is another target of enterovirus a pro (refs , ) . security proteins can inhibit host translation by mechanisms other than proteolysis. mutations of cardiovirus a (which is not a protease) alleviate virus-induced translational shut-off , . it is thought that association of cardiovirus a with ribosomes , , which seems to take place in the nucleolus , and possibly occurs through the rna-binding activity of a , might contribute to preferential use of the internal ribosome entry site (ires)-dependent viral templates. cardiovirus l was also reported to mediate translational shut-off . however, this effect could largely be due to l-mediated inhibition of nuclear export of mrna rather than to inhibition of translation per se , . on the basis of ectopic expression experiments, hepatitis a virus a has also been implicated in inhibition of cap-dependent translation , but the relevance of this observation is uncertain, as hepatitis a virus does not exert translational shut-off. the effects of security proteins on cellular transcription are less well studied, although synthesis of the mrna for cytokines and chemokines is inhibited in certain cases (see below). individually expressed poliovirus a pro was reported to cleave the general transcription factors tata-box-binding protein and cyclic amp-responsive element-binding protein (ref. ), but the biological significance of these effects is debatable , . poliovirus a pro cleaves gemin (also known as ddx ), a protein that is involved in the formation of spliceosomes . cardiovirus a has also been implicated in virus-triggered inhibition of host transcription , but this effect was not investigated further. foot-and-mouth disease virus (fmdv) l pro (refs , ) and human parecho virus a accumulate in the nucleus during the course of infection and as a result of ectopic expression, respectively, but their nuclear effects are unknown. enterovirus a pro cleaves some cytoskeletal proteins, such as cyto keratin (ref. ) and dystrophin, a protein that connects the cytoskeleton to the plasma membrane . the security proteins of several picornaviruses profoundly affect nucleocytoplasmic transport in infected cells. the targets of enterovirus a pro include nucleoporins, which are nuclear pore components that control nucleocytoplasmic exchange , . as a result, bi directional passive diffusion through the nuclear envelope is facilitated. another manifestation of the perturbation of intracellular trafficking is the suppression of nuclear export of mrnas, ribosomal rnas and u spliceosomal small nuclear rnas . passive nucleocytoplasmic diffusion of proteins is also facilitated by the cardiovirus l protein , , and this effect seems to be caused by l-triggered phosphorylation of nucleoporins , , . effects on innate immunity and viral pathogenicity one major consequence of the effects of security proteins on host metabolism is the downregulation of innate immunity. fmdv l pro suppresses interferon production , and action , , largely through the inhibition of nuclear factor-κb-dependent transcription that is caused by the degradation of the p subunit of this transcription factor , . the transcription of genes encoding various cytokines and chemokines (including tumour necrosis factor (tnf; also known as tnfa), t cellspecific protein rantes (also known as ccl ), myxovirus resistance protein and interferon regulatory factor ) is also suppressed by fmdv l pro (ref. ). poliovirus reproduction, which is largely resistant to the effects of interferon, becomes interferon sensitive in a pro mutants. introduction of the poliovirus a pro gene into interferon-sensitive emcv facilitated its replication in cells pretreated with interferon . the ability of rhinovirus a pro to cleave mitochondrial antiviral-signalling protein (mavs; also known as visa, cardif and ips ), an intermediate in the interferon generation pathway, might contribute to the insensitivity of these viruses to interferons . a pro also cleaves the catalytic subunit of dna-dependent protein kinase, which, among other activities, is involved in the induction of pro-inflammatory cytokines . cardiovirus l also suppresses interferon production by affecting the activation of interferon regulatory factor , but the exact mechanism of this interference has not yet been identified , , , . tmev l* was proposed to suppress the antiviral cytotoxic t cell response in tmev-infected mice , . in line with these observations, the functions of security proteins are less crucial for viral 'well-being' in hosts with innate immunity defects. mutations in cardiovirus l , , or a ) ), rather than one l protein. for tmev, an alternative l protein, l*, is encoded in an alternative reading frame that starts within the l coding sequence. ‡ according to the data in genbank. § values were calculated using protparam . || for the viral genera with more than one species, the level of interspecies amino acid identity (and similarity, in parenthesis) was calculated with the aid of clustal_x alignments, using utilities implemented in bioedit and the blosum similarity matrix . to focus on the core protein sequences, the internal insertions of > amino acid residues, as well as terminal insertions, were not taken into account. ¶ the peptidase_c motif was revealed by blast searches in the ncbi conserved domain database . # the conserved non-canonical zinc (zn) finger domain (with a chcc motif) was found to be present. reproductive potential, but such mutations are less detrimental for viral growth in bhk- cells, which are deficient in interferon production, than in immunecompetent cells. similarly, cardiovirus mutants lacking l grow better in mice that have a deficient interferon system than in wild-type mice , . for the l*-expressing tmev strains, functional l* is important for the ability of the virus to infect macrophages or microglia , . one of the components of the innate immune response is apoptosis, which can potentially limit viral reproduction and spread, although certain viruses can subvert the apoptotic machinery to their benefit. conflicting results have been reported on the relationship between the security proteins and the apoptotic machinery. ectopic expression of enterovirus a pro triggers an apoptotic response , , and enhances the sensitivity of cells to the apoptogenic activity of tumour necrosis factor . however, in the context of the whole genome, a pro possesses anti-apoptotic activity , . similarly, expression of tmev l in macrophage-like cells induces apoptosis, as occurs during tmev infection of these cells , whereas emcv and mengovirus l are anti-apoptotic in the context of the whole virus (at least in hela cells, in which the virus itself elicits necrotic death) . whether this apparent discrepancy is a result of the use of different assays or hosts or of the intrinsic peculiarities of cardiovirus l proteins remains unknown. tmev l* has also been reported to exhibit anti-apoptotic activity . several illuminating examples strongly suggest that security proteins can markedly modulate viral pathogenicity. fmdv lacking l is highly attenuated , . || for the viral genera with more than one species, the level of interspecies amino acid identity (and similarity, in parentheses) was calculated with the aid of clustal_x alignments, using utilities implemented in bioedit and the blosum similarity matrix . to focus on the core protein sequences, the internal insertions of > amino acid residues, as well as terminal insertions, were not taken into account. ¶ these are manually recognizable motifs. # one of the strains is reported to harbour an npr(p) motif instead of npg(p). and both l , and l* (refs , , ) notwithstanding the low level of conservation of l proteins between emcv and tmev (table ) , these proteins can be functionally exchanged with respect to their ability to inhibit interferon formation and to 'open' nuclear pores . the replacement of l in the full-length cardiovirus genome with fmdv l pro generates a virus that can overcome host defences more efficiently than its leaderless counterpart, although it has lower fitness , . such interchangeability of structurally and biochemically distinct proteins attests to the similarity of their biological functions. it is hardly by chance that viruses that encode long or multiple ls tend to have a short a or lack a altogether (assuming that a sp is not a security protein), and vice versa (fig. ) . for example, simian and porcine sapeloviruses harbour a long a and a short l, whereas avian sapelovirus encodes the longest l identified to date and the predicted a orf is very short, if it encodes a protein at all. this tendency is consistent with the notion that l and a have similar roles in virus-host interactions. as already mentioned, some picornaviruses, for example, cosaviruses, encode no security proteins. even viruses that do encode security proteins can, under certain circumstances, survive and replicate after these proteins have been inactivated or eliminated. notably, cardiovirus l , , , and l* (refs , , ) and fmdv l pro (ref. ) are not essential for viral viability, and extended deletions in a of cardioviruses , and hepatitis a virus , do not kill these viruses. furthermore, although some data suggest that poliovirus a pro has an essential replicative function , recent experiments have demonstrated its dispensability for viral viability . although this review focuses on the counter-defensive functions of security proteins, it should be kept in mind that other picornavirus proteins are also often engaged in similar functions. the targets of c pro (or its proteolytically active precursors) might include proteins that are involved in innate immunity [ ] [ ] [ ] [ ] . b and a are also important players in the virus-host struggle, being involved in the rearrangement of cytoplasmic membranes and in suppression of trafficking, secretion and antigen presentation on cellular plasma membranes , [ ] [ ] [ ] . c can also participate in some of these activities . even specialized proteins such as vpg and capsid proteins can sometimes assist in overcoming host defences. in all these cases, however, the 'security' functions are neither the main nor the conserved roles of these proteins. conversely, as well as representing a dedicated counterdefensive system, security proteins can be directly involved in viral reproduction. the products of the cleavage of eif g, and possibly of some other host proteins, by fmdv l pro and enterovirus a pro can stimulate iresdependent cell-free translation , ; the effect of a pro is partly the result of stabilization of the viral rna . the physiological relevance of these phenomena is unclear, however. the poliovirus ires dysfunction that is caused by some mutations can be compensated for by mutations in a in a host-dependent manner , suggesting the participation of host proteins. cardiovirus l was also implicated in control of ires activity, because emcv rna with deletions in the l-coding sequence exhibited a decrease in cell-free translatability . poliovirus a pro was reported to stimulate strand initiation of negative-strand rna, and this was seemingly independent of its effects on rna stability and translation . deletion of a carboxy-terminal sequence from this protein does not substantially affect its protease activity but does inhibit rna replication . deletion of the amino-terminal region notably, but incompletely, suppresses replication of the relevant replicon . similarly, deletions in a of aichi virus (a kobuvirus) inhibit viral rna replication . however, the mechanisms responsible for these effects have not been elucidated, casting doubts on whether these a proteins affect viral replication directly or by modulating host cell activities. a role for hepatitis a virus a in virion assembly and maturation has been established , . the primary cotranslational cleavage of the viral polyprotein takes place between a and b and is accomplished by the viral proteinase c pro (or its precursor), leaving the a sequence fused to the carboxyl terminus of capsid protein vp (refs , ) . in immature virions, vp retains this a extension, which is eventually cleaved off by an unidentified enzyme . the involvement of a in the maturation of some other picornaviruses cannot be excluded therefore. notably, the primary co-translational scission of the cardiovirus polyprotein generates a fusion between a and the capsid protein precursor . origins and evolution of security proteins obviously, it is not possible to construct a genealogical tree of either l or a, because they are unrelated. these proteins are not considered at all in the hypothesis on the origin of picorna-like viruses , and only the short, translation-interrupting a peptides (that is, the a sp peptides) are briefly mentioned in the proposal on picornavirus classification . there is no correlation between the nature (or even the presence) of security proteins and either the type of ires that lies upstream of the l protein or the rdrp lineage (which is generally accepted as the most reliable indicator of viral relatedness) (fig. ) . the diversity of the security proteins and their absence from many picornaviruses suggest that they are independent and late evolutionary acquisitions , . it can be speculated that the most ancient of the a molecules are the a sp peptides, as hinted by their presence in most picornavirus genera, including those belonging to different lineages (fig. ) . interruption of polyprotein synthesis after translation of the capsid proteins might be advantageous for viral reproduction. it might, for example, facilitate proper protein folding, ensure optimal kinetics of protein synthesis or control the ratios of structural to non-structural proteins, which could theoretically be achieved by incomplete translational re-initiation at the second proline residue of the npg(p) motif. it is worth noting that there is a difference in the translation factor requirements for translation of the emcv polyprotein upstream and downstream of the a- b boundary . strikingly, a sp peptides -or more accurately, the dxexnpg(p) motifs (where x is any amino acid) that are characteristic of these peptides -are found in some other picorna-like and unrelated viruses, where they seem to serve the same function [ ] [ ] [ ] . assuming that the acquisition of npg(p) was an early event, this motif might have been lost by some parechoviruses (and other picornaviruses) at a certain step of evolution . in contrast to picornaviruses, the acquisition of a sp by members of some other families of rna viruses has been proposed to have occurred at a late stage . the idea that these peptides might have originated in picornaviruses is an attractive hypothesis. the npg(p) motif can also be found in several cellular proteins, but the current data do not allow researchers to determine whether the recoding ability of this motif was a viral or cellular invention. the non-npg(p)-containing regions of the a proteins are unrelated acquisitions. cardiovirus a possesses these additional moieties in its amino-terminal region, whereas other viruses of this subset (parechoviruses, avihepatoviruses and seal picornavirus type ) contain these moieties in their carboxy-terminal regions (fig. ) . this may suggest that these moieties were acquired after the npg(p) motif. in avihepatoviruses and some parechoviruses, these newly acquired sequences harbour the h-nc motif, which is also present in the npg(p)-lacking a proteins of other parechoviruses, kobuviruses and tremoviruses (that is, viruses of different rdrp lineages) (fig. ) . a plausible hypothesis is that h-nc-containing proteins from cellular organisms were hijacked by certain picornaviruses on several independent occasions, but the possibility that a sp peptides were formed by deletions from larger a proteins cannot be ruled out. taking into account the chymotrypsin-like fold that is found in enterovirus a pro , it is reasonable to assume that this protein was derived from picornavirus c pro or a cellular protease . a similar assumption could perhaps be made for the putative sapelovirus a protease. there are no obvious clues to the possible origins of the a proteins of hepatoviruses and klasseviruses or of the non-npg(p) moieties of the a proteins of cardioviruses and seal picornavirus type . with regard to the picornavirus l proteins, one hypothesis is that the papain-like l pro proteases of aphtho viruses and erboviruses originated from cellular enzymes. no obvious relatives of other l proteins can currently be identified among cellular or viral proteins. only the origin of cardiovirus l* seems certain: the first l* probably came into being accidentally, by translation of an alternative reading frame, and was then shaped by mutations preserving the functional integrity of l, vp and vp . it is worth noting that recently identified human tmev-like cardioviruses lack this alternative reading frame . theoretically, several mechanisms could underlie the acquisition of security proteins. for example, one possibility is that viral genes were duplicated and subsequently substantially modified (such a scenario can be imagined for the origin of enterovirus a pro (ref. ) ). other scenarios are: recombination with viral or cellular rnas encoding related proteins such as proteases; conversion of non-coding rna sequences into coding sequences (which could occur through different mechanisms, such as interspecies recombination (v.i.a., a.p.g., e. v. khitrina and w. j. melchers, unpublished observations), or introduction or activation of an upstream inframe aug codon); frame shifting (or double frame shifting, in the case of a proteins); and using an alternative reading frame. unfortunately, we can only pinpoint a definite mechanism for the case of l*, for which the last mechanism has obviously been operative. the acquisition of security proteins, by whatever mechanism, required that the new 'additions' did not interfere with the function of the adjacent viral proteins -vp (or vp ) in the case of l, and vp and b in the case of a. the new proteins should either be separated from these neighbours (by self-proteolysis, the action of other viral proteases or translation interruption) or, if they remain fused, they should not impair the functions of these neighbours (as is the case with the hepatitis a virus vp - a fusion). a separate issue is the evolution of the security proteins themselves. it was proposed that the l proteins of tmev and emcv diverged during evolution to adapt to the different replication fitnesses of these viruses . the data are too scarce, however, for any generalizations at this point. l and a constitute a distinct and remarkable set of picornavirus proteins. they exhibit striking structural and biochemical diversity but (with the exception of the a sp peptides) accomplish similar biological functions by counteracting host defensive reactions. however, their abilities to solve similar problems might involve fundamentally different molecular mechanisms. this is illustrated in fig. , which summarizes the properties of the three best studied security proteins. the biological functions of enterovirus a pro and cardiovirus l are strikingly similar: inhibition of host macromolecular synthesis, permeabilization of the nuclear envelope and inhibition of active nucleocytoplasmic transport, suppression of specific innate immunity mechanisms and control of the apoptotic machinery of the host cells. no less striking is the difference in the mechanisms by which the two proteins achieve these goals. conversely, aphthovirus l pro can solve only a subset of these problems. an intriguing figure | major biological functions of the best studied but unrelated security proteins. there is a striking similarity between the functional activities of the enterovirus a protease ( a pro ) and the cardiovirus leader protein (l), but the underlying mechanisms by which these functions are carried out are fundamentally different. by contrast, the known functional activities of aphthovirus l, which is a protease (l pro ), seem to be more limited. irf , interferon regulatory factor ; nf-κb, nuclear factor-κb. question is how, and whether, the diverse security functions exhibited by a given protein (fig. ) are related to each other. the fact that mutations inactivating one function usually also impair other functions suggests the existence of a common upstream target. most of the picornavirus l and a proteins still await researchers' attention. nevertheless, the data discussed here allow us to provisionally assign security functions even to those l and a proteins that have not yet been characterized. indeed, l and a do not definitely belong to the set of essential reproductive proteins that ensure translation and replication of the viral genome (although a of hepatitis a virus assists virion maturation). we propose that the concept of security proteins is of general relevance and can be applied to viruses other than picornaviruses. the hallmarks of these proteins are as follows: structural and biochemical unrelatedness or even absence in related viruses; the dispensability of the entire protein or its functional domains for viral viability; and, for mutated versions of the proteins, fewer detrimental effects on viral reproduction in immune-compromised hosts than in immune-competent hosts. possessing one of these features would make a viral protein a good candidate security protein, whereas a combination of these features would probably confirm this designation. viruses with large dna genomes possess impressive arsenals of security proteins. the complement of security proteins is much more limited in rna viruses but is sufficient for their evolutionary success. there are also tentative examples of security proteins in non-picornavirus rna viruses. coronaviruses have several so-called accessory proteins, which are neither conserved nor essential and exhibit the capacity to suppress host innate immunity by a range of mechanisms . some, but not all, flaviviruses use ribosome frame shifting to express a non-essential non-structural protein, ns ′, mutational inactivation of which results in viral attenuation . the nss protein of the rift valley fever phlebovirus suppresses the interferon system, and its inactivation does not kill the virus but attenuates its pathogenicity . this list can readily be extended. the spectrum of picornavirus-induced diseases is extraordinarily broad. the reasons for this variability are poorly understood, although receptor compatibility and effects on viral protein and rna synthesis that are caused by differences in the availability of host factors are surely important contributors. however, the interaction between host defences and viral counterdefence is certainly one of the key factors underlying the pathogenicity of picornaviruses and other viruses. this interaction cannot be fully understood without elucidation of the roles of the security proteins. treatment and prevention of viral diseases may also markedly benefit from such elucidation. the study of security proteins is therefore an underdeveloped but highly promising research area. rna replication without rna-dependent rna polymerase: surprises from hepatitis delta virus persistent yeast single-stranded rna viruses exist in vivo as genomic rna·rna polymerase complexes in : stoichiometry giardiavirus double-stranded rna genome encodes a capsid polypeptide and a gag-pol-like fusion protein by a translation frameshift the big bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups the proposal to consider l and a proteins of picornaviruses as a distinct class of counter-defensive 'security proteins evading the host immune response: how foot-and-mouth disease virus has become an effective pathogen a review considering, among other topics, counter-defensive functions of aphthovirus l protein a second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses structure of the fmdv translation initiation site and of the structural proteins the identification of the l protein in fmdv a second protease of foot-and-mouth disease virus the two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities conservation of l and c proteinase activities across distantly related aphthoviruses genomic evidence that simian virus and six other simian picornaviruses represent a new genus in picornaviridae cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a amino acid sequence site-specific release of nascent chains from ribosomes at a sense codon equine rhinovirus serotypes and : relationship to each other and to aphthoviruses and cardioviruses sequence analysis of a porcine enterovirus serotype isolate: relationships with other picornaviruses complete genome sequence analysis of seneca valley virus- , a novel oncolytic picornavirus a highly prevalent and genetically diversified picornaviridae genus in south asian children molecular analysis of three ljungan virus isolates reveals a new, close-to-root lineage of the picornaviridae with a cluster of two unrelated a proteins molecular analysis of duck hepatitis virus type a highly divergent picornavirus in a marine mammal the cleavage activity of aphtho-and cardiovirus a proteins the position of polypeptide g on the encephalomyocarditis virus polyprotein cleavage map rna-binding properties of nonstructural polypeptide g of encephalomyocarditis virus the a proteins of three diverse picornaviruses are related to each other and to the h-rev family of proteins involved in the control of cell proliferation the discovery of cardiovirus l protein, the first leader protein to be found for picornaviruses the leader peptide of theiler's murine encephalomyelitis virus is a zinc-binding protein nmr structure of the mengovirus leader protein zinc-finger domain leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation the mengovirus leader protein suppresses a/β interferon production by inhibition of the iron/ferritin-mediated activation of nf-κb alternative translation initiation site in the da strain of theiler's murine encephalomyelitis virus a picornaviral protein synthesized out of frame with the polyprotein plays a key role in a virusinduced immune-mediated demyelinating disease the demonstration that enterovirus a pro cleaves a translation initiation factor poliovirus proteinase a induces cleavage of eucaryotic initiation factor f polypeptide p purification of two picornaviral a proteinases: interaction with eif γ and influence on translation coxsackievirus b proteases a and c induce apoptotic cell death through mitochondrial injury and cleavage of eif gi but not dap /p / nat leader protein of foot-and-mouth disease virus is required for cleavage of the p component of the cap-binding protein complex cleavage of poly(a)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro cleavage of poly(a)-binding protein by coxsackievirus a protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? genetic analysis of mengovirus protein a: its function in polyprotein processing and virus reproduction rapamycin and wortmannin enhance replication of a defective encephalomyocarditis virus virus-specific proteins associated with ribosomes of krebs ii cells infected with encephalomyocarditis virus cardiovirus a protein associates with s but not s ribosome subunits during infection encephalomyocarditis viral protein a localizes to nucleoli and inhibits cap-dependent mrna translation encephalomyocarditis virus (emcv) proteins a and bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription mengovirus leader is involved in the inhibition of host cell protein synthesis a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein inhibition of cap-dependent gene expression induced by protein a of hepatitis a virus poliovirus-encoded protease a pro cleaves the tatabinding protein but does not inhibit host cell rna polymerase ii transcription in vitro the effect of poliovirus proteinase a pro expression on cellular metabolism. inhibition of dna replication, rna polymerase ii transcription, and translation inhibition of u snrnp assembly by a virus-encoded proteinase degradation of nuclear factor kappa b during foot-and-mouth disease virus infection a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function intracellular localization and effects of individually expressed human parechovirus nonstructural proteins a proteinase of human rhinovirus cleaves cytokeratin in infected hela cells enteroviral protease a cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores differential targeting of nuclear pore complex proteins in poliovirus-infected cells rna nuclear export is blocked by poliovirus a protease and is concomitant with nucleoporin cleavage the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins nucleo-cytoplasmic traffic disorder induced by cardioviruses mengovirus-induced rearrangement of the nuclear pore complex: hijacking cellular phosphorylation machinery leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon inhibition of l-deleted foot-and-mouth disease virus replication by a/β interferon involves double-stranded rna-dependent protein kinase the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response proteinase a pro is essential for enterovirus replication in type i interferon-treated cells cleavage of ips- in cells infected with human rhinovirus proteolytic cleavage of the catalytic subunit of dna-dependent protein kinase during poliovirus infection the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production the mengovirus leader protein blocks interferon-a/β gene transcription and inhibits activation of interferon regulatory factor a theiler's virus alternatively initiated protein inhibits the generation of h- k-restricted virus-specific cytotoxicity cd t cells are important for clearance of the da strain of tmev from the central nervous system of sjl/j mice involvement of cardiovirus leader in host cell-restricted virus expression this work demonstrates that cardiovirus l is dispensable the leader polypeptide of theiler's virus is essential for neurovirulence but not for virus growth in bhk cells protein a is not required for theiler's virus replication l* protein of the da strain of theiler's murine encephalomyelitis virus is important for virus growth in a murine macrophage-like cell line influence of the theiler's virus l* protein on macrophage infection, viral persistence, and neurovirulence poliovirus a protease induces apoptotic cell death infection with enterovirus or expression of its a protease induces apoptotic cell death poliovirus protein a inhibits tumor necrosis factor (tnf)-induced apoptosis by eliminating the tnf receptor from the cell surface bypass suppression of smallplaque phenotypes by mutation in poliovirus a that enhances apoptosis a protease is not a prerequisite for poliovirus replication theiler's murine encephalomyelitis virus leader protein is the only nonstructural protein tested that induces apoptosis when transfected into mammalian cells a protein critical for a theiler's virus-induced immune system-mediated demyelinating disease has a cell type-specific antiapoptotic effect and a key role in virus persistence pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle cardiovirus leader proteins are functionally interchangeable and have evolved to adapt to virus replication fitness an attenuating mutation in the a protease of swine vesicular disease virus, a picornavirus, regulates capand internal ribosome entry site-dependent protein synthesis construction of a chimeric theiler's murine encephalomyelitis virus containing the leader gene of foot-and-mouth disease virus differential ifn-a/β production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus theiler's murine encephalomyelitis virus (tmev) l* amino acid position is important for virus persistence and virus-induced demyelination the foot-and-mouth disease virus leader proteinase gene is not required for viral replication an article showing that aphthovirus l is dispensable hepatitis a viruses with deletions in the a gene are infectious in cultured cells and marmosets analysis of deletion mutants indicates that the a polypeptide of hepatitis a virus participates in virion morphogenesis studies on dicistronic polioviruses implicate viral proteinase a pro in rna replication molecular biology of picornaviruses proteolytic cleavage of the p -rela subunit of nf-κb during poliovirus infection disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor rig-i is cleaved during picornavirus infection molecular biology of picornaviruses inhibition of cellular protein secretion by poliovirus proteins b and a coxsackievirus b inhibits antigen presentation in vivo, exerting a profound and selective effect on the mhc class i pathway role of nonstructural proteins a and b in host range and pathogenicity of foot-and-mouth disease virus early phosphatidylinositol -kinase/ akt pathway activation limits poliovirus-induced jnk-mediated cell death foot-and-mouth disease virus lb proteinase can stimulate rhinovirus and enterovirus ires-driven translation and cleave several proteins of cellular and viral origin the c-terminal domain of eukaryotic protein synthesis initiation factor (eif) g is sufficient to support capindependent translation in the absence of eif e a pro is a multifunctional protein that regulates the stability, translation and replication of poliovirus rna coding changes in the poliovirus protease a compensate for ′ncr domain v disruptions in a cell-specific manner the c-terminal residues of poliovirus proteinase a pro are critical for viral rna replication but not for cis-or trans-proteolytic cleavage replication of poliovirus rna and subgenomic rna transcripts in transfected cells aichi virus a protein is involved in viral rna replication intrinsic signals for the assembly of hepatitis a virus particles morphogenesis of hepatitis a virus: isolation and characterization of subviral particles identification and site-directed mutagenesis of the primary ( a/ b) cleavage site of the hepatitis a virus polyprotein: functional impact on the infectivity of hav rna transcripts the unique role of domain a of the hepatitis a virus precursor polypeptide p - a in viral morphogenesis picornavirales, a proposed order of positive-sense single-stranded rna viruses with a pseudo-t = virion architecture origin and evolution of viruses translational barrier in central region of encephalomyocarditis virus genome. modulation by elongation factor (eef- ) occurrence, function and evolutionary origins of ' a-like' sequences in virus genomes an article discussing viral translation-interrupting a peptides a case for ''stopgo'': reprogramming translation to augment codon meaning of ggn by promoting unconventional termination (stop) after addition of glycine and then allowing continued translation (go) euprosterna elaeasa virus genome sequence and evolution of the tetraviridae family: emergence of bipartite genomes and conservation of the vpg signal with the dsrna birnaviridae family molecular basis of viral evolution genomic features and evolutionary constraints in saffold-like cardioviruses mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation. microbiol ns ′ of flaviviruses in the japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness nss protein of rift valley fever virus induces the specific degradation of the doublestranded rna-dependent protein kinase divergent picornavirus ires elements the proteomics protocols handbook clustal w and clustal x version . cdd: specific functional annotation with the conserved domain database we thank f. van kuppeveld for critical reading of this manuscript. recent research in the authors' laboratory was supported by grants from the russian foundation for basic research (rfbr), the scientific school support program and the netherlands organization for scientific research-rfbr (nwo-rfbr). the authors declare no competing financial interests. see online article: s (figure)all links are active in the online pdf key: cord- -lbr q qh authors: chinchar, v. gregory; yu, kwang h.; jancovich, james k. title: the molecular biology of frog virus and other iridoviruses infecting cold-blooded vertebrates date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lbr q qh frog virus (fv ) is the best characterized member of the family iridoviridae. fv study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. although the broad outlines of fv replication have been elucidated, the precise roles of most viral proteins remain unknown. current studies using knock down (kd) mediated by antisense morpholino oligonucleotides (asmo) and small, interfering rnas (sirna), knock out (ko) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. in addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. in this review, we summarize current studies using not only fv , but also other iridoviruses infecting ectotherms. as described below, general principles ascertained using fv served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease. : sizes and coding potentials of iridovirid genomes. iridovirus iiv- , gq iiv- aside from differences in virion and genome sizes, the organization of virus particles within the family is generally similar. while transmission electron microscopy (tem) has been conducted using fv and other iridovirids, detailed cryo-electron microscopic (cryoem) analyses (discussed below) were performed with iiv- and provide the most detailed view of virion structure. virions exist in two forms: non-enveloped particles ~ - nm in diameter, and enveloped particles that acquire an envelope by budding from the plasma membrane. in addition to the external envelope, some iridovirids contain a fringe of fine fibers (fibrils) that extend outward from the capsid subunits [ , ] . non-enveloped particles are composed of three distinct layers: an outer capsid composed of multiple copies of an ~ kda major capsid protein (mcp), an internal lipid membrane possibly derived from the endoplasmic reticulum (er) or other cellular membranes, and an inner core containing the viral genome and associated virus-encoded proteins [ , ] . the mcp shows marked sequence conservation within all members of the family [ ] . sequence identity within the mcp gene allows primer-based pcr amplification of viral dna and provides an easy way to determine whether a given virus is a member of the family and to which genera it belongs [ ] . although the mcp is the primary structural protein and comprises % of the total virion protein content, an additional proteins have been identified following gel analysis of purified virions [ ] . while many of these proteins likely represent virus-encoded catalytic proteins that play various roles in replication, three minor capsid proteins have been identified by cryoem analysis of iiv- and designated as finger, zip, and anchor proteins [ ] . finger and zip proteins were suggested to stabilize the virus by acting as intercapsomer cross-links, whereas the anchor proteins appear to be transmembrane proteins that extend into the internal lipid membrane and provide further stabilization. in addition to these proteins, a fifth protein, a putative myristoylated protein (fv orf r), has been suggested to interact with cellular membrane fragments and play a role in virion assembly [ ] . as shown in table , iridovirid genomes range in size from - kbp. ranavirus genomes occupy the low end of this range and fall into three groups: the genomes of fv , atv, tfv, and stiv are - kbp, ehnv is kbp, whereas sgiv and giv, likely isolates of the same viral species, are kbp in size. consistent with their sizes, ranaviruses contain between and close-packed, predominantly non-overlapping orfs of amino acids or greater [ , [ ] [ ] [ ] . repetitive regions are common and may explain the high rate of recombination seen within these viruses. in addition, a small number of putative micrornas of unknown function have been detected [ ] . amino acid conservation is marked among iridovirid genes, but gene order, even within members of the same genus, is not conserved suggesting that expression is likely determined by individual promoter elements closely associated with each gene. moreover, although viral genes are expressed in an ordered temporal cascade (consisting of immediate early (ie), delayed early (de), and late (l) genes) nucleotide sequences corresponding to ie, de, and l promoters have not yet been identified. the observation that gene order is not conserved supports the view that genes can be reshuffled through recombination without adversely affecting expression. microarray analysis suggests that fv contains ie, de, and l genes [ ] . moreover, the classes assigned in that study were generally similar to those assigned to known sgiv genes in two earlier studies [ , ] . although all ranaviruses contain a core set of genes [ ] , unique genes are found within specific viral species. genes held in common (e.g., viral dna polymerase, mcp, the large and small subunits of the viral rna transcriptase, etc.) are thought to be those required for replication in all cell types, whereas those unique to a given viral species may represent specific host adaptations that contribute to virulence, host range, and immune evasion. early studies of iridovirid replication were conducted almost exclusively using fv , whereas recent studies have included additional ranaviruses such as atv and sgiv and several megalocytivirus isolates that have been linked to massive die-offs in mariculture facilities in japan and south-east asia. below we summarize events in fv -infected cells and highlight recent work that has appeared since the last comprehensive reviews [ , , , ] . fv virions, and presumably virions of all other iridoviruses, exist in two forms: non-enveloped (i.e., naked) particles and enveloped virions. although both forms are infectious, enveloped virions were shown to have higher specific infectivity [ , ] . with regard to entry, enveloped particles likely enter cells by receptor-mediated endocytosis in a ph-dependent manner and require clathrin-coated pits, whereas naked particles enter by fusion at the plasma membrane [ , ] . recently this model has been questioned by guo and co-workers who argued that tiger frog virus (tfv), a ranavirus nearly identical to fv , enters cells by an atypical, ph-dependent, caveola-mediated endocytic pathway [ ] . their conclusion was based on experiments using chlorpromazine and over-expression of a dominant-negative form of esp that inhibited assembly of clathrin-coated pits, but did not affect entry. consistent with a role for caveolae, endocytosis of tfv was dependent on membrane cholesterol and was blocked by caveolin- scaffolding domain protein. given that fv and tfv are nearly identical in nucleotide sequence [ , ] , it is surprising that their modes of entry are so different. because the reported differences in entry mechanisms between fv and tfv may be due to infection of cells from different non-physiological hosts, e.g., rats and hamsters, resolution of this issue will require direct comparison of tfv and fv entry using the above approaches coupled with tem. regardless of whether uncoating takes place at the plasma or nuclear membranes, the viral genome enters the nucleus. unlike herpesviruses, iridovirid genomes are not infectious, indicating that virion-associated proteins are required to initiate viral gene transcription [ ] . accordingly, immediate-early (ie) and delayed early (de) viral transcripts are synthesized in reactions mediated by putative virion-associated (for ie transcripts) and virus-encoded (for de mrna) transcriptional trans-activators and, at least for ie transcription, host rna polymerase ii [ ] [ ] [ ] . among the products of early transcription is a viral dna polymerase which catalyzes the synthesis of unit-sized copies of the viral genome within the nucleus [ ] . additional ie and de proteins include proteins that may play roles in blocking host immune responses such as a virus-encoded, card (caspase activation and recruitment domain) motif-containing protein (vcard), β-hydroxysteroid dehydrogenase (βhsd), and a rnase iii-like protein, catalytic proteins involved in nucleic acid synthesis (proliferating cell nuclear antigen [pcna], dna methyltransferase [dmtase], the large and small subunits of the viral homolog of cellular rna polymerase ii [vpol-iiα and -iiβ], transcription factor iis), catalytic proteins that may act to increase dttp pool sizes and influence host range (deoxyuridine triphosphatase [dutpase], deoxynucleotide kinase, the large and small subunits of ribonucleotide reductase), and proteins non-essential for replication in vitro, but needed for growth in vivo (the k protein) [ ] . following its synthesis, unit-sized viral dna is transported into the cytoplasm where it is methylated by a virus-encoded dmtase and, following a second round of dna synthesis, converted into large concatameric molecules that are thought to be the substrate from which viral genomes are derived [ ] [ ] [ ] . virion formation takes place in electron-lucent, morphologically-distinct vas. vas contain viral dna and the structural and non-structural proteins that give rise to virions, but are devoid of ribosomes, mitochondria, and other cellular organelles [ , ] . although little is known about the precise process of virion morphogenesis, by analogy to african swine fever virus and by study of various iridovirids, it appears that host membranes, perhaps derived from the er, serve as a scaffold to which capsid and shell proteins bind [ , ] . as progressively larger amounts of viral proteins bind the membrane scaffold, crescent-shaped structures that resemble icosahedral vertices are formed. ultimately both full and empty icosahedral virus-like particles are detected. however, it is unclear precisely how the genome is encapsidated. while packaging via a headful mechanism explains the circularly-permuted terminal redundancy detected within all iridovirids [ ] , it is not known whether nearly complete virions bind viral dna and internalize it through a virion portal as seen in some viral systems [ ] [ ] [ ] , or whether developing crescents/icosahedrons engulf the viral genome as they assemble. following their formation, virions remain within vas, accumulate within cytoplasmic paracrystalline arrays, or bud from the plasma membrane. at late times after infection, virions are sometimes seen within the nucleus and elongated tubular structures can be detected in vas, but these are likely artifacts that reflect breakdown of the nuclear membrane and the disruption of the assembly process due to the shortage of key structural proteins and the dysregulation of cellular and viral macromolecular synthesis. a transmission electron micrograph of an fv -infected fathead minnow cell is shown in figure a , and an enlargement of the vas, displaying full and empty virions, is shown in figure b . the identities of virus-encoded proteins involved in virion assembly remain to be determined. twelve complementation groups defective in the ability to synthesize infectious virions have been identified through study of temperature-sensitive (ts) mutants (see below), but it is unclear whether the gene products encoded by these complementation groups represent scaffold proteins that are required for virion assembly, but which are not incorporated into mature particles, authentic viral structural proteins, or virion-associated accessory proteins required for viral replication [ ] . aside from the mcp, only fv orf r, which encodes a putative myristoylated membrane protein, has been linked to virion assembly. knock down studies using either asmos or artificial micrornas [ , ] demonstrated that r was required for virion assembly, whereas in vitro studies showed r was associated with virus factories and the virion membrane [ ] . by analogy to asfv, r may play a role in recruiting er-derived membranes into virus factories where they serve as precursors of the inner viral lipid membrane and/or act as a scaffold protein. as with other nuclear and cytoplasmic, large dna viruses (ncldv) late viral gene expression is dependent upon full viral dna synthesis, and is catalyzed by a virus-encoded or virus-modified transcriptase [ ] . temperature-sensitive mutants defective in viral dna synthesis show markedly reduced levels of late gene expression as do cells treated with drugs (phosphonoacetic acid [paa], cytosine arabinoside [arac]) that block dna synthesis [ , ] . fv and other iridovirids encode homologs of the two largest subunits of rna polymerase ii and likely use these proteins, and perhaps others, to catalyze late viral gene expression [ ] . knock down of the expression of the largest subunit of the viral homolog of rna polymerase ii (i.e., vpol-iiα) results in a marked reduction in the synthesis of late viral proteins and a corresponding reduction in virion formation and supports the notion that vpol-iiα, and other viral proteins, are responsible for late viral gene expression [ ] . aside from the mcp, other late proteins tentatively involved in dna packaging and virion assembly include orf r, a putative packaging protein, and evr /alr, a protein involved in the formation of disulfide bonds [ ] . to illustrate the above, a schematic diagram of the viral replication cycle is shown in figure . [ ] . used with permission. as with other viruses, fv infection markedly inhibits host macromolecular synthesis and triggers apoptosis [ ] [ ] [ ] [ ] . both phenomena are mediated by heat-or uv-inactivated virus indicating that the inhibitory agent is a virion-associated protein [ , ] . viral infection appears to trigger activation of pkr, a double-stranded rna-activated protein kinase, which leads to the phosphorylation of the largest subunit of eukaryotic initiation factor (eif- α) and the subsequent inhibition of protein synthesis [ ] . viral translation may be spared due to the synthesis of large amounts of highly efficient viral transcripts and the presence of a virus-encoded protein, e.g., a viral homolog of eif- α (vif- α), that acts as a pseudosubstrate and prevents phosphorylation/inactivation of eif- α [ , [ ] [ ] [ ] . most ranaviruses, with the exception of fv , contain a full-sized vif- α gene [ , ] . however, because the absence of a full-sized vif- α gene in fv does not adversely affect viral protein synthesis, other viral proteins may also play roles in maintaining viral protein synthesis in infected cells. aside from the aforementioned vif- α protein, fv and other ranaviruses encode a number of putative immune evasion gene products including βhsd, vcard, and an rnase iii-like protein. by analogy to a similar protein in vaccinia virus, βhsd is thought to trigger glucocorticoid synthesis in infected animals and enhance virus replication by suppression of the overall immune response [ , ] . likewise, vcard may inhibit the induction of interferon and/or apoptosis. induction of ifn and/or apoptosis follows interaction of rig-i or mda , cellular proteins that bind dsrna, with downstream effectors such as mavs and isp- [ ] [ ] [ ] [ ] [ ] [ ] [ ] . since rig-i/mda and mavs/isp- interactions are mediated by card motifs, it is possible that a viral card-containing protein might bind either one or both of these proteins and short-circuit the ifn induction pathway [ ] . the rnase iii-like protein is found within all iridoviruses and has been postulated to block sirna-mediated interference, or to play a role in the processing of viral mrnas [ ] . alternatively, viral rnase iii could act like vaccinia virus e l and bind and/or degrade dsrna and block the activation of pkr, the induction of interferon, and the inhibition of protein synthesis [ ] . as with vaccinia virus [ ] [ ] [ ] , iridoviruses that infect ectothermic animals encode a series of proteins whose function is to ensure an adequate supply of nucleotides for viral dna synthesis. these enzymes include the two subunits of ribonucleotide reductase [ , ] , thymidine kinase [ ] , thymidylate synthase, dutpase [ ] , purine nucleoside phosphorylase (pnp), and a dihydrofolate reductase homolog [ , ] . not all of these putative enzymes are found within each viral species, and it is postulated that specific enzymes might be needed in certain hosts. for example, pnp is found only within giv and it has been suggested that because giv's natural host, the grouper, might not be able to supply the purine nucleotides required for viral replication the virus encodes its own pnp [ ] . currently there are seven completely-sequenced ranavirus genomes ranging in size from - kbp (table ) . whole genome dot plot analyses show that there are three distinct genomic organizational profiles among ranaviruses that are based on the conservation of gene order [ ] : fv -like viruses (i.e. fv , tfv and stiv), atv-like viruses (i.e. atv and ehnv) and giv-like viruses (i.e. sgiv and giv). dot plot comparisons of viruses within each group show complete co-linearity. however, comparison of atv-like ranaviruses with fv -like ranaviruses detected two large inversions, whereas comparing either of these groups to giv-like viruses revealed very little conservation of gene order [ , ] . in the latter case, gene order is so jumbled between fv /atv-like viruses and sgiv/giv-like viruses that any trace of the degree diagonal line, indicative of complete sequence alignment between genomes in dot plots, is lost. interestingly, ranavirus genomic organization is markedly different from that of poxviruses. for example, all members of the subfamily chordopoxvirinae share a conserved genomic core structure wherein gene order, with the single exception of avipoxviruses, is conserved [ ] . however, despite the conservation of gene order, global sequence alignments show that sequence identity among members of the chordopoxvirinae is only - % [ , ] . ranaviruses, on the other hand, share a much greater sequence identity (~ - % within the mcp) yet have three widely divergent genomic organizations. moreover, as additional ranavirus genomes are sequenced, we may discover novel genomic morphotypes that will elucidate the evolutionary relatedness of ranaviruses. examination of complete ranavirus genomic sequences, including whole genome dot plot and phylogenetic analyses of ranavirus core genes, suggested that ranaviruses have undergone a number of evolutionarily-recent host shifts [ , ] . phylogenetic analysis using the core iridovirid genes identified by eaton and her co-workers [ ] support whole genome dot plot analysis and indicate that fv -like viruses (fv , tfv, stiv), atv-like viruses (atv and ehnv), and giv-like viruses (sgiv and giv) cluster on separate branches of the ranavirus tree ( figure ). collectively, these data suggest that for the greater part of their evolutionary history, ranaviruses were restricted to infecting fish. in addition, the shallow branch lengths of fv -like and atv-like viruses, which infect amphibians and fish, suggest that this group of viruses has recently, at least in evolutionary terms, radiated to infect a wide-range of poikilothermic vertebrates [ ] . this hypothesis is supported by studies showing that ranaviruses are multi-host pathogens [ ] . as additional ranaviruses are sequenced, particularly those isolates that infect multiple host species, a better understanding of ranavirus evolution, host shifts, and the molecular determinants of ranavirus host range and pathogenesis will be achieved. pathological and immunological aspects of infection by fv and various iridovirids will be dealt with by chen and robert [ ] and miller et al. [ ] in this special issue of viruses. thus, only a brief overview of this topic will be provided here. although iridovirids, in contrast to the chytrid fungus batrachochytrium dendrobatidis (bd), have not been reported to drive species extinction, fv -like viruses have caused deaths in several amphibian culture facilities and in nature, and megalocytiviruses have triggered wide-spread mortality in mariculture operations in south-east asia [ , , [ ] [ ] [ ] [ ] . ranavirus infections range from inapparent to fulminant and involve a variety of tissues including the skin, kidney, liver, and intestine [ ] . using xenopus laevis as a model host, robert and his colleagues have shown that fv infection occurs in both larval and adult animals [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, whereas infections of immunocompetent adult frogs are confined to the kidney and cleared within a few weeks, infection of tadpoles or immunocompromised adults results in widespread infection that spreads to multiple internal organs and often leads to death. protection from fv infection appears to involve both humoral and cell-mediated immunity and both antiviral antibodies and cytotoxic t cells have been identified as protective. vaccination may prove useful in protecting susceptible species and vaccines have been developed to protect commercially important fish from megalocytivirus infection [ , ] . likewise, infection of bullfrog tadpoles (rana catesbeiana) with less pathogenic fv protected them against death following infection with the more pathogenic rana catesbeiana virus z [ ] . as with the above section, the ecology of ranavirus infections will be more fully discussed by miller and co-workers in their contribution to this issue [ ] . suffice it to say iridovirus infections have a marked effect on lower vertebrates. as green and co-workers observed most mortality events seen among amphibians from - were attributable to iridovirus infections [ ] . in addition, whereas lymphocystivirus and megalocytivirus infections have, to date, only been detected in fish species, ranavirus infections affect three classes of ectothermic vertebrates: bony fish, amphibians, and reptiles [ ] . results from experimental challenges as well as natural infections suggest that ranaviruses isolated from one species can not only infect different host species within the same genus, but also host species from different genera, orders, and classes. for example, amphibian ranaviruses such as bohle iridovirus, fv , and atv infect a variety of fish and amphibian species, and at least one insect virus appears to also infect reptiles [ , , , - ]. early studies with fv attempted to elucidate viral replicative events and the function of viral gene products using a variety of inhibitors, e.g., cycloheximide to confine viral gene expression to ie transcription, α-amanitin to block host rna polymerase ii activity, phosphonacetic acid to block viral dna synthesis, azacytidine to block the methylation of viral dna, flurophenylalanine to block the expression of late viral proteins, elevated temperatures ( °c) to confine viral gene expression to early transcripts and proteins, etc. (reviewed in [ , ] ). in addition, panels of ts mutants were generated and subsequently characterized [ , , ] . ts mutants proved useful in confirming and identifying various aspects of the virus life cycle, e.g., the two-stages of viral dna synthesis [ ] . ultimately, ts mutants were organized into complementation groups comprising three classes [ ] . class i mutants ( complementation groups) appeared to contain mutants with defects in virus assembly. these mutants synthesized early and late viral proteins and viral dna, but did not generate infectious virions [ ] . recently, tem analysis suggested that this class could be further sub-divided into at least two subclasses, those that failed to assemble icosahedral particles and those that formed ostensibly normal, but non-infectious, icosahedral particles at the non-permissive temperature [ ] . the remaining complementation groups appeared to contain defects in proteins associated with viral transcription (class ii) and viral dna synthesis (class iii). consistent with an early study suggesting that only early viral proteins were required for assembly site formation [ ] , tem analysis suggested that full viral dna synthesis was not required for the formation of viral assembly sites as two ts mutants that synthesized markedly reduced levels of viral dna were able to form large vas, that were devoid of viral dna, most viral proteins, and viral particles [ , ] . however, although ts mutants have contributed to our understanding of fv replication, it has not been possible to associate a given mutant phenotype with a specific viral orf, and thus link a specific viral protein with its function. to address this issue, we have recently developed approaches that target individual viral genes and have used them to determine viral gene function by observing changes in phenotype. to link specific viral proteins with their functions, viral gene expression was knocked down (kd) using antisense morpholino oligonucleotides (asmos) and gene function was inferred by changes in phenotype. asmos are dna-like macromolecules, optimally nucleotides in length, whose backbone contains morpholine rings instead of deoxyribose rings and non-ionic, phosphorodiamidate bonds in place of phosphodiester links [ ] . moreover, because phosphorodiamidate bonds are resistant to degradation by cellular nucleases, asmos are remarkably stable in cell culture [ ] . furthermore, in contrast to sirnas that sometimes trigger off-targeting effects by activating pattern recognition receptors such as toll-like receptor (tlr- ), asmos are not recognized by cellular dna receptors such as tlr- or aim- and thus do not induce innate immune responses. asmos bind complimentary sequences within the ' non-translated region, or the region immediately surrounding the aug initiation codon, of the targeted mrna and are thought to block scanning of the s ribosome by steric hindrance. in addition, asmos also block gene expression by inhibiting splicing or preventing interaction of regulatory proteins with their specific target sequence [ ] . collectively asmos have been used to block cellular and viral gene expression both in vitro and in vivo [ ] [ ] [ ] . asmos have been used to ascertain the function of several fv genes including those encoding the mcp, vpol-iiα, an kda immediate-early viral gene product ( k), a putative myristoylated membrane protein ( r), and two ie proteins of unknown function: a kda protein designated r, and kda protein termed k [ , , , ] . in an initial proof-of-concept study, sample et al. [ ] showed that asmo treatment knocked down mcp expression by greater than % and resulted in a corresponding drop in the production of infectious virions without any collateral adverse effects on the expression of other viral gene products. moreover, mcp knock down was accompanied by the appearance of atypical elements within vas suggesting that in the absence of full mcp expression aberrant viral structures, perhaps representing altered products of virion assembly, were generated. in the same study, kd of vpol-iiα was shown to result in a marked reduction in the synthesis of late viral proteins. this result provided the first formal evidence that viral homologs of host rna polymerase ii played a role in the synthesis of late viral transcripts. lastly, sample and co-workers observed that kd of the k ie protein had no observable effect on viral gene expression or replication in vitro suggesting that, at least in fathead minnow cells, k was not required for the production of infectious virions. subsequent kd studies of r, a putative virus-encoded myristoylated membrane protein, r, and k confirmed an essential role for each of these proteins in fv biogenesis [ , ] . kd of r resulted in the appearance of putative non-encapsidated viral dna cores within vas and supported the view that r plays a major role in capsid formation. kd of r and k, two ie proteins, did not affect the synthesis of other viral proteins, but resulted in a > % reduction in virion formation. tem study showed that cells treated with asmos targeting either r or k formed viral assembly sites, but failed to form virions or recognizable assembly intermediates. studies targeting the virus-encoded rnase iii-like protein (orf l), a putative ntpase (orf l), the largest subunit of ribonucleotide reductase (orf r), a putative viral membrane protein (orf l), a dna packaging protein (orf r), a putative serine/threonine kinase (orf r), a putative rad -like dna repair protein (orf r), a kda protein of unknown function (orf r), and the viral dmtase (orf r) are ongoing and have demonstrated reductions in viral yields ranging from - % [ ] . however, unlike the first study in which kd of mcp, vpol-iiα, and k was confirmed by d sds-polyacrylamide gel electrophoresis, we have been unable to identify unambiguously the targeted protein in these latest studies, and thus do not know if the partial reductions in virus yields reflect the non-essential nature of the gene product or incomplete kd of the targeted protein. to overcome this problem, recombinant viral proteins will be generated and used to produce polyclonal antibodies for western blot and immunofluorescent analyses. lastly, although asmos can be powerful tools for uncovering viral gene function, their use is potentially limited by the nature of some ranavirus mrnas. because many fv transcripts possess very short, au-rich ' non-translated regions, and because sequences surrounding the aug initiation codon may be unfavorable for targeting due to, for example, high au content or secondary structure, asmo-mediated kd approaches may not succeed with all targeted genes. in these cases, sirna-mediated kd or ko approaches, discussed below, provide alternative approaches. nevertheless, despite these limitations, asmos have provided insight into the roles of several fv genes and will continue to be a useful tool for elucidating viral gene function in vitro. together with anti-viral antibodies to confirm knock down and identify intracellular locations, these studies will broaden our understanding of the complex story of virus-host interactions. as an alternative to asmos, sirnas have also been used in a limited number of cases to knock down iridovirid gene expression [ , , , ] . while knock down has been achieved, we observed that inhibition of fv gene expression only took place if cells were infected at low multiplicities of infection, i.e., < . pfu/cell. similar to the situation in some other viral systems, the inverse relationship between multiplicity of infection and sirna knockdown suggests that a virion-associated or virus-encoded protein may block sirna-mediated knockdown by either binding or degrading sirna [ ] . to date, expression of fv transcripts encoding mcp, dmtase, and vpol-iiα have been knocked down by sirna and resulted in a marked inhibition of replication. a powerful and potentially more useful approach to elucidate viral gene function involves the generation of knock out (ko) mutants since, in contrast to transient kd triggered by asmos or sirnas, the phenotypes of ko mutants can be evaluated both in vitro and in vivo. this feature makes ko mutants especially useful for identifying viral genes that play roles in immune evasion and virulence. ko mutants have been used extensively to elucidate the function of various poxviruses genes [ ] [ ] [ ] , and methodology developed here has been adapted to ranaviruses. however, despite using poxviruses as a guide, ranavirus ko mutants, using homologous recombination to replace the targeted gene with a selectable marker, have only recently been isolated. the reasons for the difficulty in applying this approach to ranaviruses are not clear, but may reflect the presence of a ranavirus-encoded endonuclease that cleaves unmethylated dna [ , ] . if correct, introduction of unmethylated plasmid dna, bearing the selectable marker, into a virus-infected cell may lead to degradation of the majority of plasmid dna and make recombination unlikely. as was done previously in a recombinant biv vector [ , ] , positioning the selectable marker, e.g., the neomycin-resistance gene, downstream of the promoter for the k immediate early (ie) gene drove high levels of expression of the resistance gene and permitted isolation of rare recombinants. the first ranavirus ko mutant targeted the atv-encoded homolog of eukaryotic translational initiation factor α (vif- α) [ ] . as discussed above, following virus infection, host cells activate pkr, an interferon (ifn) inducible double-stranded rna activated protein kinase [ ] , that, in turn, phosphorylates the α subunit of eif- and, as a consequence, inhibits both host and viral protein synthesis. to prevent translational shut-off, several viruses, including most ranaviruses, encode an eif- α homolog that has been proposed to function as a pseudosubstrate for pkr [ ] . in vaccinia virus this homolog is designated k l, whereas in ranaviruses it is termed vif- α. although k l and vif- α differ markedly in size, they share a common sequence motif (vdrvdrekgyvdl) that is likely required for activity. in this scenario, pkr binds k l/vif- α instead of eif- α, and as a consequence eif- α is not phosphorylated and protein synthesis is maintained. to determine if atv vif- α (orf r) is functionally similar to k l, the ranavirus gene was replaced with a selectable marker, the neomycin resistance (neor) gene. to generate an atv ko mutant, neor was inserted downstream of the atv k ie promoter and this construct was bracketed with sequences identical to those in the flanking region of the targeted gene. a pcr product bearing this construct was then transfected into bf- cells and infected with wild-type (wt) atv. recombinant virus was isolated by selective growth in the presence of neomycin, and replication of the ko mutant was compared to wt virus in vitro and in vivo. the ko mutant, designated atv∆ r, replicated to titers comparable to that of wt atv in vitro, but had a small plaque phenotype. in addition, atv∆ r was -fold more sensitive to ifn than wt atv. the increased ifn sensitivity of atv∆ r was correlated with the increased phosphorylation of eif- α and the lack of pkr degradation. in contrast, wt atv degraded pkr and inhibited cellular eif- α phosphorylation. in addition, atv∆ r was less pathogenic than wt atv following infection of tiger salamanders (ambystoma tigrinum) indicating that vif- α was a likely viral virulence/immune evasion protein. thus the atv vif- α gene is a putative ifn-resistance gene that inhibits cellular innate immune responses by degrading pkr and maintaining high levels of viral protein synthesis. in an effort to improve the ko methodology, chen et al. [ ] recently developed a potentially more powerful dual selection method to generate ko mutants within fv . in this system, the gene of interest is replaced, via homologous recombination, with a gene encoding the puromycin-resistance gene fused to the gene for enhanced green fluorescent protein (egfp). in initial experiments ko mutants targeting the truncated vif- α protein (fv -∆vif- α) and the k ie protein (fv -∆ k) were generated as well as a control, "knock in" mutant in which the puromycin-resistance/egfp gene was inserted into a non-coding portion of the genome. while all three recombinant viruses grew well in vitro, the vif- α and k ko mutants, but not the control "knock in" mutant, showed a % drop in virus levels in x. laevis tadpoles. these results confirmed the previous observation that the k protein was not required for replication in vitro [ ] , and indicate that k plays a role, albeit unknown, in replication in vivo. the finding that the truncated vif- α protein of fv was also required for replication in vivo was surprising because the fv homolog of vif- α is missing the n-terminal two-thirds of this protein, including the region homologous to vaccinia virus k l and eukaryotic translational initiation factor α. inspection of the fv nucleotide sequence indicated that fv vif- α is a chimeric protein resulting from deletion and in-frame fusion of a small upstream orf with the larger, downstream vif- α orf. the resulting product contains amino acids from the n-terminus of the upstream orf and the last amino acids of the full-length vif- α protein. the reduced replication of fv -∆vif- α in vivo suggests that the c-terminal end of the fv vif- α homolog is required for full replication in vivo. in addition to the above ko mutants, a recombinant soft-shelled turtle iridovirus has recently been constructed that expresses egfp. egfp-expressing mutants may provide an alternative, simplified strategy for screening antiviral substrates through the visualization and quantification of fluorescent virus [ ] . in contrast to the above studies, a fourth approach for determining viral gene function involves the synthesis of recombinant viral proteins and assessment of their functions in vivo and in vitro. several investigators have used this approach and representative examples are discussed below. a putative homolog of fv icp was identified in sgiv [ ] . icp (mol wt . kda) is an ie gene product that is distributed predominantly within the cytoplasm but is also found within virions. a plasmid expressing icp was introduced into grouper embryonic (gp) and fathead minnow cells and stably transfected cells were characterized. over expression of sgiv icp resulted in higher cell densities and increased growth of monolayer cultures. sgiv replication was compared in gp cells transfected with an empty vector and in gp cells expressing icp . sgiv replicated more rapidly and reached titers that were -fold higher in icp expressing cells than in control cells. although conserved domains suggestive of function were not detected within icp , the authors speculate, based on the above results, that icp might encode a protein involved in cell growth control. the authors suggested that icp , like ie proteins in other viral systems, might control host cell growth by regulating the cell cycle, preventing growth arrest, or delaying apoptosis and play a critical role in promoting a proliferative environment that enhances virus replication. isknv (genus megalocytivirus) orf r encodes a viral protein with marked similarity to vascular endothelial growth factor (vvegf) [ ] . microinjection of a plasmid expressing vvegf into zebrafish one-cell embryos resulted in pericardial edema and dilation of the tail region suggesting that vvegf triggers vascular permeability. moreover, vvegf binds and up-regulates the expression of flk- and together both proteins likely contribute to the proliferative and highly vascularized nature of isknv lesions in its natural host, the chinese mandarin fish. although these results suggest that isknv orf r plays a vital role in host infection and viral replication, its precise function remains unclear. following orf virus (family poxviridae) infection, the vvegf homolog aids in scab formation and wound healing and may enhance transmission since scabs contains substantial amounts of infectious virus. a novel histone-binding protein was identified in sgiv by structural analysis of the orf l gene product [ ] . x-ray diffraction analysis of recombinant l determined the crystal structure at a resolution of . Å, and revealed that l exhibited partial structural resemblance to the histone-binding region of anti-silencing factor (asf ), a histone h /h chaperon. using recombinant l protein, interaction was demonstrated between l and histone h /h complexes and h by isothermal titration calorimetry. the authors suggested that l may be involved in both the regulation and expression of histone h and h methylation, events which allow the virus to control host cell gene expression and facilitate viral replication. sgiv orf r encodes a homolog of the fv k immediate early protein. although kd studies using an asmo targeted against k [ ] and an k knock out mutant [ ] indicated that expression of k was not required for replication in either fhm or bhk cells, replication of the k ko in xenopus laevis tadpoles was reduced about -fold compared to wt virus or a "knock in" mutant. to determine the intracellular location of the k gene product, xia et al. [ ] generated recombinant sgiv k protein and used it to produce rabbit anti- k serum. immunofluorescent assay showed sgiv k to be distributed within the cytoplasm adjacent to vas and nuclei, and western blotting using purified and disrupted virions indicated k was a non-envelope virion-associated protein. transfection of uninfected grouper cells with a vector expressing k enhanced cellular growth rates and led to an increase in the density of monolayer cultures. moreover, sgiv replication in cultures expressing k were about -fold higher than in cultures transfected with an empty vector. the authors suggest that k may be a protein that plays a role in the control of cellular growth and thus indirectly enhances sgiv replication. however, because virus yields were expressed as tcid /ml, it was not clear if the increase in sgiv yield reflected higher virus production per cell or simply equivalent cellular yields in cultures containing varying numbers of cells. rothenburg et al. [ ] cloned the vif- α gene from rana catesbeiana virus z (rcv-z) into an expression vector and examined its function in a yeast-based assay system. yeast expressing human pkr failed to grow due to the toxicity of the pkr protein. however, yeast expressing vif- α, or the vaccinia virus k l protein, suppressed the toxic effects of human pkr indicating that vif- α was functionally equivalent to the vaccinia virus protein. subsequent work showed that whereas k l was effective only against human pkr, vif- α suppressed the toxic effects of both human and zebrafish pkr suggesting that pkr antagonists evolved to protect physiologically-relevant/phylogeneticallyrelated targets. in addition, a study using vectors expressing the various domains of vif- α, i.e., n-terminal, central/helical, and c-terminal, demonstrated that the n-terminal and central/helical domains were sufficient for suppressing pkr toxicity. collectively, these results provide the first formal proof that vif- α functions as a virus-encoded pkr antagonist. recombinant versions of thymidylate synthase (lcdv-china), erv (rana grylio virus, rgv), dutpase (rgv), rnase iii (rbiv), and litaf (sgiv) were generated and evaluated for their ability to influence cellular growth, enhance virus replication, induce apoptosis, and cleave dsrna [ , , , ] . recombinant thymidylate synthase from lcdv-china was found to promote cell cycle progression and produced a transformed phenotype [ ] . antibodies directed against rana gyrlio virus (rgv) erv detected erv expression within the cytoplasm and nucleus, but not within vas; transcript analysis indicated that erv was a late viral gene product [ ] . rgv dutpase was determined to be a de gene product that was localized to the cytoplasm. ectopic expression did not enhance virus replication, however, its effect on cellular proliferation was not examined [ ] . recombinant rnase iii from rbiv cleaved dsrna, but the salt optimum for cleavage was inconsistent with a physiological effect [ ] . the authors speculated that rnase iii might play a role in the suppression of rna interference, as has been suggested for other viral dsrna-binding proteins, or could be involved in the processing of viral rna. lastly, huang et al. [ ] demonstrated that the sgiv homolog of litaf (lipopolysaccharide-induced tnf-α factor) was a de viral gene product. litaf was located within the cytoplasm and was associated with mitochondria. when over-expressed ectopically, it led to the activation of caspase and the induction of apoptosis. collectively, ts mutants, asmo-and sirna-mediated knock down, gene knock out, and recombinant protein studies are slowly elucidating the function of iridovirid genes and providing a clearer and more complete picture of how those genes enhance viral replication and modulate cellular functions. future studies of ranaviruses and other iridoviruses infecting ectothermic animals will focus on the following areas: ( ) identifying and elucidating the function of replicative genes and those contributing to virulence, host range, and immune evasion; ( ) understanding the correlates of antiviral immunity in an effort to understand the cellular and molecular basis of anti-viral immunity and to protect endangered and commercially important species from infection; ( ) determining the impact of iridovirus infections in nature, defining viral host range, identifying susceptible hosts and reservoir species; ( ) understanding how intrinsic (e.g., host immune suppression, host mhc repertoire, etc.) and extrinsic (e.g., habitat disruption, environmental stress, introduction of invasive species, etc.) factors influence the clinical outcomes of iridovirus infection. for example, determining whether iridovirids encode viral immune evasion proteins and how these proteins circumvent host immunity will highlight events at the interface of virology and immunology, and may identify possible targets for viral attenuation. successful completion of these studies will involve the interactive efforts of virologists, immunologists, population biologists, ecologists, and veterinary pathologists. the global ranavirus consortium [ ] is but one interactive group of scientists interested in the role of 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initiation factor alpha homologue the eif- alpha protein kinases, regulators of translation in eukaryotes from yeast to humans inhibition of pkr by rna and dna viruses improved knockout methodology reveals that frog virus mutants lacking either the k immediate-early gene or the truncated vif- alpha gene are defective for replication in vivo construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication identification and characterization of singapore grouper iridovirus (sgiv) orf l, an immediate-early gene involved in cell growth control and viral replication orf r, a highly abundant virion protein from singapore grouper iridovirus, is involved in serine/threonine phosphorylation and virion assembly a novel histone h binding protein orf l from the singapore grouper iridovirus (sgiv) characterization of singapore grouper iridovirus (sgiv) orf r, a putative homolog of icp involved in cell growth control and virus replication cloning, expression and subcellular distribution of a rana grylio virus late gene encoding erv homologue identification and characterization of a putative lipopolysaccharide-induced tnf-alpha factor (litaf) homolog from singapore grouper iridovirus constitutive expression of thymidylate synthase from lcdv-c induces a transformed phenoptype in fish cells we would like to thank robert sample for the electron micrographs shown in figure and the artwork depicted in figure . the work was partially supported by nsf award no. ios - . the authors declare no conflict of interest. key: cord- - mw q m authors: bomsel, morgane; alfsen, annette title: entry of viruses through the epithelial barrier: pathogenic trickery date: journal: nat rev mol cell biol doi: . /nrm sha: doc_id: cord_uid: mw q m mucosal surfaces — such as the lining of the gut or the reproductive tract — are the main point of entry for viruses into the body. as such, almost all viruses interact with epithelial cells, and make use of the normal epithelial signalling and trafficking pathways of the host cell. in addition to protein receptors, carbohydrate chains of proteoglycans and epithelial-membrane glycosphingolipids have emerged as a new class of receptors for viral attachment to the host cell. viruses enter the body through two main surfaces -the skin and the mucosal epithelia. these surfaces are covered by epithelial cells that are organized into complex structures (box ) , and this epithelial organization often dictates the mechanisms of viral entry and translocation. in this review, we explore the strategies that viruses have evolved to translocate across the epithelial barrier and to act as pathogens, according to the target-cell structure and the nature of the virus. to understand these mechanisms, we first map the pathways of viral entry. then we describe, at the molecular level, the cell receptors that allow viral attachment and entry into the cell, as well as the viral proteins that interact withand subvert -these receptors, which allows the virus to cross the cell membrane. our understanding of host-virus interactions at the molecular level has allowed the characterization of many viral receptors at the epithelial surface. the accepted model of viral entry that is achieved using a unique viral receptor has been challenged. instead, viruses are now thought to use host-cell moleculeswhich are referred to as attachment receptors or 'coreceptors' -in addition to the protein receptor, which is often renamed the 'principal' receptor. the dynamics of these receptors in the membrane is crucial because, on binding of the virus, receptors can be recruited to or excluded from transiently organized glycosphingolipidrich membrane microdomains, which are known as lipid rafts. an important class of attachment receptors has emerged recently -namely the carbohydrate chain of proteoglycans and epithelial-membrane glycosphingolipids (fig. ) . there is growing recognition of the functional importance of these biomolecules, as shown by the emergence of the field of 'glycomics' , or 'the sugar code' . indeed, oligosaccharides transfer information to complementary effector molecules, and the virus acts as a lectin by binding the carbohydrate as an attachment receptor. owing to the nature of the lectin-sugar interaction (namely charge-transfer processes that are facilitated by networks of hydrogen-bond formation), environmental factors are important. these factors relate to the characteristics of the mucosal surface, which has numerous carbohydrate groups protruding into the aqueous environment and a high ionic strength.this high ionic strength is due to the charged surface lipids and their counterions. on the viral oligomeric peptide, the lectin site is characterized by the charged amino acids and the aromatic tryptophan that surrounds the galactose ring. viruses have evolved several pathways to initiate entry through epithelial barriers. viruses can enter and infect epithelial cells by accessing the cell cytosol using one of two mechanisms -direct entry at the epithelial plasma membrane, or entry through the epithelial endocytic pathway . by contrast, some viruses do not need to infect epithelial cells to spread -they are internalized entry of viruses through the epithelial barrier: pathogenic trickery mucosal surfaces -such as the lining of the gut or the reproductive tract -are the main point of entry for viruses into the body. as such, almost all viruses interact with epithelial cells, and make use of the normal epithelial signalling and trafficking pathways of the host cell. in addition to protein receptors, carbohydrate chains of proteoglycans and epithelial-membrane glycosphingolipids have emerged as a new class of receptors for viral attachment to the host cell. surface protein that is present in latently infected people. the complex binds to the poly-immunoglobulin receptor at the basal surface of epithelial cells, and is endocytosed and delivered apically without infection . by contrast, in non-polarized cells, the entry of iga-ebv leads to infection. two ebv replicative proteins can stimulate the production of anti-ebv iga , , thereby contributing to the development of disease in latently infected people. finally, peyer's patches -specific lymphoid areas of the gastrointestinal tract -are covered by specialized epithelial cells, which are known as m cells. these patches might be one portal for the mucosal entry of poliovirus , hiv- and reovirus , by transcytosis across m cells in a receptor-mediated fashion. viruses can penetrate epithelial cells at the epithelial-cell plasma membrane directly after attachment and fusion. in polarized monostratified epithelium -where the plasma membrane is divided into two domains that have a different lipid and protein composition and different membrane dynamics -viruses usually attach and penetrate the cell cytosol preferentially at the restricted pole of the by epithelial cells and cross the epithelial barrier using transcytosis (box ), as has been described recently for the human immunodeficiency virus (hiv) , . in viruses, the genome is surrounded either solely by a capsid ('naked' virus), or by both a capsid and a membrane ('enveloped' virus; box ) . we know much about the entry mechanism of enveloped viruses, but the mechanism by which naked viruses penetrate epithelial cells is far less well understood. nevertheless, the route by which viruses enter and then infect or cross epithelial cells is not dictated solely by whether or not the virus is enveloped , , and the three main modes of entry are summarized in the text below (table ) . several enveloped and naked viruses, such as hiv- (fig. ) and poliovirus, cross simple epithelial cells by rapid transcytosis without infection. they are released -and are still infectious -at the pole of the cell that is opposite to their pole of entry, and this enables the virus to spread in the submucosa. the epstein-barr virus (ebv) uses a more complicated strategy. ebv forms a complex with mucosal immunoglobulin (ig)a that is specific for gp , a viral transcytosis a rapid and selective vesicular transcellular pathway that is characteristic of polarized epithelia. cargo is transported from one pole of the cell to the opposite pole. the cargo remains enclosed in transcytotic vesicles, which precludes access to the cytosol and therefore viral infection of epithelial cells. this receptor is expressed at the basolateral surface of epithelial cells, allowing specific transcytosis towards the apical pole of mucosal dimeric iga or pentameric igm. at the apical pole, after cleavage of the extracellular region of the receptor, which is known as secretory component (sc), the mucosal iga or igm is released with sc as secretory iga or igm, and can act as the first defence against pathogens. | proteoglycans and glycosphingolipids. a | proteoglycans are proteins that are classified by a post-translational attachment of polysaccharide glycosaminoglycan moieties, which are each composed of repeating disaccharide units. the ubiquitously expressed glycosaminoglycan heparan sulphate (shown here) is highly polymorphic, and its sulphated structural motifs are primarily responsible for its protein binding and regulatory properties. b | glycosphingolipids are anchored in the outer leaflet of the plasma-membrane bilayer by their common hydrophobic backbone, ceramide, which consists of a fatty-acid chain linked to the sphingosine base. the hydrophilic carbohydrate parts of neutral glycosphingolipids and gangliosides protrude into the extracellular space and partially cover the cell surface. galnac, (oso ), n-acetyl galactosamine sulphate; glcnso , glucosamine sulphate; glcua, glucuronic acid; idua (oso ), iduronic acid sulphate. chondroitin- -sulphate epithelial cell. some enveloped viruses attach to, and fuse with, the epithelial-cell apical membrane [ ] [ ] [ ] , whereas other enveloped viruses attach to, and fuse with, the basal membrane. importantly, viruses from the same family do not always show the same polarity of entry , , . this is due, in part, to the use of different cell receptors. however, some viruses such as poliovirus can enter at both the plasma membrane and through the endocytic pathway , (see below). both enveloped and naked viruses can enter epithelial cells by endocytosis, and they usually penetrate the host-cell cytosol by fusion from an endosome. this mode of entry makes use of specific endosomal conditions such as low ph or a high concentration of calcium. in polarized monostratified epithelium, the polarity of viral entry is a determinant for the outcome of an infection, as polarity differentially influences the processing and sorting mechanisms in apical and basolateral endosomes. however, the nature of the experimental system can change the polarity of viral entry . some enveloped viruses, such as influenza virus types a and c, bovine coronavirus and hepatitis a virus (hav) , are endocytosed at the apical pole of polarized epithelial cells, whereas vesicular stomatitis virus (vsv), which is also enveloped, is endocytosed basolaterally . for all of these viruses, however, access to the cytosol from the endosome is ph dependent. other viruses can enter by endocytosis at either of the poles in epithelial cell lines , but each route results most viruses contain two or three elements: the genome, in the form of single-stranded (ss) or double-stranded (ds) dna or rna; the capsid, which consists of viral proteins; and, possibly, an envelope, which originates from the host cell and consists of host-cell lipids that are organized as a bilayer. viral-envelope glycoproteins as well as, in some cases, selected host-cell proteins can be recruited to the envelope.'naked' viruses contain only the genetic material surrounded by the capsid. by contrast, in 'enveloped' viruses, the genome is surrounded by a capsid and is protected further by the viral envelope. [ ] [ ] [ ] at either one of the poles followed by a ph-independent translocation into the cytoplasm. the outcome is either efficient viral replication followed by host-cell lysis, or, alternatively, latent infection. in the latter case, reactivation of the latent viral genome might then occur, and lead to the infection of nearby sensory neurons and the release of new virions. these virions can then re-enter epithelial cells -but now in an highly polarized manner -by fusion at the basolateral pole . naked adenoassociated virus (aav)- enters airway epithelial cells essentially basolaterally by receptor-mediated endocytosis . its single-stranded (ss)dna is then converted to a circular genome, and its polarity of entry is reinforced by a post-endocytic barrier. by contrast, aav- that enters at the apical pole by a receptor-independent mechanism gives rise to persistent ssdna that is arrested in a transcriptionally inactive form. the ubiquitin-proteasome pathway, which is involved in the regulation of endocytosis , is instrumental in this arrest . in pluristratified epithelia or immature gastrointestinal cells, which lack tight junctions, viral entry is not polarized. instead, the viruses make use of various endocytic pathways (box ) . filovirus, influenza virus, simian virus (sv ) and measles virus are endocytosed after clustering in transient raft membrane microdomains [ ] [ ] [ ] [ ] [ ] . some enveloped viruses, such as the semliki forest virus (sfv) and sindbis virus, as well as some naked viruses, including jamestown canyon (jc) polyoma virus, parvovirus, adenovirus- , two of the picorna viruses -human echovirus- (e- ) and the minor group of human rhinoviruses (hrv) -and foot and mouth disease virus (fmdv), use receptor-mediated endocytosis through the clathrin-coated-vesicle pathway [ ] [ ] [ ] [ ] [ ] . once they are in endosomes, the surface proteins of viruses such as influenza, fmdv, vsv, sindbis virus or sfv undergo a conformational change that is dependent on a mildly acidic ph, and they can then disrupt the endosomal membrane. alternatively, other viruses, such as the poliovirus , and the major group of hrv , translocate from the endosome into the cell cytosol in a ph-insensitive manner, before gaining access to the lysosome -a compartment that is deeper in the endocytic pathway , . a ca + -dependent, but ph-independent, endocytosis and virusuncoating model has been proposed for the rotavirus that would apply equally to other viruses, whether they are enveloped or naked. the differences in the viral-entry pathways are due largely to the nature of the molecular interactions between the viral components and target-cell receptors. viral pathogenesis arises from mechanisms that have been developed m cell 'membranous' or 'microfold' cell. this is a specialized epithelial cell covering the lymphoid peyer's patches in the gut. m cells can internalize macromolecules and microorganisms and deliver them to the underlying lymphoid tissue. endosome a membranous transport vesicle that is involved in endocytosis. a protein heterocomplex that connects neighbouring simple epithelial cells and controls the barrier function of the tight mucosal surface. an invagination of the plasma membrane that is surrounded by clathrin, a cytosolic protein that is formed by a triskelion of three heavy and three light chains. triskelions assemble into a polyhedral lattice to form the clathrin coat. process of viral infection is difficult to assess. indeed, at the molecular level, the kinetics of each virus-host-cell interaction is dependent on its association constant, the concentration of viral ligands and of host-cell receptors that are available to interact at any given moment, and the nature of the target cell. to attach to the surface of the target cell, an increasing number of enveloped or naked viruses have been described as acting as lectins, by using a peptide of their envelope or capsid proteins, respectively, that has a lectin site. they compete with endogenous lectins to bind epithelial-cell-surface carbohydrates, which act as attachment receptors. viral surface proteins are multimeric, they have several lectin sites, so they can interact with several receptor molecules at a time at the host-cell surface. such clusters of lectin sites have a much higher affinity for oligosaccharides than their monomeric counterparts . this multimeric interaction interferes with lipid organization and dynamics, and stabilizes raft microdomains at the epithelial-cell membrane. the lipids in these domains differ from other membrane lipids in their lateral diffusion in the membrane, and they can be separated in vitro, owing to their insolubility in detergent. the rafts are small, mobile, unstable and they probably fluctuate in their size and composition as a result of molecular interactions at the cell surface. attachment receptors and viral receptors can be recruited to, or excluded from, membrane microdomains such as rafts, clathrin-coated pits or caveolae , . this remodelling of the host-cell membrane is a determinant of the mechanism of virus entry and signal transduction in the host epithelial cell . the carbohydrate moiety of host-cell glycoproteins, glycosphingolipids and proteoglycans has emerged as a widely used virus-attachment receptor . sialyloligosaccharides. influenza virus contains two major surface proteins that are involved in viral entry -haemagglutinin and neuraminidase -which bind and cleave sialyloligosaccharide, respectively. balanced haemagglutinin and neuraminidase activities are crucial for efficient viral binding to the cell surface, and for viral replication. influenza virus haemagglutinin is one of the best-known viral lectins. its trimeric organization increases its affinity for sialyloligosaccharide and allows the virus to bind to the surface of the epithelial cell . the epithelial molecules that contain this sialyloligosaccharide carbohydrate receptor remain unknown, although they could be glycoproteins or glycosphingolipids . on endocytosis of the influenza virus, haemagglutinin is cleaved into haemagglutinin and haemagglutinin . whereas low endosomal ph exposes the haemagglutinin fusionpeptide domain, haemagglutinin mediates viral fusion with the endosomal membrane . similarly, human jc virus and sendai virus, as well as sialyloligosaccharide-dependent strains of rotavirus and reovirus, also attach themselves to epithelial cells to block or abuse normal cell processes and, as with bacteria , the surface proteins of enveloped or naked viruses bind to host-cell molecules that have receptor functions. so, viruses mimic the natural ligand of these receptors and interfere with their signalling to promote viral entry into the cell and the spread of infection (box ) . the classical concept of viral receptors has been superseded by new data, which indicate that the binding and entry of viruses is a multi-step process that involves the recognition of, and attachment to, the epithelial-cell surface. this is followed either by penetration of the host-cell cytosol, with infection of the cell, or by transcytosis. each step involves many host-cell receptors. these receptors range from ubiquitous cell-surface-associated carbohydrate moieties of membrane glycoproteins, proteoglycans or glycolipids -which are inserted in the dynamic bilayer of the target-cell membrane and usually act as attachment receptors -to cell-specific transmembrane proteins, which can mediate numerous different steps. these many steps are not independent of each other, but their spatio-temporal sequence in the . both contain viruses that use either the chemokine receptor ccr (r virus; found in most acutely infected patients, and therefore thought to be the main vector of infection) or cxcr (x virus; found later in patients, as the disease progresses) for fusion and/or infection. in the upper small intestine, galactosyl-ceramide (galcer) + /ccr + /cxcr − epithelial cells endocytose cell-free r virus at the lumenal surface in a galactosyl ceramide/ccr -receptor-mediated mechanism, whereas cellfree x viruses cannot enter these cxcr epithelial cells . alternatively, r (or x )-infected mononuclear cells bind to the epithelial cell and induce the polarized budding of newly formed r (or x ) viruses, which are rapidly endocytosed through galcer that is present in raft microdomains , . hiv transcytoses across epithelial cells to the serosal surface, where fusion of the transcytotic vesicles releases virus into the lamina propria. here, r viruses infect the ccr + /cxcr + /cd + lamina propria t lymphocyte, but not the ccr − /cxcr − /cd + intestinal macrophage . by contrast, the infection of intestinal cxcr + /cd + t cells by x viruses that are transcytosed from x -infected cells is blocked by stromal-cell-derived factor (sdf)- that is present in the mucosa assembled in rafts for virus attachment and entry into the epithelial cell. this virus-cell interaction, which involves several species of viral surface protein, probably stabilizes raft microdomains, allowing signal transduction in the epithelial cell and endocytosis of the virus. a galactosylceramide/sphingomyelin-binding motif, which is similar to that found in hiv- gp , is also found on prion protein and the amyloid-β peptide that is involved in alzheimer's disease . proteoglycans. another class of carbohydrate attachment receptors used by viruses are found on proteoglycans. proteoglycans are proteins that are classified by the post-translational attachment of polysaccharide glycosaminoglycan moieties (fig. ) . glycosaminoglycan chains provide the initial docking sites for viruses to bind to eukaryotic cells. the ubiquitously expressed glycosaminoglycan heparan sulphate is highly polymorphic, and its sulphated structural motifs are responsible primarily for its protein binding and regulatory properties, as shown recently for respiratory syncytial virus (rsv). during the past decade, proteoglycans have emerged as key players in the regulatory network of the cell . depending on the length of the glycosaminoglycan chain, and on the sulphated structural motifs, a single glycosaminoglycan chain can bind many viral ligands on a single virion . as the cell attachment receptors for numerous enveloped and naked viruses, the glycosyl epitopes of the epithelial-cell-surface proteoglycans mediate virus adhesion, in turn initiating signal transduction as described for the glycosynapse . hsv- uses glycosaminoglycan as a receptor for entry into epithelial cells as well as into primary neuronal cells. there are five hsv- glycoproteins -gb, gc, gd, gh and gl -and two of these (gb and gc) mediate the attachment of the virus to cellular heparan sulphate , . human herpesvirus (hhv- ), which is associated with kaposi's sarcoma, has a broad cellular tropism that includes epithelial cells. this broad tropism might be due, at least in part, to interaction of the viral surface glycoproteins with heparan sulphate . enveloped rous sarcoma virus (rsv) and adenovirus- and - (ref. ), as well as several naked viruses including echovirus, aav- , human papilloma virus (hpv)- , hpv- and fmdv (refs , , ( ) ( ) ( ) ( ) , interact with heparan sulphate proteoglycans, which facilitates the attachment and infectivity of the virus. the use of heparan sulphate as an alternative receptor is likely to be the result of an adaptation to growth in cell lines . paradoxically, the binding of a virus to heparan sulphate might prevent the virus from reaching the cell surface. indeed, heparan sulphates are present in proteoglycans in the extracellular matrix, and these noncellular matrix structures can bind viruses. the basal lamina, for example, is a barrier to the spread of hsv- from, and back into, epithelial cells. in addition, certain bodily fluids contain heparin, heparan sulphate and heparin-binding proteins, all of which can compete with and inhibit the binding of viruses to cell-surface heparan sulphate . through sialyloligosaccharides, which are either from glycosphingolipids or proteoglycans . glycosphingolipids. several enveloped and naked viruses have recently been shown to interact specifically with a defined carbohydrate moiety of glycosphingolipids (fig. ; table ). glycosphingolipids -which are characteristic components of eukaryotic plasma membranes -are a highly polymorphic class of lipids and are principal components of the apical plasma membrane of epithelial cells in the gastrointestinal and urinary tracts, myelin and neuroepithelial cells . glycosphingolipids are anchored in the outer leaflet of the plasma membrane bilayer by their common hydrophobic backbone, ceramide, which consists of a fatty-acid chain that is linked to the sphingosine base. the hydrophilic oligosaccharide residues of neutral glycosphingolipids and gangliosides protrude into the extracellular space and, together with the membrane glycoproteins and proteoglycans, they constitute the glycocalyx of the cell surface . the multimeric glycoprotein subunits gp and gp (refs , ) of the hiv- envelope both attach to the cell-membrane glycosphingolipid, galactosylceramide. indeed, d-galactose is particularly eligible for stacking to the aromatic ring system of gp owing to the van der waals interactions and the presence of a set of polarized c-h bonds (c-h/π-electrons) on one side of the galactose ring. interestingly, hiv- and herpesvirus can enter neural cells (which are also polarized) and mucosal epithelial cells, both of which are rich in glycosphingolipids . hiv- (ref. ), naked ebola and marburg viruses (through their capsid proteins) and measles virus , require glycosphingolipids to be as an alternative, primary cells can be used. however, the purification of primary cells results in the destruction of epithelial-barrier function, and polarity is lost because primary cells cannot re-form tight junctions in vitro. biopsies that are mounted at the interface between two chambers maintain their epithelial architecture and barrier function for a few hours. recently, sheets of epithelial cells with intact cell junctions have been purified from the gastrointestinal tract, but the lifespan of these cells is less than one day. a new direction for the experimental models has emerged by taking into account the interaction of epithelial cells with submucosal cells. pringault's group succeeded in reconstructing an epithelial barrier similar to those that cover lymphoid organs, which included m cells. they did this by co-culturing transformed intestinal epithelial cells with primary intraepithelial lymphocytes. such co-culture systems preserve the barrier function of monostratified primary epithelial cells. hela (cervical carcinoma) cells have been widely used as pluristratified epithelium, which is mainly because they grow well and are easily transfectable. however, they differ considerably from primary cells, which are difficult to maintain in culture as an organized tissue. sialyloligosaccharide an oligosaccharide chain that is linked to a terminal sialic acid (n-acetyl neuraminic acid). glycosaminoglycan the polysaccharide moiety of proteoglycans, which is added posttranslationally and is composed of repeating disaccharide units. one of the glycosaminoglycan parts of proteoglycans, this is a long, polyanionic carbohydrate chain that consists of a repeating disaccharide unit. glycosynapse a membrane structure that provides a connection between two cells, and is involved in a glycosylation-dependent celladhesion/recognition processes. www.nature.com/reviews/molcellbio r e v i e w s epithelial cell surface, but hpev- then enters the host cell through the clathrin-dependent endocytic pathway . as an alternative, viral proteins might use other motifs to bind rgd-sensitive integrins in an rgdindependent fashion (that is, the viral protein does not act as a physiological counter-ligand). for example, rotaviral vp protein binds the rgd-sensitive integrin-α β , which is basolaterally expressed (or, alternatively, it binds integrin-α v β ), as a post-attachment receptor through the gde(a) (glycine-aspartic acid-glutamic acid/alanine) amino-acid motif of the viral protein , , . when both of these integrins are expressed at the epithelial-cell surface, they work together to promote viral entry . the rotaviral vp protein interacts with integrin-α x β through a grp (glycine-arginine-proline) motif, and integrin-α β through an lvd (leucine-valine-aspartic acid) amino-acid motif . direct interaction between vp and vp has been observed. adenoviruses use also integrin-α v as a receptor to mediate their endocytosis, but binding of the virus to the integrin activates a signalling pathway that is distinct from the physiological one . echoviruses bind to integrinα β , and during viral entry, caveolin and integrin-α β co-localize with e- capsid proteins and migrate into the perinuclear area in the cell . other adhesion molecules, such as intercellular adhesion molecule (icam ), function as viral receptors on epithelial cells. the major group of hrv uses icam as the viral receptor . hrvs have a cleft that encircles the fivefold axes of icosahedral symmetry, which accommodates the amino-terminal domain of icam . another widely used class of receptor consists of components of the epithelialcell tight junctions. the use of such receptors to penetrate epithelial-cell cytosol at the epithelial cell-cell junction implies that the junction complex is disrupted, with an immediate effect on the integrity of the epithelial barrier. whereas the interaction of viral components with components of the epithelial tight junction has been described biochemically , how viruses access such receptors -which are 'hidden'in the tight junction in vivo -is difficult to clarify, despite recent studies on cell lines . two cell-surface glycoproteins -the coxsackievirus and adenovirus receptor (car) and the junction-adhesion molecule (jam) -were identified as transmembrane components of the tight junction in epithelial cells, as well as being entry receptors for coxsackie virus/adenovirus and reovirus, respectively , . owing to the localization of car at the tight junction, infection by both adenovirus and coxsackievirus in vivo could require the destruction of the tight-junction complex for the virus to be able to access its receptor, as is the case in vitro . such destruction might be involved in regulating tissue-specific inflammatory responses to viral infection . coxsackievirus binds car through a 'canyon' at the surface of the virus. by finally, the glycosylation state of epithelial-cell-surface proteins and lipids varies with the differentiation, ageing and activation of the cell, and such modulation of surface carbohydrates has an evident effect on the susceptibility of epithelial cells to viral infection . several classes of protein receptor, which often show a polarized distribution, are used opportunistically by viruses to attach to and infect cells. a protein receptor either mediates these sequential steps by itself, or it has to cooperate with attachment receptors. as mentioned above, few studies describe the mechanism of such cooperativity. integrins are a class of surface molecules that are used by several viruses (enveloped and naked) to attach to and infect epithelial cells. one class of integrin, the cellular role of which is to maintain cell-cell contact, is expressed basolaterally in cell culture and in gut tissue adjacent to the tight-junction complex on the basolateral pole. however, other integrins, such as α β , are expressed apically in crypt and villus enterocytes throughout the intestine. integrins that are expressed at the cell surface bind to ligands that are referred to as disintegrins. these disintegrins contain motifs of several amino acids that are specific to each integrin. one of these motifs -rgd (arginine-glycine-aspartic acid) -is specific to a set of integrins that are known as rgd-sensitive integrins. disintegrin motifs are found in viral surface proteins, which allows the virus to bind integrins, and thereby interferes with the bona fide ligand. a restricted set of rgd-sensitive integrins often seems to be used by viruses , . several viruses bind rgd-sensitive integrins in an rgd-dependent manner . for example, the rgd motif of the hhv- envelope glycoprotein b interacts with integrin-α β to allow attachment of hhv- to (and the infection of) epithelial cells and use of the integrin signalling pathway . the rgd-containing capsid protein vp of fmdv attaches to the integrin-α v β , which is expressed on primary epithelial cells, then uses the signalling pathways that are initiated at the integrinβ cytoplasmic domain . the rgd-containing capsid protein vp of human parechovirus (hpev- ) also interacts with integrins -α v β and α v β -on the viruses can contact the epithelium as two forms -as cell-free viral particles or as an infected cell. most studies have been carried out using cell-free particles. however, the importance of infected cells has been revisited. it is known empirically that target-cell infection is usually more efficient using cell-associated viruses, but the mechanism remains unclear. recent data on human immunodeficiency virus (hiv) , and epstein-barr virus (ebv) indicate that, by interacting with the epithelial target cell, infected cells might start to bud newly formed cell-free viral particles. these particles interact differently with epithelial cells than does an isolated cell-free virus inoculum. this might be due to the differences in the viral-envelope composition or in the contact between the infected cell and epithelial cells. enterocyte an intestinal epithelial cell that is organized in monostratified layers. family , , cd (ref. ; together with its co-receptor epithelial moesin), and the n-aminopeptidase cd (refs , ) , which can dimerize and probably transduce signals. the glycosylphosphatidylinositol (gpi)-anchored complement regulatory protein daf (decay acceleration factor) is widely used by enteroviruses, including echoviruses, human enterovirus (ev)- , coxsackievirus types b and a (ref. ), as well as other gpi-anchored proteins. these gpi-anchored proteins, which are localized in apical rafts, can be endocytosed by a rho-gtpase-dependent mechanism (fig. ) . so, as shown recently for e- , they can mediate internalization of the virus in the endosome before translocation into the epithelial-cell cytosol . molecules that are expressed basolaterally include the transferrin receptor. by contrast, viruses can use non-polarized protein receptors , and so they need a polarized carbohydrate co-receptor for entry. the interaction between a cellular host and a viral pathogen is an important field of research, both for unravelling polarized membrane trafficking and for understanding epithelial pathology (for example, see contrast, the terminal knob portion of the fibre protein of human adenovirus (hadv)- (ref. ) binds to car. this allows the viral capsid penton base protein to bind cell-surface integrin-α v β and its subsequent endocytosis in clathrin-coated vesicles . nectin (also known as poliovirus-related protein or hvec) is a ca + -independent cell-adhesion molecule that is localized at cadherin-based intercellular junctions. it is used by α-hsv- and α-hsv- for entry and infection after attachment to epithelialcell heparan sulphate proteoglycan. in reovirus infection, human jam -an integral membrane protein that organizes the tight junctions of epithelial cellsbinds the head domain of the viral σ outer-capsid protein. it binds at the basolateral (serosal) pole of the epithelial layer, after reovirus has undergone transcytosis across m cells . jam functions as a serotype-independent receptor that can mediate virus attachment and infection . other classes of polarized receptor. several protein receptors have a polarized distribution, which allows viral entry into epithelial cells, or cell resistance to infection when a virus enters epithelial cells by the opposite pole. apical viral receptors include the carcino-embryonic antigen during the first cycle of rotavirus replication in mucosal epithelial cells, the synthesis of rotaviral proteins in the cell cytoplasm leads to an increase in the plasma-membrane permeability to ca + , to activation of regulatory mechanisms and to an increase in the concentration of ca + in the endoplasmic reticulum (er). the increased concentration of cytosolic ca + in infected cells promotes the activation of ca + -dependent enzymes, which in turn induces cell lysis and the release of viral proteins and viral progeny. non-structural protein (nsp)- might act as a viral enterotoxin on as-yet-uninfected cells to induce secretory diarrhoea through a | ca + -dependent secretion by intestinal cells, b | ca + -dependent secretion of peptides and amines to stimulate the enteric nervous system (ens), and c | further activation of epithelial-cell chloride (cl -) secretion by the ens. in parallel, released virus infects downstream absorptive cells. this will lead to a massive cell death and, as a consequence, reduction of the absorptive surface of the intestinal epithelium and an osmotic component of diarrhoea . various forms of the virus along the rotavirus-maturation pathway are shown: dlp, double-layer particle; imp, intramembrane particle; ins( , , )p , inositol , , -trisphosphate; plc, phospholipase c; serca, sarcoplasmic/endoplasmic-reticulum ca + -atpase; tlp, triple-layer particle. cell type directly involved in the pathology -rather than receptor molecules transfected in standard cell-line models -could help. it will also be important to define the environmental parameters that influence binding of the virus to its receptors, and interaction with co-receptors, especially when considering lectin-sugar interactions. the use of new techniques that involve dynamic fluorescence microscopy will help, both for mapping the viral pathway inside epithelial cells and also for studying signalling in epithelial cells. the first step of viral attachment to the cell -through either proteoglycan or glycosphingolipids -raises the question of the specific role of the glycosyl epitope, not only in viral attachment, but also in further transduction of signals. the final glycosylation state of glycoproteins and lipids varies with cell differentiation, maturation or ageing , and it could govern the specificity of the virus-carbohydrate interaction that is involved in viral entry into epithelial cells. lipid microdomains participate in the clustering of viral proteins on the viral membrane and cell receptors on the cell membranes, which favours virus-cell interactions. the spatio-temporal correlation of the different steps of the virus attachment to carbohydrates of the cell surface, and their further interaction with additional protein cell receptors or co-receptors, remains to be established. clearly, epithelia are more than just a physical barrier. they are a dynamic host defence system with sensor molecules, signalling circuits and effector molecules that coordinate and execute a graduated reaction to microbes. we are now on the verge of learning more about the molecules and pathways that are involved in these epithelial responses. it seems clear that no real correlation can be made between a family (or even a type) of virus and a defined mechanism of interaction with the epithelial cell.viruses from families as different as naked and enveloped viruses can use the same attachment receptors , (for example, carbohydrates or proteins). conversely, closely related viruses from the same family might use completely different attachment receptors that will dictate the intracellular fate of the virus , . even the same virus might use the same attachment receptor but different protein receptors depending on the type of epithelial cell, which extends its infectious potential . the cellular features of the epithelial target therefore seem to be essential if we are to describe the mechanisms of a viral interaction with an epithelial cell. these features are the epithelial trafficking pathways and the epithelial-specific protein and lipid composition that are associated with their signalling pathways. this description emphasizes how viruses act as pathogens -that is, by subverting the normal epithelial cell function to their own benefit. we are just beginning to understand the molecular interactions between viral and cellular components. however, the factors that control the finely regulated specificity that is observed in vivo for each virus, for a defined target cell, remain unclear. the signalling that is induced when a virus binds its receptors should be compared with the pathways that are activated when the natural ligands bind these same receptors. entry receptors for many viruses have now been described, and it will be important to correlate data from both cell lines and primary cells, despite the pitfalls inherent in each system (box ) . in this regard, studies that use the primary cell a cell that is isolated directly from living tissues instead of transformed cells. the sugar code: functional lectinomics virus entry and release in polarized epithelial cells transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier primary intestinal epithelial cells selectively transfer r hiv- to ccr + cells an ex vivo system using human primary epithelial cells to show the selective filter function of an epithelial barrier during a viral infection break ins and break outs: viral interactions with the cytoskeleton of mammalian cells epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin a-mediated entry by epstein-barr virus epstein-barr virus-encoded lmp and cd mediate il- production in epithelial cells via an nf-κb pathway involving tnf receptor-associated factors detection of epstein-barr viral dna internal repeats in the nasopharyngeal mucosa of chinese with iga/ebv-specific antibodies immunofluorescence analysis of poliovirus receptor expression in peyer's patches of humans, primates, and cd transgenic mice: implications for poliovirus infection transepithelial transport of hiv- by m cells is receptor-mediated intestinal m cells: a pathway for entry of reovirus into the host human coronavirus e infects polarized airway epithelia from the apical surface entry and release of measles virus are polarized in epithelial cells polarized entry and release in epithelial cells of black creek canal virus, a new world hantavirus human cytomegalovirus infection of caco- cells occurs at the basolateral membrane and is differentiation state dependent a first step toward understanding membrane fusion induced by herpes simplex virus disruption of adherens junctions liberates nectin- to serve as receptor for herpes simplex virus and pseudorabies virus entry entry of poliovirus into cells is blocked by valinomycin and concanamycin a uptake of poliovirus into the endosomal system of hela cells interactions between rotavirus and gastrointestinal cells influenza a viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice infection of polarized cultures of human intestinal epithelial cells with hepatitis a virus: vectorial release of progeny virions through apical cellular membranes herpes simplex virus glycoprotein d bound to the human receptor hvea centripetal transport of herpes simplex virus in human retinal pigment epithelial cells in vitro entry of human cytomegalovirus into retinal pigment epithelial and endothelial cells by endocytosis role of apical and basolateral membranes in replication of human cytomegalovirus in polarized retinal pigment epithelial cells herpes virus infection of rpe and mdck cells: polarity of infection polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia the ubiquitin-proteasome system and endocytosis endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly lipid rafts and signal transduction caveolar endocytosis of simian virus is followed by brefeldin a-sensitive transport to the endoplasmic reticulum, where the virus disassembles major histocompatibility complex class i molecules mediate association of sv with caveolae jc virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells entry of human parechovirus the clathrin endocytic pathway in viral infection the structure and function of a foot-andmouth disease virus-oligosaccharide receptor complex this work tracks the entry of comparatively different viruses of the same family into cells by cellfractionation analysis a clear review on the role of calcium in viral infection of the epithelial cell, and the viral soluble virulence factors pathogenic trickery: deception of host cell processes structural basis of lectin-carbohydrate recognition an important review on the characteristics of lectin-carbohydrate interactions that rely on the structure and oligomerization state of the lectin peptide, as well as on the nature and conformation of the carbohydrate a novel cell entry pathway for a daf-using human enterovirus is dependent on lipid rafts cell biology of virus entry herpesviruses and heparansulfate: an intimate relationship in aid of viral entry reference uses herpesviruses to emphasize the role of heparan sulphate -a ubiquitous component of cell proteoglycans -in viral entry into host cells receptor binding and membrane fusion in virus entry: the influenza hemagglutinin broad distribution of the jc virus receptor contrasts with a marked cellular restriction of virus replication sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope among the various ganglioside specificities for different rotavirus strains, a common carbohydrate minimal structural element is shown to be required for binding of these strains using naked reovirus as an example, this paper shows the cooperativity of carbohydrate attachment and protein receptors for virus interaction with epithelial cells sorting of newly synthesized glycosphingolipids to the two surface domains of epithelial cells the cell-to-cell transfer of information through the synapse, which is characteristic of neural cells, has recently been extended to other cell types such as immunological synapses. the importance of carbohydrates in such cellular functions is underlined human immunodeficiency virus and host cell lipids. interesting pathways in research for a new hiv therapy secretory iga specific for a conserved epitope on gp envelope glycoprotein inhibits epithelial transcytosis of hiv- hiv- gp envelope residues - exposed on native virus act as a lectin to bind epithelial cell galactosyl ceramide synthetic soluble analogs of galactosylceramide (galcer) bind to the v domain of hiv- gp and inhibit hiv- -induced fusion and entry lipid sorting in epithelial cells identification of a common sphingolipidbinding domain in alzheimer, prion, and hiv- proteins glycosaminoglycan sulfation requirements for respiratory syncytial virus infection heparan sulfate: decoding a dynamic multifunctional cell regulator an informative review on the structure and function of heparan sulphate viral and cellular requirements for entry of herpes simplex virus type into primary neuronal cells human herpesvirus envelope glycoprotein k . a interaction with the target cells involves heparan sulfate the fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types and echoviruses bind heparan sulfate at the cell surface membrane-associated heparan sulfate proteoglycan is a receptor for adenoassociated virus type virions junction adhesion molecule is a receptor for reovirus human papillomavirus infection requires cell surface heparan sulfate role of the cytoplasmic domain of the β-subunit of integrin α (v) β in infection by foot-and-mouth disease virus initial interaction of herpes simplex virus with cells is binding to heparan sulfate integrin α (v) β ( ) mediates rotavirus cell entry rgd and other recognition sequences for integrins integrin α β (cd c/ ) is a cellular receptor for kaposi's sarcoma-associated herpesvirus (kshv/hhv- ) entry into the target cells vla- (α β ) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells entry of rotaviruses is a multistep process adenovirus endocytosis via α (v) integrins requires phosphoinositide- -oh kinase internalization of echovirus in caveolae viral evolution toward change in receptor usage: adaptation of a major group of human rhinovirus to grow in icam- -negative cells this characterization of the cellular function of the coxsackievirus and adenovirus receptor (car) sheds light on the polarity of the entry of these viruses entry of α-herpesviruses mediated by poliovirus receptor-related protein and poliovirus receptor coronavirus infection of polarized epithelial cells quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors assymetric budding of viruses in epithelial cells: a model system for study of epithelial polarity interaction of the poliovirus receptor with poliovirus endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton gpi-anchored proteins are delivered to recycling endosomes via a distinct cdc -regulated, clathrinindependent pinocytic pathway conversion by peyer's patch lymphocytes of human enterocytes into m cells that transport bacteria the role of cell-to-cell transmission in hiv infection cell-to-cell contact as an efficient mode of epstein-barr virus infection of diverse human epithelial cells biological parameters of hiv- infection in primary intestinal lymphocytes constitutive expression of stromal derived factor- by mucosal epithelia intercellular trafficking and protein delivery by a herpesvirus structural protein role of tight junctions of polarized epithelial mdck cells in the replication of herpes simplex virus type the spread of herpes simplex virus type from trigeminal neurons to the murine cornea: an immunoelectron microscopy study neutrophil chemotaxis induced by corneal epithelial cells after herpes simplex virus type infection human cytomegalovirus infects caco- intestinal epithelial cells basolaterally regardless of the differentiation state patterned entry and egress by epstein-barr virus in polarized cr -positive epithelial cells epstein-barr virus gh is essential for penetration of b cells but also plays a role in attachment of virus to epithelial cells vaccinia virus preferentially enters polarized epithelial cells through the basolateral surface specific interaction of hiv- and hiv- surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside gm respiratory syncytial virus induces selective production of the chemokine rantes by upper airway epithelial cells respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus immunocytochemical colocalization of specific immunoglobulin a with sendai virus protein in infected polarized epithelium polarized budding of measles virus is not determined by viral surface glycoproteins measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells identification of the α integrin as a candidate receptor for papillomaviruses influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins vesicular stomatitis virus glycoprotein does not determine the site of virus release in polarized epithelial cells rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration rotavirus infection of cultured intestinal epithelial cells induces secretion of cxc and cc chemokines reovirus receptors and apoptosis identification of carbohydrate-binding domains in the attachment proteins of type and type reoviruses retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor inhibition of clathrin-dependent endocytosis has multiple effects on human rhinovirus serotype cell entry a novel membrane protein is a mouse mammary tumor virus receptor coronaviruses in polarized epithelial cells infection of polarized epithelial cells with enteric and respiratory tract bovine coronaviruses and release of virus progeny mouse hepatitis virus strain a is released from opposite sides of different epithelial cell types antigenic and molecular analyses reveal that the equine rotavirus strain h- is closely related to porcine, but not equine, rotaviruses: interspecies transmission from pigs to horses? we apologize to our colleagues for omitting references in this review because of space limitations. we thank b. wecksler and v. david for their editing of this manuscript. this work was supported by agence nationale de recherche sur le sida (anrs) and sidac-tion/ensemble contre le sida funds to m.b. the following terms in this article are linked online to: entrez: http://www.ncbi.nlm.nih.gov/entrez/ haemagglutinin | neuraminidase | gb | gc | gd | gh | gl swiss-prot: http://www.expasy.ch/ car | caveolin | cd | cd | daf | icam | integrin-α v | integrin-β | jam | moesin | nectin access to this interactive links box is free online. key: cord- -y gjh authors: wilson, m.r. title: meningitis, viral date: - - journal: encyclopedia of the neurological sciences doi: . /b - - - - . - sha: doc_id: cord_uid: y gjh this article provides an overview of the pathogenesis, epidemiology, causes, clinical presentation, laboratory diagnosis, and treatment of the most common causes of viral meningitis in the united states. it also summarizes other infectious and noninfectious causes of lymphocytic or aseptic meningitis. the concept that agents other than bacteria can invade the central nervous system (cns) began with the emergence of poliomyelitis as an epidemic infection and, subsequently, with the realization that similar meningeal inflammation and cerebrospinal fluid (csf) pleocytosis occurred in as many as % of patients with mumps parotitis. that meningitis could be caused by other 'filterable agents' (i.e., viruses) was demonstrated by rivers and scot, who in recovered lymphocytic choriomeningitis virus (lcmv) from the csf of an affected patient. it is now known that a wide and constantly changing variety of viruses may invade the cns to produce meningitis or encephalitis. viral meningitis is important in three respects. first, viral meningitis must be differentiated from the much more dangerous condition, bacterial meningitis: until this is accomplished, patients presenting with signs and symptoms of meningitis must be considered medical emergencies, and antibiotic treatment for presumptive bacterial meningitis must be instituted. second, viral meningitis, although rarely fatal, may produce clinical impairment that can persist for weeks to months, especially in the immunosuppressed patient. finally, 'lymphocytic' or 'aseptic' meningitis may be caused by agents other than viruses, and the possibility of other, more readily treated (and sometimes more dangerous) conditions must be kept in mind when diagnosing a patient with presumed viral meningitis. before meningitis can occur, the causative agent must first penetrate the body from the external environment and then gain entry to the cns across the blood-brain barrier (bbb). entry of the virus into the host may occur by gastrointestinal inoculation (as is the case with the enteroviruses), cutaneous inoculation (as occurs with the arthropod-borne agents), the respiratory route (as is the case with mumps), or transmucosal penetration or intravenous inoculation (as occurs with human immunodeficiency virus (hiv)). early workers in the field of viral cns infections believed that invasion of the nervous system occurred by the spread of viruses along neurons, as is the case with rabies virus. currently, however, it is known that the majority of viral meningitis results from hematogenous dissemination of virus following symptomatic or clinically inapparent systemic infection. penetration across the bbb may occur at the choroid plexus or through meningeal capillaries. exceptions to this include meningitis following genital herpes simplex infection, meningitis associated with herpes zoster, and mollaret's meningitis, in which reactivated infection of herpes simplex virus type (hsv- ) within the dorsal root ganglia leads to repeated episodes of meningitis. viral replication in the meninges, superficial brain or spinal cord parenchyma, and the ventricular system elicits an inflammatory response, which is predominantly lymphocytic. this results in an alteration of the bbb so that protein levels increase within the csf. unlike bacteria or fungi, however, viral replication does not lower glucose within the csf, nor does it usually results in altered transport of glucose across the bbb. thus, in contrast to bacterial, mycobacterial, or fungal infections, csf glucose concentrations during viral meningitis are usually normal. meningitis is the most common phenotype of a neuroviral infection, and it is a far more common condition than bacterial or fungal meningitis. approximately cases of lymphocytic or presumed viral meningitis occur each year in the united states although numbers are difficult to ascertain because it is not a reportable illness. csf pleocytosis has also been reported in individuals infected with measles, mumps, and hiv without signs and symptoms of meningeal irritation. similar asymptomatic cns involvement probably occurs with other viruses as well. although viral meningitis affects all age groups, it is predominantly a disease of childhood. the agents causing viral meningitis can be divided into three broad groups: ( ) common agents of viral meningitis, including the enteroviruses, arthropod-borne agents, and hsv- ; ( ) less common agents, including hiv, mumps, lcmv, human herpesvirus (hhv- ), and parvovirus b ; and ( ) agents known to cause lymphocytic meningitis only in rare cases. outside the united states, new viruses like toscana virus and chikungunya virus are emerging causes of viral meningitis. in addition, a number of nonviral, and occasionally noninfectious, conditions may cause a clinical syndrome indistinguishable from viral meningitis. enteroviruses account for approximately % of cases in which the causative virus is identified ( table ) . enteroviruses are small, nonenveloped single-stranded positive-sense ribonucleic acid (rna) viruses within the family picornaviridae. although more than serotypes of enteroviruses have been identified, coxsackievirus a and echoviruses e , e , e , e , and e account for % of all cultured isolates of csf. poliovirus, although no longer found in developed countries due to mass vaccination efforts, still causes aseptic meningitis and paralytic disease in countries like pakistan, afghanistan, and nigeria where cultural taboos, lack of development, and war have stymied vaccination efforts. enteroviruses are disseminated by fecal-oral spread, and cases in developed countries cluster during summer months when sanitation tends to be most relaxed. recent studies employing polymerase chain reaction (pcr) methods, however, confirm older observations that enteroviral cns infections occur throughout the year, and many previously undiagnosed cases of viral meningitis occurring during winter months are also caused by these viruses. coxsackieviruses, echoviruses, and enteroviruses may cause encephalitis and, rarely, paralytic disease, especially in neonates and the immunosuppressed. this was vividly illustrated in with the enterovirus outbreak in cambodia that killed more than children. arthropod-borne viruses, or arboviruses, include agents from several different families whose hosts are typically small mammals or birds. they spread to humans through the bite of an arthropod vector ( table ) . although these agents are most commonly considered to cause encephalitis, all of them, with the exception of eastern equine encephalitis, more frequently cause meningitis. the most common domestic arthropodborne agents associated with viral meningitis include st. louis encephalitis virus, the california/lacrosse group of viruses, colorado tick fever, and west nile virus, which caused an explosive outbreak when it first arrived in the united states in and more recently in . these agents, like enteroviruses, have a peak incidence in summer and early fall. the exception to this rule is colorado tick fever, which is more frequently transmitted in the spring and early summer. herpesviridae are enveloped, double-stranded dna viruses. hsv- and hsv- , varicella-zoster virus (vzv), epstein-barr virus (ebv), cytomegalovirus (cmv), and hhv- all cause viral meningitis. of these, however, only hsv- has been associated with a significant number of cases. older data suggest that hsv- accounts for - % of viral meningitis cases. recent work employing pcr suggests that it may be the most common cause of viral meningitis in adult women. hsv- most often causes meningitis following primary genital infection. occasional cases may also follow primary genital infection with hsv- . patients with recurrent (mollaret's) meningitis often have recurrent infection due to hsv- . csf pleocytosis occurs during both chicken pox and herpes zoster with or without skin lesions; this pleocytosis is usually asymptomatic but may occasionally be associated with meningitic symptoms. hsv- is also the most common cause of nonepidemic viral encephalitis, commonly producing an asymmetric lesion in the anterior and medial temporal lobes. in the immunosuppressed and elderly, vzv can cause a multitude of other cns syndromes including stroke due to vasculitis, herpes zoster ophthalmicus, myelitis, encephalitis, and cranial neuropathies. cmv can cause ventriculitis and radiculitis in patients with hiv. lastly, hhv- is now appreciated to produce a bilateral medial temporal lobe encephalitis in bone marrow transplant patients. hiv has been associated with both acute and persistent lymphocytic meningitis. onset is most commonly at the time of seroconversion, and for the astute clinician, this represents an opportunity to make an early diagnosis of hiv during a period of time when the patient's viral load is very high, and thus, is at high risk for transmission. the course may be uniphasic, chronic, or, occasionally, recurrent. in recurrent cases that happen despite the patient achieving systemic virologic control with antiretroviral medications, the differential diagnosis should include the possibility of cns hiv escape either due to a resistant strain of hiv having evolved in the cns compartment or due to inadequate cns penetration of antiretroviral medications. hiv rna can be identified in csf using reverse transcriptase-polymerase chain reaction (rt-pcr) methods, which also allow an assessment of viral burden and the resistance mutation profile. although hiv itself typically causes a csf pleocytosis of less than cells/mm with a as discussed previously, hsv- , hhv- , vzv, cmv, and ebv occasionally cause meningitis. mumps virus, like measles virus, is a paramyxovirus, containing a single-stranded negative-sense rna genome. before the advent of the mumps vaccine, mumps was the most common cause of viral meningitis, accounting for more than % of isolates. currently, mumps virus meningitis is rare in developed countries. the virus is still a common cause of cns infection in underdeveloped countries and countries like japan in which vaccination programs have been suspended due to vaccine-induced cases of lymphocytic meningitis. in these countries, mumps virus is an important cause of sensorineural deafness. in experimental animals infected in utero, mumps can cause aqueductal stenosis, and there are approximately human cases of this in the literature. lcmv is an arenavirus containing a single-stranded ambisense rna genome. wild and laboratory mice are the natural hosts, and there is recent evidence that snakes harbor these viruses as well. lcmv is associated with human cases of meningoencephalitis as a consequence of exposure to laboratory or wild mice, and in rare epidemics it is associated with pet hamsters. cases are more common in impoverished areas with poor hygiene. important outbreaks of fatal lcmv meningoencephalitis have also occurred in clusters of solid organ transplant patients infected by organs from asymptomatic donors. lcmv meningitis typically occurs during autumn and early winter, and it has been suggested that this reflects more extensive mouse-human contact as mice move inside to escape winter weather. in studies before , the virus was thought to account for - % of cases of viral meningitis. in recent years, reports of meningitis due to lcmv have been rare. however, congenital lcmv infection is a significant, often unrecognized cause of chorioretinitis, hydrocephalus, microcephaly or macrocephaly, and mental retardation. acquired lcmv infection likewise may be an underappreciated illness. the meningitis caused by lcmv may be extremely persistent and has been associated with symptoms and csf abnormalities lasting for months. acquired lcmv infection may also be associated with encephalitis, transverse myelitis, a guillain-barré-type syndrome, and both transient and permanent hydrocephalus. parvovirus b most commonly causes an acute febrile illness, accompanied by erythema infectiosum. the virus can also produce meningitis and meningoencephalitis in both immunocompetent and immunocompromised patients. the combination of rash and signs of meningeal irritation may mimic acute meningococcal infection. other items to always consider in the differential of rash and meningitis include west nile virus, syphilis, rickettsial infection (i.e., rocky mountain spotted fever), enteroviruses, lyme disease, human granulocytic anaplasmosis, and human monocytic ehrlichiosis, many of which are treatable. csf findings are typical of viral infection. occasionally, csf may be normal. rare causes of viral meningitis include influenza a and b viruses, parainfluenza viruses, rotaviruses, coronaviruses, measles virus, and adenoviruses. lastly, although jc virus is typically associated with progressive multifocal leukoencephalopathy, it can also cause viral meningitis, especially in the immunosuppressed. onset of viral meningitis may occur following a symptomatic, systemic illness, or as an isolated event following inapparent systemic infection. patients can present with fever ( %), headache ( %), photophobia, neck stiffness ( %) and/or back pain. significant alteration of consciousness is far less common than in bacterial meningitis. immunocompromised patients may have even more subtle exam findings and histories, and there should be a low threshold for further investigation with lumbar puncture in these cases. seizures or focal neurological signs are unusual although cranial neuropathies are seen with certain viruses like vzv, hsv, west nile virus, and hiv. focal signs should raise concerns about a concomitant viral encephalitis or a focus of infection, such as a brain abscess. patients are usually uncomfortable but do not appear severely ill. physical examination may reveal evidence of systemic illness, including rash, lymphadenopathy, pharyngitis, or splenomegaly, depending on the infectious agent. neurological examination commonly reveals nuchal rigidity with the patient unable to touch chin to chest. resistance to passive neck flexion and kernig's and/or brudzinski's signs may be present but are inconsistent. both signs may be absent in milder cases. a useful test of nuchal rigidity is to ask the patient to touch forehead to knee; this will often be positive when all other tests of meningeal irritation are questionable or absent. papilledema is rare. routine blood studies may reveal a lymphocytic leukocytosis. liver function tests may be elevated if there is hepatic involvement. the most important diagnostic test in viral meningitis is the lumbar puncture. this should be preceded by head magnetic resonance imaging (mri) or, less optimally, computed tomography if focal signs are present, there is significant altered mental status, or if there is any suspicion of increased intracranial pressure. spinal fluid will usually show mildly elevated opening pressure, lymphocytic pleocytosis, elevated protein, and normal glucose ( table ) . the cell count is usually less than cells/mm . protein is usually in the range of - mg/dl. exceptions exist to this csf formula, however. cell count may be as high as cells/mm . during the first - h of infection, csf may contain a mixture of polymorphonuclear leukocytes and lymphocytes with even a polymorphonuclear predominance early in infections with west nile virus, hsv, and vzv. glucose concentrations, although usually % of blood values, may be significantly depressed: this has been reported with meningitis due to herpes zoster, mumps, and lcmv. return of csf parameters to normal may be extremely prolonged following viral meningitis, and isolated reports have described persistent csf pleocytosis and elevated protein over periods of weeks to months. before the advent of pcr, diagnosis of viral meningitis was difficult and often an exercise in futility: viruses may take considerable time to grow in culture, and many viral agents cannot be readily grown from csf. viral serologies comparing acute and convalescent sera have been used for retrospective diagnosis, and serological diagnosis can be accelerated by comparing serum and csf antibody titers to identify the synthesis of specific antiviral antibodies within the cns; however, serological tests only rarely allow rapid enough diagnosis to direct therapy. the advent of pcr methods has revolutionized the diagnosis of both meningitis and encephalitis. pcr for enteroviruses, hsv- , hsv- , vzv, and cmv are readily available in many laboratories, and pcr diagnosis of other agents is often available through larger commercial laboratories or the centers for disease control. although pcr is highly specific, it has limited sensitivity in certain circumstances. it is relatively insensitive in the first days of infection with hsv as well as after day of infection. the overall sensitivity of pcr for vzv is %; if vzv is suspected, immunoglobulin g (igg) and immunoglobulin m (igm) antibodies should always be sent from the csf as well. in the case of hiv, rt-pcr methods are available not only for diagnosis but also for determining viral load and resistance mutations. even with the use of pcr, the causative agents in many cases of viral meningitis remain undiagnosed. viral meningitis should be considered in the differential diagnosis of any patient presenting with headache, photophobia, and neck stiffness. however, the presence of these findings also makes it mandatory to exclude bacterial infection. although patients with viral meningitis are less severely ill, bacterial meningitis may also appear mild in its early stages. conversely, patients with viral meningitis may deteriorate. many other infectious conditions can also cause lymphocytic meningitis. these include secondary syphilis, leptospirosis, brucellosis, infections by mycobacterium tuberculosis, lyme disease caused by borrelia burgdorferi, infections due to ehrlichiae or, rarely, other rickettsial agents, mycoplasma pneumoniae, and fungi (particularly cryptococcus neoformans, histoplasma, blastomyces coccidioides, and candida). tuberculous and fungal meningitis are often, but not always, accompanied by a significant decrease in csf glucose. lyme meningitis may produce csf findings identical to those seen in viral meningitis. however, erythema migrans, multiple cranial neuropathies and polyradiculopathies are common features of lyme meningitis. similarly, patients with lyme meningitis tend to have fewer white blood cells (mean, vs. /mm ) and a significantly greater percentage of mononuclear cells than patients with viral meningitis. both m. tuberculosis and m. pneumoniae are difficult to culture but are readily detectable by pcr; pcr tests for ehrlichiae are in limited use. noninfectious etiologies can also cause a lymphocytic or aseptic meningitis including side effects of a number of medications like nonsteroidal anti-inflammatory drugs, serum immunoglobulins, carbamazepine, lamotrigine, and trimethoprim sulfamethoxazole. recurrent meningitis can occur in patients with periodic leakages from dermoid or epidermoid cysts abutting the meninges. in such patients, the diagnosis can be made by a detailed mri examination of the brain and the spinal cord. lastly, autoimmune disease can manifest as a lymphocytic meningitis, sometimes representing the initial presentation of systemic lupus erythematosus and sarcoidosis. it can also be seen associated with sjö gren's syndrome and rheumatoid arthritis, particularly in patients who take nonsteroidal anti-inflammatory drugs. most cases of viral meningitis are self-limited, and antiviral therapy is usually not indicated. controlled studies of antiviral csf during the first h of viral meningitis may contain a mixture of lymphocytes and polymorphonuclear leukocytes. in these cases, in contrast to bacterial meningitis, csf glucose is usually normal and follow-up lumbar puncture h later will often but not always show lymphocytes only. c positive gram stain requires approximately colony-forming units (cfu)/ml of csf. approximately % of gram stains will be positive if csf contains cfu/m. prior antibiotic treatment will reduce this amount by %. abbreviations: afb, acid-fast bacillus; csf, cerebrospinal fluid; pcr, polymerase chain reaction. agents in viral meningitis have not been reported in detail. recent data from controlled studies presented in abstract form, however, suggest that virological and clinical improvements are better in patients with severe enteroviral meningoencephalitis treated with the antiviral agent pleconaril than with placebo. similarly, depending on the severity of illness, consideration should be given to the therapy for hsv meningitis with acyclovir or similar agents. use of antiviral agents in the treatment of viral meningitis is still essentially experimental and must be balanced against the severity of disease and complications of the therapy. an exception to this is hiv meningitis, in which diagnosis of hiv infection is in itself an indication for highly active antiretroviral therapy. viral meningitis is almost always a self-limiting disease. recovery may occur within days. however, symptomatic illness is not infrequently prolonged, and patients may require weeks or months to return to full health. permanent neurological deficits or intellectual impairment are rare and, if present, should prompt a more thorough workup for nonviral etiologies of meningitis. see also: encephalitis, viral. herpesviruses, human. meningitis, bacterial. meningitis, fungal aseptic meningitis, a disease of diverse etiology: clinical and etiologic studies on cases the rational clinical examination. does this patient have acute meningitis lymphocytic choriomeningitis virus: reemerging central nervous system pathogen cerebrospinal fluid in central nervous system infections prospective analysis of cases of enteroviral meningitis: interest of systematic genome detection in cerebrospinal fluid irrespective of cytologic examination results lymphocytic choriomeningitis virus: a neglected pathogen of man viral infections of the nervous system diagnosis and surveillance of herpes simplex virus infection of the central nervous system the outbreak of west nile virus infection in the new york city area in cerebrospinal fluid findings in aseptic versus bacterial meningitis laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex pcr screening assay viral meningitis clinical significance of enteroviruses in serious summer febrile illnesses of children treatment of potentially life-threatening enterovirus infections with pleconaril prolonged fever caused by parvovirus b -induced meningitis: case report and review key: cord- - rp py authors: chow, kuan-chih; hsiao, cheng-hsiang; lin, tze-yi; chen, chi-long; chiou, shiow-her title: detection of severe acute respiratory syndrome–associated coronavirus in pneumocytes of the lung date: - - journal: am j clin pathol doi: . /c edu raqbtxbhce sha: doc_id: cord_uid: rp py previous reports have indicated that patients with severe acute respiratory syndrome (sars)–associated coronavirus infection could develop atypical pneumonia with fulminant pulmonary edema. however, the target cells of sars viral infection have not been characterized in detail. we report the pathologic findings of the lung in cases of sars. chest radiographs at to weeks of infection revealed an atypical pneumonia with pulmonary consolidation, a clinical characteristic of sars infection. the presence of the sars virus was determined by nested reverse transcription–polymerase chain reaction (rt-pcr), and the infected cells were identified by in situ hybridization in open-lung biopsy and postmortem necropsy specimens. expression of sars virus–encoded rna was detected in all cases by rt-pcr, and the sars viral signal was localized in pneumocytes by using in situ hybridization. severe acute respiratory syndrome (sars) is an acute infectious disease that affects primarily the lower respiratory tract, with clinical manifestations of atypical pneumonia with dry cough, persistent fever, progressive dyspnea, and, sometimes, the abrupt deterioration of lung function [ ] [ ] [ ] [ ] [ ] and the ensuing oxygen deprivation-associated systemic organ failures. , at this stage, the disease can be lethal. postmortem pathologic examination showed that fulminant pulmonary interstitial infiltrate, substantial pulmonary edema, and extensive pulmonary consolidation with alveolitis, formation of hyaline membrane, and the presence of desquamated alveolar epithelial cells, which corresponded well with the progression of clinical symptoms, frequently were observed. , an elegant study by ksiazek et al further demonstrated that in addition to the dispersed alveolar epithelial cells, foamy macrophages and multinucleated giant cells were abundant in the damaged alveolus as well. inoculation with bronchoalveolar lavage fluid from these patients can induce not only cytopathic changes of vero e cells, but also formation of multinucleated syncytial cells, an index of viral infection. the subsequent analysis of extracellular particles from the supernatant of cytopathically altered vero e cells by negative-stain electron microscopy revealed the characteristic coronavirus particle. however, the results of nucleotide sequence alignment indicated that these isolates were quite different from any known coronavirus, - a positive singlestranded rna virus. because the virus is sars infection-specific, it was named sars-associated coronavirus, or sars virus. nevertheless, the target cells of sars viral infection and the essence of multinucleated giant cells are not well characterized. in the present study, we used nested reverse transcription-polymerase chain reaction (rt-pcr) for the immediate determination of sars viral infection, and the presence of the sars viral signal was identified by using in situ hybridization. three patients with sars infection were identified in the china medical university hospital, taichung, and the national taiwan university hospital, taipei, taiwan, from march to may . case was a -year-old man, who had fever and dry cough days after his return from guangdong, china. six days later, the symptoms of severe respiratory distress developed, and the man was admitted immediately to the hospital. owing to his critical condition, endotracheal intubation was undertaken on admission, and open lung biopsy was performed to resolve the cause of pulmonary distress days after there was no response to palliative treatment. the treatment did not include antiviral or steroid regimens. interestingly, the patient's condition improved, and the endotracheal tube was removed days after surgery. the patient recovered substantially and was discharged weeks later. case was a -year-old man in whom fever and dry cough developed days after the visit by a relative from hong kong, china. three days later, severe respiratory symptoms ensued, and he was admitted to the hospital. the patient responded poorly to combination therapy with steroids and ribavirin, and his condition became critical days after admission. although an endotracheal tube was inserted when his condition became critical and the use of a mechanical ventilation device was initiated in a week, the patient died days later. case was a -year-old woman in whom fever and dry cough developed to days after accidental contact in a sars-infested hospital. two days later, the patient developed symptoms of severe respiratory distress and was transferred immediately to a sars specialty hospital. nevertheless, the patient did not respond to the combination therapy with steroids, ribavirin, and intravenous immunoglobulin, and her condition worsened days after admission. although endotracheal intubation and a mechanical ventilation device were used to attempt to improve the pulmonary condition, the patient died in days. the presence of the sars virus in biopsy samples was determined primarily by using nested rt-pcr. [ ] [ ] [ ] [ ] [ ] the rna in situ hybridization procedure was described previously. briefly, a -µm section from a paraffin-embedded tissue sample was deparaffinized in xylene, dehydrated, and predigested with . mg/ml of nuclease-free proteinase k (boehringer mannheim, mannheim, germany) at room temperature for minutes. the slide was washed with distilled water, rinsed with % ethanol, and air dried. fluorescein isothiocyanate-conjugated antisense probe ( ng/ml in % formamide, × standard saline citrate, and . % dry milk) to different regions of the sars viral genome was placed over each tissue section. the probes ❚table ❚ were synthesized by mission biotech (taipei, taiwan), and the sequence was according to genbank/ay . . the sample was denatured at °c on a thermal plate for minutes and then moved to a moist chamber at °c for hours. following extensive washing with × standard saline citrate, the hybridization product was detected by using anti-fluorescein isothiocyanate antibodies conjugated with alkaline phosphatase (dako a/s, copenhagen, denmark). the chromogenic development was processed in a mixture of -nitroblue tetrazolium and -bromo- -chloro- -indolphosphate (boehringer mannheim). the slide was counterstained with methyl green. positive staining was recognized under the microscope as brownish purple granules. probes to other viruses, eg, epstein-barr virus, type i human t-cell lymphotropic virus, cytomegalovirus, enterovirus , and parvovirus b , [ ] [ ] [ ] that were used in previous studies were included to determine the specificity of the sars viral probes. a specimen of nasopharyngeal carcinoma with positive serologic results for the epstein-barr virus was used as a positive control for the in situ hybridization procedure. paraffin-embedded sections of normal counterpart of the lung and lung specimens from patients with lung cancer or acute respiratory distress syndrome (ards) were used as negative controls. immunohistochemical characterization of cd was performed on paraffin-embedded sections of biopsy and necropsy tissues by using an lsab method (dako, carpinteria, ca). the chromogenic reaction was visualized by peroxidase-conjugated streptavidin and aminoethylcarbazole (sigma, st louis, mo). slides were counterstained with mayer hematoxylin or methyl green, and positive staining was recognized under a microscope as crimson granules. sars viral infection was detected preliminarily by using rt-pcr in affected lung tissue samples from open lung biopsy (case ) and necropsies (cases and ) ❚table ❚. sars viral rna, however, was not detected in peripheral blood samples. histopathologically, in addition to the presence of various degrees of alveolar deformation and tissue fibrosis in the infected lung, cells with enlarged cytoplasm frequently were detected ❚image a❚ and ❚image b❚. the number of enlarged cells increased with the progression of disease severity. moreover, in the acute phase (case ), these enlarged cells (image a) resembled alveolar macrophages and, possibly, activated type ii pneumocytes ❚image c❚. in the chronic phase (case ), the cytoplasm of these cells became highly vacuolated and, sometimes, laminated (image b). the nuclei, however, became more convoluted. further characterization showed that these cells were not immunoreactive to cd , a bona fide marker of activated histiocytes ❚image a❚, and suggested that, alternatively, they might be of pneumocyte origin, in particular, the type ii pneumocyte. by using antisense oligonucleotide probes specific to the nucleic acid binding protein-encoding (n) and membrane protein-encoding (m) regions of the sars viral genome, the sars viral signal was detected in these enlarged cells ❚image b❚. no signal to other viral probes was detected in these enlarged cells (data not shown). in addition, the viral signal was not detected in the neighboring fibroblasts, capillary endothelial cells, or bronchial epithelial cells ❚image c❚. nevertheless, when antisense oligonucleotide probes specific to the replicase-encoding (rep) region of the sars viral genome were used to determine the presence of virus, the intensity of the in situ hybridization signal decreased substantially compared with that detected by probes to the n and m regions ❚image d❚ ❚table ❚. interestingly, liver cells were negative for the sars viral signal; so were muscle cells, myocardial cells, endothelial cells, and splenocytes. the obvious local inflammation was found only in the lung tissues. although marked inflammation was observed in the infected tissues, transudate and exudate were found mainly around epithelial cells that were positive for the sars viral signal ❚image a❚. no transudate or exudate was identified in the neighboring regions, which had abundant macrophages ❚image b❚. by detecting the viral signal in diseased lung tissue by combining rt-pcr and in situ hybridization, we found that these patients had contracted sars viral infection during the epidemic outbreak of sars in taiwan. although an infection-associated pathologic lesion was shown basically in every part of the lung by radiographs, pathologic evaluations showed that mortality-related tissue damage was located mainly in the lower respiratory tract , ; this finding corresponded well not only with our observation that the sars virus infected mainly pneumocytes (in particular, type ii pneumocytes that are major sources of surfactant, the essential element to maintain the normal function of the alveolus), but also with the progression of clinical symptoms. in fact, in an elegant review, ware and matthay described in detail the symptoms of ards, which are quite similar to those found in patients with sars viral infection, such as alveolar filling, pulmonary consolidation, and atelectasis revealed by radiographs and the presence of diffuse alveolar deformation, alveolar exudate, hyaline membrane, and tissue fibrosis detected by pathologic examination. disease-related mortality, like that of patients with sars, also correlated with advanced tissue fibrosis of the lung and, possibly, persistent hypoxemia and oxygen deprivation-related organ failure. , it therefore is worth noting that abnormalities of the production and, probably, the composition of surfactant could be attributed mostly to the dysfunction of gas exchange, alveolar deformation, and oxygen deprivation-mediated systemic organ failure in patients with ards or sars. , moreover, based on the characteristics of positive-strand rna genome, replication mode, and infection route, , the sars virus would prefer infecting cells that have a membrane receptor for the virus and are ready for protein synthesis, ie, type ii pneumocytes of the lung. our pathologic findings supported such speculation by showing that the sars virus could infect mainly type ii cells and the infected type ii cell might not only reduce the production of bona fide surfactant but also increase the expression product of the viral protein. , interestingly, amino acid sequences further indicated that the sars viral protein might contain several potential chemokine-like motifs, in particular, within the replicase polyprotein ab region. (the amino acid sequence was according to genbank/p .) the potential chemokinelike motifs are listed in ❚table ❚. as noted, chemokines, a superfamily of small (about % of the mature chemokines consist of only - amino acid residues) cytokines, are chemoattractants and activators of specific types of leukocytes. most of the family members have at least conserved cysteine residues that can form intramolecular disulfide bonds, and, based on the position of the first cysteine residues, these chemokines are categorized into the cxc subfamily (α) that has amino acid residue between the first conserved cysteines and the cc subfamily (β), in which the first cysteine residues are in the chronic phase, the cytoplasm of enlarged cells became highly vacuolated (arrows) and, sometimes, laminated (arrowhead). the nuclei, however, became more convoluted (h&e, × , ). c, the type ii pneumocytes (arrows) and alveolar macrophages (arrowheads) in the normal lung. aminoethylcarbazole was used to detect alveolar macrophages (crimson red cytoplasmic precipitates) (aminoethylcarbazole, methyl green counterstain, original magnification × ). adjacent to one another. the cxc chemokines are chemotactic for neutrophils and lymphocytes. on the other hand, the cc chemokines are chemoattractive only for monocytes and lymphocytes, not neutrophils. [ ] [ ] [ ] in fact, the profuse infiltrate of macrophages in the infected lung and the atrophic white pulp of the spleen considered together with the lack of lymphocytes in the damaged lung clearly indicate that sars viral infection-specific product(s), which a b ❚image ❚ characterization of the enlarged cells by immunohistochemical and in situ hybridization methods. a, the enlarged and multinucleated cells (arrows) were not immunoreactive to cd , a bona fide marker of activated macrophage (a representative cell is indicated by the arrowhead). cd expression was detected by immunohistochemical analysis (cd , hematoxylin counterstain, original magnification × ). b, by using in situ hybridization and antisense oligonucleotide probes specific to the nucleic acid binding protein-encoding (n) and membrane protein-encoding (m) regions of the severe acute respiratory syndrome (sars) viral genome, the sars viral signal was detected in the enlarged and multinucleated cell (arrow, purple blue precipitates). the viral signal also was detected in a cluster of oval-cuboidal cells (arrowhead) and suggested that they might be of pneumocyte origin, in particular, the type ii pneumocyte. c, the viral signal was not detected in the neighboring fibroblasts, capillary endothelial cells, activated macrophages, foamy macrophages (arrowheads), or bronchial epithelial cells (arrows). d, when antisense oligonucleotide probes specific to the replicase-encoding region of the sars viral genome were used to determine the presence of virus, the intensity of the in situ hybridization signal in enlarged and multinucleated cells (arrows) decreased substantially, to about / , of that detected by the probes to the n and m regions (compared with the intensity of the viral signal in image b). b-d, following localization of the sars viral signal by in situ hybridization, immunohistochemical analysis with cd was used to identify macrophages (b-d, cd , methyl green counterstain; b, × ; c, × ; d, × ). are potentially monocytotactic and lymphotoxic, could be expressed in an atypical manner. it is worth noting that the amino acid sequence of sars viral replicase also contains papain-like endopeptidase (nsp ) and c-like endopeptidase motifs. [ ] [ ] [ ] [ ] with potential chemokine-like motifs and intrinsic enzyme activity, it is likely that sars viral products would elicit an unconventional immune response that resembles the acute phase of histiocytic necrotizing lymphadenitis, with a dramatic increase of the cd + histiocyte population ❚table ❚ sars, severe acute respiratory syndrome; +, positive; +++, strong positive; -, negative; ±, intermediate. * antisense oligonucleotide probes specific, respectively, to the replicase-encoding region (rep) and the nucleic acid binding protein-encoding (n) and membrane protein-encoding (m) regions of the sars viral genome were used to determine the viral signal by in situ hybridization. intensity of the viral signal was measured by scanning of the specific cell types following in situ hybridization. † the interval between the onset of fever and the sample collection. ‡ although case was traced epidemiologically as the origin of outbreak in taiwan, the subject was not contagious at the time when open-lung biopsy was performed. in the clinical behavior of sars infection, the patient is most contagious when the fever persists; after fever subsides, the patient becomes less contagious or not contagious. locally. in the chronic phase, the extent of cd + histiocytes decreased considerably, and foamy histiocytes became predominant. in this phase, the rep signal in enlarged and multinucleated cells became limited. unlike histiocytic necrotizing lymphadenitis, though, no karyorrhectic bodies were detected within the cytoplasm of infected epithelial cells or histiocytes, and these pathologic appearances would lower the possibility of adenovirus or cytomegalovirus involvement, in which karyorrhexis, necrotizing exudates, and intranuclear inclusion bodies are abundant. moreover, these pathologic observations also corresponded well with the clinical behavior of the disease, in which patients were most contagious when the fever persisted. on the other hand, the infected lesion limited to the lower respiratory tract, ie, the type ii pneumocyte in the alveolus, further correlated with dry cough, and the symptoms would be less consistent with infection by the influenza virus, parainfluenza virus, hantavirus, or respiratory syncytial virus that could broadly infect the bronchiolar epithelium. our results demonstrate that in the sars virus-infected lung, the viral signal is defined mostly within epithelial cells, in particular, pneumocytes. the impact of sars viral expression on mortality could be via the downregulation of surfactant production, which in turn leads to alveolar filling, alveolar collapse, tissue fibrosis, and pulmonary dysfunction. expression of the sars viral rep region, in which certain regions resemble a chemokine motif, would further result in an increased histiocyte population and, hence, deteriorated local inflammation. undoubtedly, both hypotheses remain to be verified, and the effect of sars viral infection on host gene regulation, in particular, the "cytokine dysregulation" that might be induced aberrantly in the macrophages and the surrounding tissues of the afflicted lung, deserves more in-depth study. it is clear, however, that rep expression can be associated directly or indirectly with the severity of sars disease and, possibly, the mechanism of tissue dysfunction, and this in and of itself should provide an interesting target for discovering and designing a novel therapeutic protocol for sars viral infection, if the disease should persist and recur. identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong the clinical pathology of severe acute respiratory syndrome (sars): a report from china coronaviridae: the viruses and their replication virus infection in patients with histiocytic necrotizing lymphadenitis in taiwan: detection of epstein-barr virus, type i human t-cell lymphotropic virus, and parvovirus b virus infection in pediatric patients with congenital anemia in taiwan: detection of erythrovirus b , epstein-barr virus and cytomegalovirus detection of cytomegalovirus infection in a patient with febrile ulceronecrotic mucha-habermann's disease the acute respiratory distress syndrome sars coronavirus: a new challenge for prevention and therapy the cytokine handbook the biology of chemokines and their receptors calling in the troops: regulation of inflammatory cell trafficking through innate cytokine/chemokine networks macrophage activation through ccr -and cxcr -mediated gp -elicited signaling pathways lung pathology of fatal severe acute respiratory syndrome viral infections from the key: cord- -x jpsnxg authors: mokili, john l; rohwer, forest; dutilh, bas e title: metagenomics and future perspectives in virus discovery date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: x jpsnxg monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires both adequate preparedness and quick response. identifying the causative agent of a new epidemic is one of the most important steps for effective response to disease outbreaks. traditionally, virus discovery required propagation of the virus in cell culture, a proven technique responsible for the identification of the vast majority of viruses known to date. however, many viruses cannot be easily propagated in cell culture, thus limiting our knowledge of viruses. viral metagenomic analyses of environmental samples suggest that the field of virology has explored less than % of the extant viral diversity. in the last decade, the culture-independent and sequence-independent metagenomic approach has permitted the discovery of many viruses in a wide range of samples. phylogenetically, some of these viruses are distantly related to previously discovered viruses. in addition, – % of the sequences generated in different viral metagenomic studies are not homologous to known viruses. in this review, we discuss the advances in the area of viral metagenomics during the last decade and their relevance to virus discovery, clinical microbiology and public health. we discuss the potential of metagenomics for characterization of the normal viral population in a healthy community and identification of viruses that could pose a threat to humans through zoonosis. in addition, we propose a new model of the koch's postulates named the ‘metagenomic koch's postulates’. unlike the original koch's postulates and the molecular koch's postulates as formulated by falkow, the metagenomic koch's postulates focus on the identification of metagenomic traits in disease cases. the metagenomic traits that can be traced after healthy individuals have been exposed to the source of the suspected pathogen. john l mokili , forest rohwer , and bas e dutilh , monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires both adequate preparedness and quick response. identifying the causative agent of a new epidemic is one of the most important steps for effective response to disease outbreaks. traditionally, virus discovery required propagation of the virus in cell culture, a proven technique responsible for the identification of the vast majority of viruses known to date. however, many viruses cannot be easily propagated in cell culture, thus limiting our knowledge of viruses. viral metagenomic analyses of environmental samples suggest that the field of virology has explored less than % of the extant viral diversity. in the last decade, the cultureindependent and sequence-independent metagenomic approach has permitted the discovery of many viruses in a wide range of samples. phylogenetically, some of these viruses are distantly related to previously discovered viruses. in addition, - % of the sequences generated in different viral metagenomic studies are not homologous to known viruses. in this review, we discuss the advances in the area of viral metagenomics during the last decade and their relevance to virus discovery, clinical microbiology and public health. we discuss the potential of metagenomics for characterization of the normal viral population in a healthy community and identification of viruses that could pose a threat to humans through zoonosis. in addition, we propose a new model of the koch's postulates named the 'metagenomic koch's postulates'. unlike the original koch's postulates and the molecular koch's postulates as formulated by falkow, the metagenomic koch's postulates focus on the identification of metagenomic traits in disease cases. the metagenomic traits that can be traced after healthy individuals have been exposed to the source of the suspected pathogen. direct-count epifluorescence and transmission electron microscopy have shown that viruses are highly abundant in most environments. bergh et al. demonstrated that l of seawater can contain as many as virus-like particles (vlps) [ ] , approximately times more than the number of prokaryotes. terrestrial environments often have vlps per gram. by extrapolation from the estimated number of prokaryotes in different environments [ ] , viruses are the most abundant entities in the biosphere totaling an estimated number of . Â , . Â , . Â , and . - . Â in the open ocean, in soil and in oceanic and terrestrial subsurfaces, respectively. in the human holobiont, the human cells are outnumbered -fold by bacteria and -fold by viruses. viral acquisition starts early in life in utero or perinatally during the first few weeks after birth as demonstrated by studies of the gut viral communities in infants. while no vlps could be detected in the earliest infant stool samples, there were $ virus particles per gram wet weight of feces by the end of the first week [ ] . the majority of these vlps appear to be bacteriophages, the bacteria-infecting viruses [ ] [ ] [ ] . culture techniques have been the gold standard for the detection of viruses for over a century. despite the knowledge gained using the cultivation of viruses in cell culture, the consensus is that we have barely begun to chart the viral world, which is the 'dark matter' of the biological universe and a rich source of future discoveries [ ] . since the vast majority of viruses are not easily cultivatable, exploration of this dark matter requires culture-independent methods with larger detection coverage than culture. while the sequencing of the s fragment of the small subunit of the ribosomal rna (rrna) gene has a proven track record for the detection of known and novel cellular organisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] , this technique is not applicable to viruses because they lack the gene. indeed, viruses do not share any common gene that could similarly qualify as a unified phylogenetic marker [ ] . metagenomics is an alternative culture-independent and sequence-independent approach that does not rely on the presence of any particular gene in all the subject entities. this approach was originally developed as a tool for 'functional and sequence-based analysis of collective microbial genomes contained in environmental samples' [ , ] . early metagenomic studies analyzing the genetic content of environmental samples yielded the identification of metabolic traits, the characterization of organisms and the discovery of new antibiotics and enzymes [ ] [ ] [ ] [ ] [ ] . metagenomic studies now encompass a wide scope of research fields including marine environmental research, plant and agricultural biotechnology, human genetics and diagnostics of human diseases. accordingly, the number of metagenomics papers in peer-reviewed journals has increased greatly since ( figure a ). the scope of applications for metagenomics will likely widen from environmental microbiome studies to routine clinical diagnostics for palliative care of patients, public health, industry and beyond. the first application of metagenomics to the field of virology was in the analysis of the viral communities sampled at two near-shore marine locations in san diego [ ] . since then, it has been used to survey viruses in numerous environments including freshwater, marine sediment, soil and the human gut. figure b shows an overview of diverse areas where the metagenomic approach has been applied for virus discovery since . the success of these studies relied upon the advances observed in the past decade in the area of sequencing technology and in bioinformatics. although the fundamental concept of metagenomics has not changed, several technical advances have proven valuable for the discovery of previously unidentified, uncultured viruses. while metagenomics originally depended upon cloning for the analysis of doublestranded dna genomes [ , , , ] , high-throughput sequencing technologies can now be applied to all types of genomes, including single-stranded dna and rna [ ] . , , , , , [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , ] . m: main characterization method used: ngs, high-throughput sequencing using gs flx or gs titanium platform; sg-sanger, shotgun library with sanger sequencing method. s: sample, symbols used for sample type: ip, insect pool; sb, skunk brain; int: intestine; panc: pancreas; hf: human feces; se, sewer effluent; ms: marine sediment; nasopharyngeal aspirates (npa). historically, diseases caused by viruses have been known before the discovery of their causative agents. the acquired immunodeficiency syndrome (aids), poliomyelitis, cervical cancers, and burkitt's lymphoma were identified before their causative agents. whereas poliomyelitis was documented in ancient egyptian literature as early as approximately bc [ ] , poliomyelitis virus was not discovered by landsteiner and popper until [ ] . descriptions of clinical conditions likely to have smallpox have been found in ancient literature from egypt ( - bc), china ( bc) and india ( bc)-long before both jenner's discovery of smallpox vaccination and the later isolation of variola virus [ ] [ ] [ ] . the future perspectives in virology appear that, the metagenomic approach will generate a plethora of genetic information from unknown and potentially infectious agents, some of which could be associated with human diseases. the discovery of viruses will start to precede the characterization of the diseases they cause, well before the pathogenicity of these agents is defined. at this turning point in history, important questions need to be answered. for example, how far has this new viral metagenomics discipline evolved in its first decade? what has been learned so far that can be applied to viral discovery and the forecasting of future viral outbreaks? in this article, we review virus discovery techniques with a focus on metagenomic approaches that employ high-throughput sequencing technologies to characterize novel viruses. before the advent of molecular methods, many techniques including filtration, tissue culture, electron microscopy (em), serology and vaccination have been used for the detection of viruses. in , ivanovski demonstrated the presence of infectious agents, coined 'virus' by beijerinck in , in filtrate of infected leaves passed through a chamberland filter. this marks the discovery of the tobacco mosaic virus [ ] and the birth of a new era in virology. until then, the field of virology was not clearly defined. the instrumentation, from the discovery of tissue culture to modern molecular biology methods, has shaped the field and helped to discover many viruses. since the invention of the technique of tissue culture in and the propagation of poliovirus in animal cells in , cultivation of viruses has remained the gold standard for virus discovery for over a century [ ] [ ] [ ] . despite the achievements made by the culture technique, several limitations have hindered the discovery and detection of viruses in routine laboratory settings. virus propagation requires the development of controlled conditions that mimic the natural ecosystem shared between viruses and their hosts [ ] . the invention of the electron microscope in provided the first visual proof of a virus. however, this technique is relatively expensive, tedious and lacks both sensitivity and specificity. alternatively, serology can provide a hint of the acquisition of novel viruses -as was the case for hepatitis c virus [ , ] -before the viral agents have been cultured or viewed by electron microscopy. the immune sera method has shown little value for virus discovery. the inoculation method, however, not only helped to identify novel viruses, but also was used as an immunization method to confer crossprotection against closely related viruses. indeed, the cowpox-based inoculation developed by jenner in was the first effective vaccine against an infectious disease. nearly two centuries later, this strategy was used to eradicate smallpox. however, it is unlikely that jenner's method would pass the scrutiny of modern ethical review boards for vaccine or virus discovery [ ] . the trends in clinical virology practices show gradual substitution of the traditional virus discovery methods with novel molecular biology technology. nevertheless, traditional and the newer molecular biology techniques to isolate, identify, and characterize viruses play complementary roles in the viral discovery effort. for a comprehensive list and detailed description of molecular methods used for virus discovery, readers are referred to reviews by delwart [ ] and tang [ ] . here, we focus on the viruses discovered using these methods and their future applications in clinical microbiology and public health settings. two types of molecular methods have been used for the virus discovery effort: sequence-dependent and sequence-independent methods. sequence-dependent methods, including pcr using consensus primers and hybridization methods such as microarrays, require the knowledge of the nucleic acid for the detection of novel viruses. indeed, consensus sequences of previously known viruses have been used to identify novel viruses including highly divergent clades of human immunodeficiency virus [ ] , simian retroviruses [ ] [ ] [ ] [ ] , and hepatitis e virus [ ] . however, pcr using consensus primers based on previously characterized viruses have little or no value in detecting completely novel viruses. the microarray techniques were first introduced in to monitor the expression of multiple genes simultaneously [ ] . for virus discovery, microarrays can be prepared with probes that hybridize known viral sequences and potentially novel viruses with sufficient sequence similarity. the method has been applied to detect a wide range of known viruses as well as novel highly divergent viral taxa [ ] . microarray screening has led to the identification and characterization of a novel gammaretrovirus, xenotropic murine leukemia virusrelated virus (xmrv), in prostate tumors [ , ] . subsequent studies did not confirm these initial findings [ , ] , which points to potential limitations of the method. another example of a well-known virus discovered with microarrays is sars-cov, a highly divergent coronavirus discovered amid a worldwide outbreak of the severe acute respiratory syndrome (sars) in [ ] . reproducibility of results between microarray tests is frequently poor [ ] . unlike pcr and microarrays, the sequence-independent viral metagenomic approaches do not rely on prior knowledge of viruses in the samples. the suppression subtractive hybridization (ssh) and representational difference analysis (rda) are examples of sequence-independent virus discovery methods. ssh was used first to study gene expression [ ] and was later applied to investigate the etiology of diseases of unknown origin [ ] . by hybridizing dna obtained from patients and control subjects, nucleic acid from an unknown pathogen(s) can be detected [ ] [ ] [ ] . use of rda led to the discovery of human herpes simplex virus type (hhv ) [ ] , torque teno virus (ttv) [ ] , gbv-a, gbv-b viruses [ ] and a novel highly divergent murine norovirus [ ] . this method lacks sufficient sensitivity to detect viruses when the viral burden is low or when the dna sequence of the suspected etiological agent is not clearly distinguishable from the control sample [ ] . sequence-independent single-primer amplification (sispa) circumvents the viral load limitation of ssh. although there are several variations to the original protocol published by reyes et al. [ ] , the main strategy of sispa is to exploit the sensitivity and the specificity of pcr amplification using primers that bind oligonucleotide fragments ligated to any putative viral dna materials in the sample. sispa has been modified to allow the detection of both dna and rna viruses after the removal of genomic and contaminating nucleic acids [ ] . the sispa method was used successfully for the discovery of hepatitis e virus [ , ] , norwalk virus [ ] , human astrovirus [ , ] , and parvoviruses and [ ] . another sequence-independent technique, the viral metagenomics (described in detail below), provides superior capability to detect known and unknown viruses than the traditional and molecular sequence-dependent and sequence-independent methods. compared to virus discovery approaches outlined above, viral metagenomics is less biased. potentially, any viruses in the samples, culturable or unculturable, known or novel can be readily detected with the viral metagenomic approach. viral metagenomic methods have evolved significantly since they were first developed. in early studies [ , , , ] , preliminary sample preparation involved shearing of dna and cloning. these steps were required in order to obtain sufficient dna given the low amount of viral dna in environmental samples ($ mg/ l of sea water). because viral dna often contains modified nucleotides and because some viral genes (e.g. holins and lysozymes) are toxic to cells, the dna was randomly sheared to produce small fragments before cloning [ , , , ] . the process of sample preparation has since been streamlined and the sequencing speed increased with the advent of high-throughput sequencing technologies. the replacement of cloning with highthroughput methods has revolutionized metagenomics. there are several high-throughput sequencing platforms commercially available that vary by the sequencing principle, the sequencing speed, the cost and read length. an overview of a typical viral metagenomic protocol that can be used in a virus discovery study is provided in figure . essentially, a metagenomic analysis involves three main steps: ( ) sample preparation, ( ) high-throughput sequencing and ( ) bioinformatic analysis. below we provide an outline of each of these steps. more detailed descriptions have been previously published [ ] . sample preparation. theoretically, any type of sample can be analyzed using the metagenomic approach, including seawater [ ] , blood [ ] , horse feces [ ] , stool [ , [ ] [ ] [ ] [ ] , marine sediments [ ], coral tissues [ , ] , and hot springs [ ] . because viral genomes are relatively short, bacterial or eukaryotic nucleic acids can severely interfere with the isolation and detection of viral dna or rna that typically represents only a small fraction. thus, removal environmental virology high throughput sequencing flow chart for the generation of a viral metagenome using highthroughput sequencing. of non-viral nucleic acid is necessary [ , ] . homogenization, filtration and ultracentrifugation are often necessary to concentrate the viral particles present in the sample ( figure ). to ensure that viruses are not lost during the virus preparation, epifluorescence microscopy with sybr-gold staining is used on aliquots of samples obtained after the homogenization, filtration, and chloroform treatments to monitor the presence of vlps [ ] . chloroform treatment followed by dnase digestion is used to remove contaminating dna. the chloroform disrupts mitochondrial, bacterial and eukaryotic membranes, thereby exposing non-viral dna to the subsequent nuclease treatment [ , ] . unfortunately, chloroform treatment may also cause enveloped viruses to lose their protective lipid membrane, thereby rendering their dna subject to dnase digestion [ ] . moreover, dnase treatment does not always completely eliminate non-viral dna in the sample [ , ] . after extraction, dna may need to be amplified with random primers [ , ] . the whole transcriptome amplification (wta) kit can be used for the synthesis of cdna from viral rna [ ] . single virus genomics (svg) was introduced by allen and collaborators to selectively isolate viruses before sequencing [ ] . svg uses flow cytometry to sort viruses based on a method originally described by brussard et al. [ ] . following the sorting, dna of different sizes is immobilized in agarose gel, and then amplified using the multiple displacement amplification (mda) method. the svg approach can also be applied to rna viruses provided a reverse-transcription step is inserted between the flow cytometry and mda. high-throughput sequencing. early metagenomic applications involved the generation of shotgun libraries and direct sequencing of the total dna content using the sanger enzymatic dideoxy-sequencing method. this approach permitted the discovery of novel phages in marine environments [ , ] . the sanger technique had been the standard method for sequencing since it was first described in [ ] . development of the 'next-generation' sequencing platforms offered the combined advantages of speed, automation and high-throughput, thereby increased sequencing capabilities by a factor of to a million relative to the sanger technology. the illumina/solexa and roche next-generation sequencing platforms have been used most often in virus discovery (figure ). the illumina/solexa method is based on sequencing-by-synthesis chemistry using fragments of the sample dna ligated to oligonucleotide adapters. the adapters on a solid support act as primers for dna polymerase to incorporate reversible terminator nucleotides, each labeled with a different fluorescent dye. a typical sequencing run can generate up to gigabases of data with an average read length of - nucleotides [ ] . the sweetpotato badnavirus and the sweetpotato mastrevirus are examples of viruses discovered using the illumina/solexa sequencing platform [ ] . the flx titanium pyrosequencer commercialized by roche has been the most used for the discovery and characterization of novel viruses (http://www. .com/ publications-and-resources/publications.asp?postback= true). this platform was used for the identification of an uncharacterized mycovirus [ ] , solenopsis invicta virus [ ] , merino walk virus and a new arenavirus [ , ] , among others (figure b) . for sequencing, dna is fragmented and ligated to biotinylated specific linkers. the complex dna/linkers fragment is attached to streptavidin-coated beads that anchor the dna inside a droplet of water and pcr reagents in oil emulsion. each fragment is first amplified to produce the template for sequencing reaction. sequencing is carried out by annealing primers to the linker portion of the template complex, followed by the incorporation of nucleotides by dna polymerase, which facilitates the extension of the complementary dna. the pyrophosphate released by this process is measurable by the production of light [ , ] . the roche system measures the pyrophosphate released as the result of nucleotide incorporation during dna synthesis mediated by dna polymerase. the amount of light released is proportional to the intensity of the light signal captured by a charge-coupled device (ccd) camera, which then converts light signals into digital data [ , ] . a typical optimum run using a pyrosequencer yields about one million reads with an average length of - nucleotides, totaling about . gigabases. bioinformatic analyses. the analysis of the copious data generated by high-throughput sequencing is the most challenging aspect of metagenomics. an inherent difficulty in assigning taxonomic designations to viral sequences is that there is no universally homologous nucleic acid component present in all viruses that can be used to build phylogenetic trees -a factor that also fuels the debate over whether or not viruses belong in the tree of life [ , [ ] [ ] [ ] [ ] . in most metagenomic studies, sequences generated by high-throughput sequencing are queried by homology search tools to previously documented sequences stored either in a local database or in public databases such as the genbank. unfortunately, homology searches against known sequences in genbank cannot characterize unknown viruses (figure ). the analysis of metagenomic libraries requires fast computation and the right algorithms to characterize sequences as belonging to putative viruses. to ensure that bioinformatic analyses are performed only on high quality data, the reads are typically processed through a software pipeline to remove any background sequences including host and bacterial dna that had not been removed by the filtration, chloroform, and dnase i treatments [ ] [ ] [ ] . the resulting sequence reads are assembled with strict parameters to generate contigs, each made of sequences derived from the same organism quasi-species. using a stringent assembly parameter is critical to avoid sequence chimerization. the contigs sequences are then compared to the genbank non-redundant nucleotide database using blast [ ] or usearch [ ] . note that using a database containing only viral sequences will not be able to identify bacterial, archaeal or eukaryotic sequences and lead to an overestimation of the fraction of unknowns (see below). with the increasing number of data generated from different studies, there is a need for a cross-metagenome meta-analysis [ , ] . this is particularly important because of the diversity of different viral metagenomic protocols and the lack of standard algorithm for downstream data analysis. the following items should be included in any report on viral metagenomic studies: firstly, the sequencing platform and its version number; secondly, raw sequence data accession numbers in a public database; thirdly, details about the bioinformatic analysis, including the homology search tool and the database being used to assign the taxonomy, and their versions; fourthly, a list of known and previously unknown viruses found, clearly showing if the 'novel' viruses are new strains of a previously described species or completely different viruses; and fifthly, causality evidence if any. the most intriguing aspect of viral metagenomics is the fact that a large number -usually the majority -of sequences has no significant similarity to anything known. in this review, we refer to these sequences as the 'unknown' (figure a) . a typical human or environmental viral metagenome can contain between % and % unknown sequences (figure ) searches (figure b ). depending on how they are viewed, the unknowns can represent either a formidable challenge or a treasure trove for virus discovery. although researchers often tend to consider the unknowns as 'junk,' these sequences could be a valuable blueprint for the discovery of novel viruses [ , , ] . thus far, there is a lack of suitable bioinformatic methods to characterize the unknown sequences. a tentative solution is to compare the sequences between samples in order to at least gain some insight about the viral entities that are shared between them. a program such as phaccs (phage communities from contig spectra) can be used to assess the biodiversity of uncultured viral communities by mathematically modeling the community structure using the contig spectrum of metagenome assemblies [ ] . this method was extended to assess crossassemblies of reads from different samples [ ] , providing a homology-independent tool for the comparison of metagenomes with a high proportion of unknown sequences. although phaccs may provide a glimpse of the composition and difference between metagenomes, it has limited value for the characterization of novel viruses. two tools can be used to predict whether unknown sequences are from bacteriophages undergoing lytic and lysogenic lifestyles. one such tool described by deschavanne et al. [ ] compares the genome signatures of query sequences against those of their host genome in order to identify host-phage relationship and information about the phage lifestyle. the second method, phacts, depends on residual homology between the putative unknown sequence and sets of randomly selected viral proteins from known viruses (k mcnair et al., phacts: a computational approach to classifying the lifestyle of phages, unpublished data). alternatively, viruses may be classified by basic sequence properties. for instance, the circularity of the contig, its oligonucleotide profile [ ] , and the open reading frame (orf) structure (s akhter et al., phispy: a novel algorithm for finding prophages in bacterial genomes that combines similarity-based and composition-based strategies, under review) may all provide clues whether the unknown sequence could be from a potential novel virus. these properties can be combined into a prediction network used to classify viruses into lifestyle groups or taxonomic clades. although newly discovered viruses are often labeled 'novel,' the question remains whether these sequences represent truly novel viruses or ancient viruses that simply have never been observed before. the age of a sequence has traditionally been determined by multiple alignments of query sequences with their homologs and by calculating the divergence times from a common ancestral node on a phylogenetic tree. dates can be estimated using either a molecular clock [ ] or by assigning a calibration date to a specific node in the tree based on fossil or other evidence [ ] [ ] [ ] . for viral metagenomic sequences, however, building a phylogenetic tree is itself problematic because often the sequenced reads may represent non-overlapping subregions of an unknown viral genome. moreover, there is no fossil data available to calibrate the age of nodes in the tree. a promising approach might be to estimate divergence times from assembled viral contigs. de novo assembly allows non-overlapping regions to be combined into a single consensus sequence. for a given molecular clock, snp analysis of the contributing reads could provide an estimation of how long ago the sequenced reads diverged. such estimates may be critical when addressing the question of the origin of a newly identified infectious agent. until recently, virus discoveries were made in the context of disease etiology. thus, virus discovery studies were biased mainly because of the use of convenient samples available from patients. because of the difficulties involved, the investment of efforts and resources required to isolate viruses often could not be justified outside the disease context. it is likely that the context of the diseases has also led to the misconception that all viruses are pathogenic. this dogma was challenged by the discovery of viruses such as torque teno virus (ttv) and hepatitis g virus (gbv-c), originally associated with post-transfusion hepatitis [ , [ ] [ ] [ ] , and then were subsequently shown be classical examples of viral commensals [ , ] . the widely accepted notion that viruses act as obligatory pathogens is beginning to give way to the concept that viruses can be part of the normal flora of the human body. considering their high abundance in the gastrointestinal tract, on skin and even in blood and lungs [ ] it is unlikely that viruses could only be pathogenic without any benefits for their hosts. the abundance of viruses, particularly phages, in the lung -an environment previously thought to be sterile -may reflect their beneficial role in keeping bacterial populations in check [ ] . the pathogenicity of the gbv-c has shifted to a more radical designation as a 'good' virus in cases of coinfection with hiv. indeed, gbv-c has been associated with a more favorable prognosis for patients with hiv infection by slowing the progression to aids [ , ] . similarly, dengue virus, a known pathogen, has been shown to limit hiv- replication and to reduce the viral load [ ] . these examples need to be taken into account when metagenomic approach is applied to virus discovery. the characterization of a novel virus can be easily achieved in silico with limited bioinformatics tools but the determination of causation may not always be trivial. the causality is not always conclusive even when the suspect virus is found in the scene of the crime. in other words, finding a virus in a sample from a patient with an illness of unknown etiology and even demonstrating the association does not always prove causation. for this reason, strict guidelines proposed by robert koch and later modified by rivers [ ] have been used to assign causality to infectious agents. one of koch's postulates requires that the candidate etiological agent be isolated from a diseased organism and grown in pure culture. however, many viruses cannot be propagated by culture techniques [ ] . new molecular biology techniques have been used for virus discovery bypassing the prerequisite of the koch's postulates. for instance, the merkel cell polyomavirus (mcv) was identified as the causative agent of merkel's cell carcinoma without satisfying all of the requisites of koch's postulates [ ] . similarly, the sea turtle tornovirus was associated with fibropapillomatosis using a culture-independent metagenomic approach [ ] . the methodological shift, from culture to metagenomics, will likely create a paradigm shift in the demonstration of disease causation. in many instances koch's postulates will no longer be satisfied if culture techniques are used to prove causality. falkow [ ] proposed the modified koch's postulates which uses molecular methods to monitor the role played by genes in distinct bacterial virulence. to satisfy the revised molecular koch's postulates, a strong association must be established between the phenotype or property under investigation and the pathogenic members of a genus or pathogenic strains of a species. the gene of interest should be found in all pathogenic members of the genus or species but be absent in nonpathogenic strains. at best, the nonpathogenic strains could carry the gene with critical mutations that could render the strain non-virulent. however, new molecular methods do not always distinctively characterize virulence genes and make a clear association with a disease of unknown etiology. this could be because genes can be expressed at different time-points during infection. genes can be turned on and off and may require intrinsic factors in order to trigger the disease process. alternatively, we propose the metagenomic koch's postulates, which focus on the identification of metagenomic traits in disease subjects. the metagenomic traits are molecular markers such as sequence reads, assembled contigs, genes or full-genomes that can uniquely distinguish diseased metagenomes from those obtained from matched healthy control subjects (figure ) . the metagenomic traits found in diseased patients can be monitored in healthy individuals exposed to the suspected infectious agent. although this novel approach requires separation or isolation of remaining co-occurring disease candidates (figure . ) , it does not necessarily require the isolation of the pathogen in tissue culture or pure culture media unlike the original koch's postulates. therefore, the genetic make-up of the agent responsible for a disease can provide early clues before its isolation by tissue culture. the modified metagenomic koch's postulates proposed in this paper require that: firstly, the diseased metagenome be significantly different from the metagenome constructed with the same sample type obtained from a healthy matched control subject. the suspected metagenomic traits must be present and more abundant in the diseased subject compared to matched control (figure . ) . secondly, inoculating a healthy individual with a sample from a diseased subject must result in disease state (figure . ). differential metagenomic traits in step ( ) recovered in the newly induced diseased subject may be the biomarker of the candidate etiological agent; and finally, selective inoculation of samples from the disease subject (in step ) must induce disease in another healthy control subject if the metagenomic contains the trait associated with the etiological agent of the disease, or phenotype under investigation (figure . ) . assuming that the metagenomic trait 'e' (figure . ) is a contig sequence from a previously unknown and unculturable virus, its early identification using the metagenomic approach could spearhead the effort to generate diagnostic assays such as elisa and pcr, well before the isolation and the characterization of the viruses by culture techniques. fulfilling this metagenomic model of the koch's postulates is possible when one or multiple viral agents are involved in disease causation. with the original koch's postulates or the modified molecular koch's postulates, it is difficult enough to prove causality with one suspected agent using the culturing prerequisite. the complexity is even greater when multiple viruses are involved in the causation of a disease. a similar approach, the sirna-ome used previously by kreuze et al. [ ] led to the detection of etiological viruses causing diseases in plants despite the low copy number of the suspected traits [ ] . the modified metagenomic koch's postulates could also be tested in human diseases such as the murine mink cell leukemia caused by a c-type retrovirus, named the mink cell focus-inducing virus (mcfiv) [ ] . mcfiv requires the cooperative interaction with other viruses to increase its propensity to cause leukemia [ ] . the burkitt's lymphoma caused by others epstein-barr virus (ebv) in regions holoendemic for plasmodium falciparum, the etiology of malaria [ ] . metagenomics could become the future method of choice enabling the simultaneous analysis of multiple agents in a sample and assessment of the association and disease causality without the limitations imposed by culture techniques [ , , ] . the etiology of many diseases remains unknown. these ailments are collectively defined as diseases of unknown etiology when all conventional testing laboratory techniques are unsuccessful. yet, the diseases with unknown origin have high rates of morbidity and mortality. for example, as many as % of cases of the infantile diarrhea, which alone claims $ . million fatalities annually, have no known specific causative agent [ ] . infantile diarrhea, the pyrexia of unknown origin, influenza-like illnesses, chronic fatigue syndrome, alzheimer's disease, various forms of tumors such as diffuse large b-cell lymphoma and many other diseases of unknown origin can benefit directly from the metagenomic technology. the success of metagenomics in identifying novel viruses in a wide variety of samples opens doors to new application areas particularly in public health and the prevention of infectious diseases. although the metagenomic technology is not yet part of the routine diagnostics, results from clinical virology research provides valuable proof of concepts for a new era in clinical virology practices. for example, finkbeiner et al. analyzed samples from children using metagenomics and identified a large number of known eukaryotic viruses as well as sequences from putatively novel viruses [ ] . another study identified a corona-like virus, the human cosavirus e (hcosv-e ), in a child with acute diarrhea [ ] . these initial studies identified promising viral candidates to establish the etiology in these cases of diarrhea. the pandemic of influenza a ( h n ) provided proof of concept in that metagenomics was effective to rapidly characterize the full genome of the flu virus [ ] . using the metagenomic approach, palacios et al. discovered an arenavirus in samples which had tested negative by culture, pcr, serology and a microarray assay using oligonucleotide probes from a wide range of infectious agents [ ] , suggesting a potential causative agent for unexplained cases of posttransplantation death. in another study, towner et al. described a new ebola virus responsible for an outbreak of a hemorrhagic fever in the district of bindibugyo, uganda [ ] . rapid identification of these agents would provide the blueprint for the development of therapeutic regimen or preventive vaccine. prevention is better than cure. potentially, a single or multiple jump of an animal virus to humans can have serious consequences. one way to prevent infectious diseases is through vaccine development. but the development of a vaccine takes time and demands a huge amount of resources. preventing the introduction of an unknown virus to human populations is rather a farreaching goal unless the methods of virus identification and characterization are put in place. a simple and practical strategy would be to assess the danger posed by viruses that thrive in animals and could cross to human through zoonosis. zoonosis is a source of up to % of emerging infectious diseases in humans [ ] . as such, cross-species transfer from animals to humans has serious repercussions not only in public health but also in the socio-economical and political stability [ , [ ] [ ] [ ] [ ] [ ] . the detection and characterization of novel viruses are of paramount importance in the forecasting of future outbreaks of viral diseases in humans. surveying natural reservoirs for potential zoonotic infection [ ] and human populations such as bush meat hunters who are exposed to animals could help prevent major outbreaks before the wide spread of viruses to human population. data obtained in early identification of viruses are valuable for forecasting new emerging and re-emerging viral epidemics. the experience gained from studying marine environments and hostile mine environments can be applied in public health programs that seek to determine the normal viral population and monitor changes in different geographical settings. we have termed such an approach as public health viral metagenomics surveillance (phvms). viral metagenomics surveillance is defined as the survey of the functional and taxonomic signatures representing the viruses normally circulating within that population in the absence of noticeable epidemics. in the event of a zoonotic outbreak, these functional and taxonomic signatures of the virome will likely show detectable shifts. figure shows a hypothetical rank abundance curve for six viruses (a-f). the introduction of a highly pathogenic species (g) can be expected to result in a disruption of the normal virome, including the appearance of opportunistic viral infections (h). using phaccs analysis [ ] , several parameters can be compared between the normal and disturbed viromes including the total number of viral species (richness) and their relative abundance (evenness). another approach would be to determine the normal virome, a background viral metagenome to refer to in case of an outbreak. lessons learned from studies of bacterial microbial metagenomes suggest that different environments often have different microbial signatures [ ] , including the functional metabolic information, the nucleotide usage, proportion of different species. disrupting key metabolic processes of an environment can lead to disruption of the balance in that ecosystem. similarly, the viromes in different human populations in different locations may display functional profiles characteristic of their respective environment, lifestyle and viruses circulating in each region. the magnitude of disturbance of the virome profile will depend on the fitness and virulence of the newly introduced pathogens and the immune fitness of the host. the viral communities in two different metagenomes can be compared using xipe [ ] . this statistical approach was developed for comparing metagenomic sequences derived from samples collected from the sargasso sea and from acid mine drainage and was able to accurately predict the physiology, metabolic potential and ecology of each ecosystem [ ] . during the last decade, we have witnessed the emergence of metagenomics as a powerful novel tool with endless areas of applications in virology. epidemiological data suggest that novel viruses are likely to be introduced into the human population through zoonosis [ , ] . also, the danger of intentional introduction of viruses through bioterrorism cannot be ignored. viral metagenomics is a powerful, fast and sensitive technique available for identifying viruses including those that cannot be detected by conventional culture and sequence-dependent methods. monitoring of emerging infectious diseases using a metagenomic approach. a hypothetical example of the potential use of the public health viral metagenomics surveillance (phvms) approach for virus discovery based on comparison of viromes sampled before (i) and during (ii) an epidemic. depicted here are the rank abundance curves for viral species (a-h), where g represents a newly introduced, highly pathogenic species and h a less virulent virus. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest high abundance of viruses found in aquatic environments prokaryotes: the unseen majority consider something viral in your research analysis of a marine picoplankton community by s rrna gene cloning and sequencing comparing bacterial communities inferred from s rrna gene sequencing and shotgun metagenomics bacterial s sequence analysis of severe caries in young permanent teeth a renaissance for the pioneering s rrna gene a comparison of random sequence reads versus s rdna sequences for estimating the biodiversity of a metagenomic library metagenomics -the key to the uncultured microbes a census of rrna genes and linked genomic sequences within a soil metagenomic library the phage proteomic tree: a genomebased taxonomy for phage metagenomics: genomic analysis of microbial communities biotechnological prospects from metagenomics opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms next-generation dna sequencing techniques a history of poliomyelitis. yale studies in the history of science and medicine the discovery of the poliovirus smallpox and its eradication the greatest killer -smallpox in history discovery of the first virus, the tobacco mosaic virus: or ? methods to detect infectious human enteric viruses in environmental water samples role of cell culture for virus detection in the age of technology rapid viral diagnostic techniques delwart el: viral metagenomics a very comprehensive description of metagenomic methods and important benchmarks achieved in the virus discovery effort isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome an assay for circulating antibodies to a major etiologic virus of human non-a, non-b hepatitis ethical reflections on edward jenner's experimental treatment metagenomics for the discovery of novel human viruses a comprehensive review describing metagenomic methods and important benchmarks achieved identification of a novel clade of human immunodeficiency virus type in democratic republic of congo a novel simian immunodeficiency virus from black mangabey (lophocebus aterrimus) in the democratic republic of congo characterization of a novel simian immunodeficiency virus (sivmonng ) genome sequence from a mona monkey (cercopithecus mona) isolation and partial characterization of a lentivirus from talapoin monkeys (myopithecus talapoin) a novel simian immunodeficiency virus (sivdrl) pol sequence from the drill monkey, mandrillus leucophaeus isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis quantitative monitoring of gene expression patterns with a complementary dna microarray viral discovery and sequence recovery using dna microarrays identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r q rnasel variant prostate cancer: xmrv -contaminant, not cause? no association of xenotropic murine leukemia virus-related viruses with prostate cancer reliability and reproducibility issues in dna microarray measurements efficient isolation of genes differentially expressed on cellulose by suppression subtractive hybridization in agaricus bisporus virus discovery by sequenceindependent genome amplification suppression subtraction hybridization (ssh) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus suppression subtractive hybridization: a versatile method for identifying differentially expressed genes identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology identification of two flavivirus-like genomes in the gb hepatitis agent stat -dependent innate immunity to a norwalk-like virus sequence-independent, single-primer amplification (sispa) of complex dna populations metagenomics and the molecular identification of novel viruses viruses in the faecal microbiota of monozygotic twins and their mothers hepatitis e virus (hev): the novel agent responsible for enterically transmitted non-a, non-b hepatitis the isolation and characterization of a norwalk virus-specific cdna identification of a novel astrovirus (astrovirus va ) associated with an outbreak of acute gastroenteritis detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species laboratory procedures to generate viral metagenomes an excellent compilation of standard operating procedures to perform metagenomic analysis on different types of samples the marine viromes of four oceanic regions method for discovering novel dna viruses in blood using viral particle selection and shotgun sequencing analysis of the virus population present in equine faeces indicates the presence of hundreds of uncharacterized virus genomes multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses viral diversity and dynamics in an infant gut rna viral community in human feces: prevalence of plant pathogenic viruses viral communities associated with healthy and bleaching corals metagenomic analysis of stressed coral holobionts assembly of viral metagenomes from yellowstone hot springs using pyrosequencing to shed light on deep mine microbial ecology microbes and health sackler colloquium: metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity extraction of high molecular weight genomic dna from soils and sediments rapid amplification of plasmid and phage dna using phi dna polymerase and multiply-primed rolling circle amplification assessment of whole genome amplification-induced bias through highthroughput, massively parallel whole genome sequencing whole transcriptome amplification for gene expression profiling and development of molecular archives single virus genomics: a new tool for virus discovery flow cytometric detection of viruses dna sequencing with chainterminating inhibitors complete viral genome sequence and discovery of novel viruses by deep sequencing of small rnas: a generic method for diagnosis, discovery and sequencing of viruses arbovirus detection in insect vectors by rapid, highthroughput pyrosequencing isolation and characterization of solenopsis invicta virus , a new positive-strand rna virus infecting the red imported fire ant, solenopsis invicta a new arenavirus in a cluster of fatal transplant-associated diseases genomic and phylogenetic characterization of merino walk virus, a novel arenavirus isolated in south africa parallel tagged sequencing on the platform targeted high-throughput sequencing of tagged nucleic acid samples the history of pyrosequencing a new method of sequencing dna the not so universal tree of life or the place of viruses in the living world reasons to include viruses in the tree of life viral genomes are part of the phylogenetic tree of life there is no such thing as a tree of life (and of course viruses are out!) quality control and preprocessing of metagenomic datasets fast identification and removal of sequence contamination from genomic and metagenomic datasets tagcleaner: identification and removal of tag sequences from genomic and metagenomic datasets basic local alignment search tool the minimum information about a genome sequence (migs) specification get the most out of your metagenome: computational analysis of environmental sequence data cloning of a human parvovirus by molecular screening of respiratory tract samples metagenomic analysis of coastal rna virus communities identification of a third human polyomavirus metagenomic characterization of chesapeake bay virioplankton a metagenomic survey of microbes in honey bee colony collapse disorder metagenomic and small-subunit rrna analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil biodiversity and biogeography of phages in modern stromatolites and thrombolites viral genome sequencing by random priming methods metagenomic analysis of human diarrhea: viral detection and discovery novel borna virus in psittacine birds with proventricular dilatation disease rapid identification of known and new rna viruses from animal tissues next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa the complete genome of klassevirus -a novel picornavirus in pediatric stool discovery of a novel single-stranded dna virus from a sea turtle fibropapilloma by using viral metagenomics novel anellovirus discovered from a mortality event of captive california sea lions metagenomic analysis of viruses in reclaimed water novel circular dna viruses in stool samples of wild-living chimpanzees novel picornavirus in turkey poults with hepatitis the fecal virome of pigs on a high-density farm systematic artifacts in metagenomes from complex microbial communities metagenomics: facts and artifacts, and computational challenges phaccs, an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information the use of genomic signature distance between bacteriophages and their hosts displays evolutionary relationships and phage growth cycle determination metagenomic signatures of microbial and viral metagenomes molecular dating in the evolution of vertebrate poxviruses genomic fossils calibrate the long-term evolution of hepadnaviruses fossil record of an archaeal hk -like provirus r s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock detection of a novel dna virus (ttv) in blood donors and blood products prevalence of gbv-c and hepatitis g virus variants in patients with fulminant hepatic failure in japan a prospective study of transfusion-transmitted gb virus c infection: similar frequency but different clinical presentation compared with hepatitis c virus transfusion transmission of highly prevalent commensal human viruses chronic viral hepatitis in hemodialysis patients metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals persistent gb virus c infection is associated with decreased hiv- disease progression in the amsterdam cohort study gbv-c/hepatitis g virus (hgv) rna load in immunodeficient individuals and in immunocompetent individuals decrease in human immunodeficiency virus type load during acute dengue fever viruses and koch's postulates sequence-based identification of microbial pathogens: a reconsideration of koch's postulates clonal integration of a polyomavirus in human merkel cell carcinoma significant paradigm change and a challenge to the existing koch's postulates and the proposal to use molecular methods to assign etiology to infectious agents a virus-virus interaction circumvents the virus receptor requirement for infection by pathogenic retroviruses etiology of endemic burkitt's lymphoma deep sequencing analysis of rnas from a grapevine showing syrah decline symptoms reveals a multiple virus infection that includes a novel virus the prostate cancer-associated human retrovirus xmrv lacks direct transforming activity but can induce low rates of transformation in cultured cells identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea a metagenomic analysis of pandemic influenza a ( h n ) infection in patients from north america newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda risk factors for human disease emergence emerging disease: looking for trouble bushmeat hunting, deforestation, and prediction of zoonoses emergence emergence of unique primate t-lymphotropic viruses among central african bushmeat hunters naturally acquired simian retrovirus infections in central african hunters applying the theory of island biogeography to emerging pathogens: toward predicting the sources of future emerging zoonotic and vector-borne diseases functional metagenomic profiling of nine biomes an application of statistics to comparative metagenomics new dna viruses identified in patients with acute viral infection syndrome novel, divergent simian hemorrhagic fever viruses in a wild ugandan red colobus monkey discovered using direct pyrosequencing we are grateful to merry youle for helpful suggestions and editing of the manuscript. jm was supported by a grant from the ucsd center for aids research (niaid p ai ) and moores ucsd cancer center (nci p ca ). bed was supported by the dutch science foundation (nwo) veni grant ( . . ). key: cord- -r k u ro authors: yu, xia; sun, shanshan; shi, yu; wang, hao; zhao, ruihong; sheng, jifang title: sars-cov- viral load in sputum correlates with risk of covid- progression date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: r k u ro nan the pandemic of coronavirus diseases (covid- ) imposes a heavy burden on medical resources [ ] . whether there is correlation between viral load and disease severity has not been clarified. in the study, we retrospectively collected the virological data, as well as demographic, epidemiological clinical information of patients with confirmed covid- in a single hospital in zhejiang province, china. we compared the baseline viral loads between severe patients and those mild to moderate at admission and also between those developing severe disease during hospitalization and those not. we studied patients with confirmed covid- who were admitted from january , , to march , , in the first affiliated hospital of zhejiang university. the sputum specimens were collected from the lower respiratory tract of each patient at admission and the levels of viral nuclei acid were determined by a real-time pcr (rt-pcr) approach and indicated by the cycle threshold (ct) values of rt-pcr assays [ ] . other demographic, epidemiological and clinical information were collected and inputted into a pre-designated electronic data collection form. all patients followed up to march , . all the statistical analyses were performed using graphpad prism (graphpad software inc.; san diego, ca, usa) and spss . (spss inc.; chicago, il, usa). of the patients, were severe on admission. of the other mild-moderate cases at admission, cases became severe during hospitalization. the demographic, epidemiological and clinical information was shown in table . all patients were tested for sars-cov- nucleic acid on sputum specimens from the lower respiratory tract at admission. as shown in fig. a , severe patients had significantly lower ct values than mildmoderate cases at admission ( vs. , p = . ), suggesting a higher viral load in the lower respiratory tract. furthermore, a higher viral load was observed in sputum specimens from patients who became severe during the hospitalization than those did not ( vs. , p = . ). as shown in fig. b , the ct values of rt-pcr assays negatively correlated with the probability of progression to severe type in all the patients representing mild-tomoderate at admission. we found that the viral load of the sputum specimen in the lower respiratory tract tested at baseline is closely related to the severity of covid- . more importantly, patients with a higher baseline viral load are more likely fig. a comparison of baseline sputum viral load between severe and mild-to-moderate patients at admission or between those becoming severe and those did not during the hospitalization. b relationship between the estimated probability of disease progression during the hospitalization and baseline sputum viral load. viral load is indicated by the ct value of rt-pcr assay. the asterisk indicates a p value < . to become severe. this finding apparently justifies the concept that early antiviral treatment, if effective, would reduce the risk of progression and thereby the mortality, which has been demonstrated in influenza [ ] . in our study, sputum specimens were used, instead of nasopharyngeal and oropharyngeal swabs because it has been shown that samples from lower respiratory tract generally contain a higher level of viral load than nasopharyngeal and oropharyngeal swabs [ ] and acquiring swabs is uncomfortable for patients. in summary, we found a positive association between sputum viral load and disease severity as well as risk of progression. potential association between covid- mortality and health-care resource availability sars-cov- viral load in upper respiratory specimens of infected patients viral load of sars-cov- in clinical samples publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions j.s conceptualized the idea and designed the study. x.y and s.s drafted the manuscript and j.s revised it. x.y, s.s, y.s, h.w, and r.z participated in the data collection, analysis, and interpretation. the authors read and approved the final manuscript. this work was supported by grants from chinese national natural science foundation (no. and ) and the fundamental research funds for the central universities. the datasets and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request. this study is reviewed and approved by the ethics committee of the first affiliated hospital of zhejiang university. following a full explanation of the study, written consent was obtained from each patient or his/her authorized representatives. not applicable. the authors declare that they have no competing interests.received: march accepted: april key: cord- -wjf t vp authors: brister, j. rodney; ako-adjei, danso; bao, yiming; blinkova, olga title: ncbi viral genomes resource date: - - journal: nucleic acids res doi: . /nar/gku sha: doc_id: cord_uid: wjf t vp recent technological innovations have ignited an explosion in virus genome sequencing that promises to fundamentally alter our understanding of viral biology and profoundly impact public health policy. yet, any potential benefits from the billowing cloud of next generation sequence data hinge upon well implemented reference resources that facilitate the identification of sequences, aid in the assembly of sequence reads and provide reference annotation sources. the ncbi viral genomes resource is a reference resource designed to bring order to this sequence shockwave and improve usability of viral sequence data. the resource can be accessed at http://www.ncbi.nlm.nih.gov/genome/viruses/ and catalogs all publicly available virus genome sequences and curates reference genome sequences. as the number of genome sequences has grown, so too have the difficulties in annotating and maintaining reference sequences. the rapid expansion of the viral sequence universe has forced a recalibration of the data model to better provide extant sequence representation and enhanced reference sequence products to serve the needs of the various viral communities. this, in turn, has placed increased emphasis on leveraging the knowledge of individual scientific communities to identify important viral sequences and develop well annotated reference virus genome sets. recent outbreaks of ebolavirus ( , ) and middle east respiratory syndrome coronavirus (mers-cov) ( , ) clearly demonstrate the power of sequence analysis in viral surveillance, host reservoir identification and public health policy debate. as these viruses have filled media headlines, their genome sequences have spilled into international public databases. such real time analysis promises to fundamentally alter our understanding of viral biology and significantly impact public health responses to viral dis-ease, but it also places renewed emphasis on public research infrastructure that is necessary to support the storage and analysis of sequence data. this infrastructure includes primary databases that together comprise the international nucleotide sequence database collaboration (insdc) ( ) , genbank ( ) , european molecular biology laboratory's european bioinformatics institute (embl-ebi) ( ) , and dna database of japan (ddbj) ( ) , and reference databases like the viralzone resource at the swiss institute of bioinformatics (http://viralzone.expasy. org) ( ) and the viral genome resource at national center for biotechnology information (ncbi) (http://www. ncbi.nlm.nih.gov/genome/viruses/) ( ) . whereas primary databases are archival repositories of sequence data, reference databases provide curated datasets that enable a number of activities, among them are transfer annotation to related genomes ( ) ( ) ( ) , sequence assembly and virus discovery ( ) ( ) ( ) ( ) , viral dynamics and evolution ( ) ( ) ( ) and pathogen detection ( , ( ) ( ) ( ) . the ncbi viral genomes project was established in response to the growing need for a public, virus-specific, reference sequence resource ( ) . the project catalogs all complete viral genomes deposited in insdc databases and creates so-called refseq records for each viral species. each refseq is derived from an insdc sequence record, but may include additional annotation and/or other information. accessions for refseq genome records include the prefix 'nc ', allowing them to be easily differentiated from insdc records. for example, the refseq genome record for enterobacteria phage t has the accession nc but was derived from the insdc record af . typically, the first genome submitted for a particular species is selected as a refseq, and once a refseq is created, other validated genomes for that species are indexed as 'genome neighbors'. as such, the viral refseq data model is taxonomy centric, or more specifically, species centric, and all refseq records and genome neighbors are indexed at the species level. this model requires both the demarcation of individual viral species and the grouping of genome sequences into defined species. virus genome type refseq genome segments total genome segments total insdc sequences dsdna viruses, no rna stage dsrna viruses ssdna viruses ssrna negative-strand viruses ssrna positive-strand viruses, no dna stage retro-transcribing viruses a the table does not include influenza virus sequences. these sequences are stored in a specialized database ( , ) . there are now validated viral and viroid genome segments deposited within insdc databases, not including influenza sequences, which are stored in a specialized database ( , ) . this figure represents a nearly fold increase since (figure ), and this rise reflects both steady increases in the number of novel viruses sequenced--as measured by the number of refseq genome segments--and a large increase in the number of genome neighbors, i.e. genome sequences belonging to viral species already represented by a refseq (figure ). as shown in table , refseq genome segments are distributed among all viruses, but genome neighbor segments are concentrated among smaller, ssdna, rna, and retro-transcribing viruses. although many of these neighbor genomes are concentrated among human pathogens, there are also several viruses of agricultural importance with high numbers of sequenced genomes ( table ). while most of the viruses in table are well studied in the laboratory, many other sequenced viruses are not. the refseq data model for most organisms underscores the importance of very well annotated reference sequence records ( ) . unfortunately, a minority of viral systems are experimentally well defined, so there is often little primary data on which to base genome annotations. in some cases, sequence homologies allow the transfer of annotation from experimentally defined to poorly characterized genomes ( ) ( ) ( ) . yet, often genomes are annotated by purely ab initio processes ( ) ( ) ( ) . given the difficulty of implementing a purely well annotated representation of viral genome sequences, the viral refseq model has evolved into a more flexible approach that includes both reference and representative sequences. reference refseq records provide sources of well annotated sequence features, whereas representative records provide coverage of extant sequence variation. the comment 'reviewed refseq' is added to refseq records to highlight those that include additional annotation, and as of this writing, there are reviewed viral refseq records, including references for several human pathogens, such as human immunodeficiency virus (nc ), measles virus (nc ) and poliovirus (nc ) and several other important viral systems such as enterobacteria t (nc ), enterobacteria t (nc ) and tobacco mosaic virus ( ) ( ) . moreover, some viral communities are developing well defined subspecies classification such as the genotyping schemes for hepatitis b virus and hepatitis c virus ( ) ( ) ( ) . these genotyping schemes can provide an important framework for the interpretation of genome sequence data ( ) , and more communities are expected to develop genotyping schemes in the coming years. finally, there are cases when the best characterized viral isolate is a laboratory variant, and it may be important to create multiple refseq records in order to provide both experimentally annotated references and sufficient sequence representation of circulating isolates. together these cases highlight the need for both reference genome sequences that capture the best possible annotation and representative genome sequences that capture important intraspecies variation or define subspecies categories. therefore the viral refseq model has expanded to include both reference and representative genome sequences to better serve community needs. the rising pace of viral discovery has a number of implications for data processing by the viral genomes group. viral taxonomy within the ncbi taxonomy database is based on the list of valid species names and classifications provided by the international committee for the taxonomy of viruses (ictv) ( , ) . when the viral genomes project was initiated, there were many more viral species recognized by the ictv than viral refseq genome sequence records ( figure ). however, as the rate of viral genome sequencing has increased over the past decade, so too has the pace of viral discovery. as a result many refseqs are made from viruses clearly distinct from existing ones but without of- ficial taxonomy designation. taxonomy also affects the interpretation of genome sequence data, and technical difficulties encountered when sequencing the termini of some ssrna and ssdna viruses often lead to differing community standards for 'complete genomes' ( ) . this means that some difficult to sequence genomes are considered complete if they include the entire coding region but are missing some terminal sequence. improved methods may eventually resolve this issue ( ) , but in the meantime it would be useful for communities to define completeness standards with regard to current technology. in addition to manual selection based on genome length, the taxonomy of both refseq genome records and insdc genome neighbor records are validated. indeed, given that many novel virus genome sequences are submitted before analysis by the ictv (see figure ), validation of taxonomy assignment is a major facet of curation. taxonomy is important to the overall usability of ncbi viral genome resources, and when properly implemented, creates a framework for groups of related sequences. using standards established by individual ictv study sections ( ) and published reports, the taxonomy of each viral genome is validated and updated as necessary. newly submitted viral genomes without official ictv assignment are placed with 'uncharacterized' taxonomy bins that are easily distinguished from those recognized by the ictv. often little information is included in the insdc sequence record and a growing number of sequences do not include any linked publications. using sequence analysis and comparative genomics, every attempt is made to place new genomes into a family (i.e. the 'uncharacterized' bin associated with a specific family) or lower order classification bin. however, some genomes are very distinct from previously characterized ones and only higher order classification is possible. reference viral refseq records are generally curated by biologists using in-house annotation tools and the scientific literature as guides. a panel of viral genome advisors from outside ncbi bolsters curation efforts by offering expert guidance or taking responsibility for specific refseq records themselves. this approach is used for the maintenance of adenovirus and herpesvirus refseq records ( ) and could be extended to other virus genomes ( ) . these efforts considered, the growing number of viral genomes submitted to insdc databases and the rapid pace of scientific discovery make maintenance of up-to-date references difficult. therefore collaboration with scientific communities is critical to providing accurate annotation. sometimes these collaborative efforts are directed at curating a single refseq record, and all of the reviewed refseq records mentioned in the previous section were curated in collaboration with experts from individual viral communities. other times these collaborations are more extensive and touch many sequence records. for example, overlapping gene annotations were corrected on refseq records from virus families (arteriviridae, arteriviridae, bunyaviridae, caliciviridae, circoviridae, disistroviridae, flavoviridae, luteoviridae, paramixovridae, parvoviridae, picornaviridae, potyviridae, reoviridae, togaviridae) as directed by experimental or predictive analysis ( , ) . a new emphasis has been placed on initiating annotation collaborations at the beginning of a large genome sequencing program so that reference annotations, isolate naming schemes and other standards can be established prior to sequence submission ( ) ( ) ( ) . these collaborations often include members of the uniprot viral protein annotation program ( ) ( ), and/or curators from sequencing centers and other databases ( ) in addition to members of the relevant viral communities and effectively ensure both well annotated references and consistently annotated insdc sequence records. such arrangements underscore the extensive impact of viral genome annotation issues--from public databases to sequencing centers to individual researcher communities--and were formalized within the viral genome annotation working group, which brings together stakeholders and provides a forum for the discussion of annotation issues ( , ) . in addition to protein annotation and isolate naming issues, this group is working to define standards for viral genome sequence data. as the number of viral sequences has risen, so has the demand for curated metadata describing sequences. the viral genomes group has implemented two models designed to capture and standardize metadata. in the first model exemplified by the virus variation resource, host, isolation country and other important metadata are parsed from individual sequence records, mapped against vocabulary lists and standardized ( , ) . sequences can then be searched using these standardized metadata terms. currently, only a small subset of viral sequences are included in the virus variation resource, including those for influenza, dengue and west nile viruses, but the ultimate goal is to expand this semi-automated model to include more viruses. the second model captures and standardizes host information for all viruses, and whenever a new refseq record is created, a manually curated 'viral host' property is assigned to the relevant species within the ncbi taxonomy database. the property defines higher order, biologically relevant taxonomic host groups--algae, archaea, bacteria, diatom, environment, fungi, human, invertebrates, plants, protozoa and vertebrates--and enable sorting and selection of sequences within the ncbi taxonomy (http://www. ncbi.nlm.nih.gov/taxonomy) and viral genomes resource. for example searching the ncbi taxonomy database with the term 'vhost fungi'[properties] (quotes included) will return a list of taxonomy groups comprised of viruses that infect fungi. users can then select the 'genome' database from 'find related data' link on the taxonomy search page to view all viral genomes associated with viruses retrieved from the search. in cases where a virus infects multiple types of organisms, multiple terms are assigned, for example 'invertebrates, plants'. to search ncbi taxonomy for viruses that infect multiple hosts simply include 'and' between search terms, for example 'vhost invertebrates' [properties] and 'vhost plants' [properties] (quotes included). the current distribution of assigned viral host terms is shown in figure . the ncbi viral genome resource can be accessed at www.ncbi.nlm.nih.gov/genome/viruses/. on this home page, users will find ftp links where users can download accession list of all viral and viroid genomes (refseq and genome neighbors) and the complete viral and viroid ref-seq dataset. perhaps the central features of the resource are the viral and viroid genome browsers. these tables list all viral and viroid species represented by a reference sequence and include links to genome neighbor sequences. users can navigate to specific taxonomic groups and sort the table by viral host type. once a dataset has been defined by taxonomy and host types, users can download the resultant table, the list of refseq accessions in the table, or a list that includes refseq and genome neighbor accessions as well as taxonomy and viral host information. several specialized viral resources and tools are also linked through the viral genomes resource home page. these include specialized resources for influenza, dengue and west nile and other viruses that are part of the virus variation resource (http://www.ncbi.nlm.nih.gov/genome/viruses/variation/) ( , , ) . the link to the retrovirus resource (http://www.ncbi.nlm.nih.gov/genome/viruses/retroviruses) provides access to the retrovirus genotyping tool and hiv- , human interaction database ( , ) . these tools are designed to assist retroviral researchers in the identification and classification of sequences and to document hiv- and human protein and replication interactions through a searchable interface. finally, there is a link to the pairwise sequence comparison tool (pasc) (http://www.ncbi.nlm.nih.gov/sutils/pasc), a blast-based tool with graphical output that can be used to establish taxonomic classification criteria of some viruses and classify viruses with newly sequenced genomes ( , ) . both refseq records and other genomes for species are linked throughout ncbi resources and can be used in a variety of operations. among these, the refseq dataset can be used to reduce the redundancy of blast searches (http://blast.ncbi.nlm.nih.gov/blast.cgi) ( ), providing fewer, higher quality sequences within search results. to restrict nucleotide blast searches to include only viral refseq genomes, employ the 'choose search set' options in the blast search interface ( ): select 'reference genomic sequences (refseq genomic)' in the database field and enter 'viruses' in the 'organism' field text box. for protein blast searches, the viral refseq protein set can be used by selecting 'reference proteins' (refseq proteins) in the database field and entering 'viruses' in the 'organism' field text box. data derived from viral refseqs are also used to support a number of other databases including gene ( ) and protein clusters ( ) . each species that includes a refseq can be found in the genome database (http://www.ncbi.nlm.nih.gov/genome) ( ) . this resource can be searched by taxonomy names, and retrieved genome records include links to all refseqs for that species. each individual genome record also includes links to neighbor sequences for that species under 'related information', and these can be viewed by selecting the 'other genomes for species' option. these links display all genome neighbor records in the nucleotide database where they can be viewed and/or downloaded. genome neighbor records can also be retrieved from multiple genome records using the 'find related data' options, allowing the user to search for an entire viral family or similar and then retrieve all genome neighbor records defined by the original search criteria. simply select 'nucleotide' in 'database' pull down menu and 'other genomes for species' from the 'option' pull down menu to return all genome neighbors for all the species listed in the search results. as the sequencing revolution continues to gather steam, and the rate of viral genome sequencing increases, reference databases will be pressed to serve growing community needs. meeting these will require further collaboration with individual viral communities and across public databases. data models will also need to shift to better represent the extant sequence universe and provide better standardized sequence annotation. once annotated, large-scale genome sequence data will need to be presented in ways that facilitate human data sorting and discovery operations. this will require semiautomated metadata capture and standardization, as well as innovative interfaces and tools that leverage metadata in discovery operations. many of these approaches and processes are currently being tested within the ncbi virus variation resource ( ) where users can readily find sequences based on specific, standardized sequence descriptors, greatly improving the accessibility and utility of viral sequence data. while currently limited to a handful of human pathogens, our intent is to expand the virus variation data model to include more viruses from more viral communities. this should open up a number of possibilities and will support the aggregation and retrieval of sequences based on community-defined criteria like genotypes or complete genome sets as is currently possible for influenza virus sequences ( , ) . the growing cloud of viral genome sequences also poses significant barriers to the maintenance of reference genome records. the pace of experimental discovery and the number and breadth of viral genomes make it increasingly difficult to provide well annotated, up-to-date reference sequences. to counter, we must leverage community knowledge and activities against the goal of better refseq viral resources and must collaborate with viral communities to maintain well annotated reference sequences, develop community-accepted gene and protein naming standards and define community-established subspecies classification schemes. though collaborations have been initiated within d nucleic acids research, , vol. , database issue some communities ( , ( ) ( ) ( ) ) , they need to be scaled to include more groups. as a public resource, we serve a range of communities--from the public health to the basic research--and rely on them to both better inform our mission and help support it. only by engaging our stakeholders and working together on shared goals can we provide the rigorous resources necessary to support viral sequence data activities. emergence of zaire ebola virus disease in guinea--preliminary report genomic surveillance elucidates ebola virus origin and transmission during the outbreak middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study the international nucleotide sequence database collaboration the european bioinformatics institute's data resources ddbj progress report: a new submission system for leading to a correct annotation viralzone: recent updates to the virus knowledge resource ncbi reference sequences (refseq): a curated non-redundant sequence database of genomes, transcripts and proteins flan: a web server for influenza virus genome annotation vigor extended to annotate genomes for additional different viruses vigor, an annotation program for small viral genomes evaluation of alignment algorithms for discovery and identification of pathogens using rna-seq identification of a novel polyomavirus from patients with acute respiratory tract infections klassevirus , a previously undescribed member of the family picornaviridae, is globally widespread a highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting molecular epidemiology of contemporary g p[ ] human rotaviruses cocirculating in a single u.s. community: footprints of a globally transitioning genotype going viral: next-generation sequencing applied to phage populations in the human gut pathseq: software to identify or discover microbes by deep sequencing of human tissue virusfinder: software for efficient and accurate detection of viruses and their integration sites in host genomes through next generation sequencing data a cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples national center for biotechnology information viral genomes project virus variation resource--recent updates and future directions ncbi reference sequences (refseq): current status, new features and genome annotation policy improving gene annotation of complete viral genomes identification of proteins associated with murine cytomegalovirus virions microbial virus genome annotation-mustering the troops to fight the sequence onslaught imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches molecular identification of hepatitis b virus genotypes/subgenotypes: revised classification hurdles and updated resolutions consensus proposals for a unified system of nomenclature of hepatitis c virus genotypes expanded classification of hepatitis c virus into genotypes and subtypes: updated criteria and genotype assignment web resource is there any value to hepatitis b virus genotype analysis? the ncbi taxonomy database virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses rapid cdna synthesis and sequencing techniques for the genetic study of bluetongue and other dsrna viruses a new approach to determining whole viral genomic sequences including termini using a single deep sequencing run herpesvirus systematics evolution of viral proteins originated de novo by overprinting overlapping genes produce proteins with unusual sequence properties and offer insight into de novo protein creation uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family filoviridae virus nomenclature below the species level: a standardized nomenclature for laboratory animal-adapted strains and variants of viruses assigned to the family filoviridae the universal protein resource (uniprot) in vipr: an open bioinformatics database and analysis resource for virology research towards viral genome annotation standards virus variation resources at the national center for biotechnology information: dengue virus the influenza virus resource at the national center for biotechnology information a web-based genotyping resource for viral sequences human immunodeficiency virus type , human protein interaction database at ncbi pairwise sequence comparison (pasc) and its application in the classification of filoviruses improvements to pairwise sequence comparison (pasc): a genome-based web tool for virus classification blast: a more efficient report with usability improvements ncbi blast: a better web interface database resources of the national center for biotechnology information the national center for biotechnology information's protein clusters database we would like to thank vyacheslav chetvernin, boris fedorov, sergey resenchuck, igor tolstoy, tatiana tatusova and jim ostell for their development and support. key: cord- -xv tcugl authors: reina, giacomo; peng, shiyuan; jacquemin, lucas; andrade, andrés felipe; bianco, alberto title: hard nanomaterials in time of viral pandemics date: - - journal: acs nano doi: . /acsnano. c sha: doc_id: cord_uid: xv tcugl [image: see text] the sars-cov- pandemic has spread worldwide during , setting up an uncertain start of this decade. the measures to contain infection taken by many governments have been extremely severe by imposing home lockdown and industrial production shutdown, making this the biggest crisis since the second world war. additionally, the continuous colonization of wild natural lands may touch unknown virus reservoirs, causing the spread of epidemics. apart from sars-cov- , the recent history has seen the spread of several viral pandemics such as h n and h n flu, hiv, and sars, while mers and ebola viruses are considered still in a prepandemic phase. hard nanomaterials (hnms) have been recently used as antimicrobial agents, potentially being next-generation drugs to fight viral infections. hnms can block infection at early (disinfection, entrance inhibition) and middle (inside the host cells) stages and are also able to mitigate the immune response. this review is focused on the application of hnms as antiviral agents. in particular, mechanisms of actions, biological outputs, and limitations for each hnm will be systematically presented and analyzed from a material chemistry point-of-view. the antiviral activity will be discussed in the context of the different pandemic viruses. we acknowledge that hnm antiviral research is still at its early stage, however, we believe that this field will rapidly blossom in the next period. t he current emergence caused by sars-cov- is dramatically changing the everyday life of all of us. due to the high globalization, new viruses can spread all over the world much faster than ever, infecting the communities worldwide. the current technologies and measurements are able to sensibly slow down the infection spread. however, their cost is tremendously high, impacting the healthcare systems and causing the shutdown of industries and the lockdown of the population. the impact of sars-cov- on the worldwide economy is estimated to be a − % decline, making this the biggest crisis since the world wars. a possible vaccine is hopefully expected to come in − years, originating a buffer period pretty much uncertain for many people. vaccination is the only way known to accelerate the flock immunity without causing further death by this pandemic. contemporary history has seen the spread of other viral pandemics such as h n flu ( − ) , h n flu ( ), hiv (peak reached between and ), sars ( ), while mers ( to now) and ebola ( to now) viruses are in a prepandemic phase. the continuous colonization of wild nature lands may touch unknown virus reservoirs causing the spread of contagious epidemics. due to these facts, there is a clear urgency in the development of viral treatments to avoid the risk of new pandemics. in particular, the possibility to have smart antiviral tools able to efficiently disinfect surfaces, block the viral spreading, enhance the survival of infected people, and boost immunization are highly desirable. in particular, more investments in the next years are expected in the antiviral research. hard nanomaterials (hnms) have been extensively studied for many types of applications including drug delivery, bioimaging, and biosensing. − the use of nanomaterials in biomedical research is highly developed, reaching in some cases clinical approval. despite that, nanomaterials have been mainly developed for cancer therapy, while scarce attention has been spent on their application in viral infections. the continuous virology research has more and more increased into viral replication machinery, allowing the preparation and rationalization of more sophisticated vaccine formulations and viral inhibitors. the use of hnms may be one of the keys to provide more effective biomedical agents with a wide spectrum of activity in viral pandemics. an increasing number of reports describe how hnms can be successfully applied to block viral spread. hnms can be used at different stages of viral infection: blocking viral entry, hampering interaction with infected host cells, and modulating immune responses. due to their core composition (e.g., metal oxides, noble metals), hnms can have an important antiviral activity inactivating some specific proteins of the capsid or dysregulating radical homeostasis in the virus particles. additionally, surface functionalization can sensibly increase hnm antiviral activity, enabling the mimicking of host cells or enhancing the targeting efficiency. in this review, we will critically analyze different strategies for the application of hnms as antiviral agents. the rational and the synthetic strategies will be highlighted and correlated to different relevant examples. particular attention will be paid on the mechanisms of antiviral action and on hnm applicability and efficacy. for the sake of clarity, this review is divided into three parts, namely: blocking viral entry, antiviral activity in host cells, and stimulation of immune system. we have focused on the different stages of infection. in the section dedicated to blocking viral entry, the application of hnms for surface disinfection and inactivation of the virus prior to interaction with host cells will be described. subsequently, the interaction of hnms after internalization into host cells will be addressed, stressing their antiviral delivery features and their activity in viral replication blockage, leading to host cell survival. then, activation of immune response induced by hnms, triggering the innate and the adaptive (e.g., nanovaccines) immunity, will be presented. the different adopted strategies will be correlated to the nanomaterial core (e.g., composition, size, shape) and surface (e.g., chemistry, surface charge) properties. finally, limits (e.g., unknown long-term toxicity), advantages (e.g., high and wide spectrum virucidal activity), and perspectives will be discussed with particular attention to the applications in viral pandemics (e.g., hiv, sars, and influenza viruses). this review is addressed to material and biomaterials scientists who are interested in antiviral research. we acknowledge that application of hnms as antiviral agents is still in the early stages; however, we believe that the research on this topic is going to grow soon. thus, with this contribution we genuinely hope to inspire researchers in the preparation of smart and efficient hnm antiviral agents. the last decades have been characterized by increasing investigation on viruses. in particular, their surface charge, protein composition, and host cell entry mechanism have been elucidated, allowing to formulate the first-generation wide spectrum antivirals. blocking the viral entry is one of the most common known antimicrobial procedure to stop infections at the early stage. in this context, antiviral materials have been used for surface disinfection or for epidemic limitation in humans and animals. due to their high surface to volume ratio, composition, and tunable surface chemistry, hnms are now more and more studied as powerful agents in blocking viral entry. as for other biological interactions, the attachment and entry of viruses into host cells are mediated by multivalent interactions between the surface of the virus and cell surface receptors. nanomaterials can display multivalency that makes them able to compete with the host cells on virus attachment, limiting their infectivity. the mechanism of antiviral actions relies on the inactivation of the capsid proteins. as a matter of fact, hnms have been used for: ( ) blocking target proteins for viral entry, ( ) capsid protein oxidation, ( ) mimicking cell surface, and ( ) mechanical rupture of viruses ( figure ). these strategies target proteins and mechanisms of entry common in most of the viruses, thus allowing the preparation of wide spectrum antiviral agents. in this section, the application of different hnms as powerful inhibitors of viral entry will be discussed. noble nanoparticles. noble nanoparticles (nps), made of gold and silver, are attractive as antiviral agents for their surface functionalization versatility and their capacity to cleave disulfide bonds. their use in disinfection has been extensively studied for different types of viruses. the morphology and the size of the nps play a crucial role in their ability to efficiently interact with the capsids and in their toxicity for the organism. these nanomaterials are characterized by a very large specific surface area (inversely proportional to the particle diameter). as the particle size becomes smaller and smaller, the percentage of surface atoms increases, creating many unsaturated bonds due to lack of neighboring atoms. as a consequence, agnps and aunps have unstable atoms with high surface energy. this kind of structure provides a lot of contact adsorption sites and reaction points for further modifications. these chemical features allow to easily combine surface np atoms with other atoms through chemical bonds. besides the composition of the metal core, several studies have pointed out the importance of the control of the surface chemistry. the surface groups can: ( ) stabilize nps in the biological media, ( ) insert targeting agents, and ( ) enhance the circulation time inside the body. antiviral efficiency can be also enhanced by the multivalency effect, where highly branched ligands are used to locally augment the local concentration of the targeting molecules. in this section the main strategies and results for silver (agnps) and gold (aunps) nanoparticles in blocking viral entry will be critically discussed. silver nanoparticles. many studies have shown that naked agnps have a good effect on the control and prevention of a variety of viral diseases (table ) . however, the antiviral mechanism of nanosilver is still unclear. the antiviral action is associated with the following mechanisms: nanosilver can prevent the virus from entering the host cells and inhibit the virus from binding to the cell receptor, thereby stopping the virus from infecting the targeted cells. agnps may be able to bind the viral surface protein and inhibit the interaction between the virus and the cell membrane receptors (figure , left). however, it has been also reported that agnps can inactivate the virus through denaturation of surface proteins containing cysteine and methionine residues present on the viral capsid, in a similar way reported for bacteria. for example, agnps smaller than nm were shown to interact with the sulfur-bearing residues of gp glycoprotein knobs distributed on the lipid membrane of hiv- virus, preventing the virus from binding to cd receptor site on the host cells, thus inhibiting the viral infection. by means of a viral adsorption assay, it was shown that the agnp mechanism of anti-hiv action is based on the inhibition of the initial stages of the hiv- cycle. to demonstrate that the antiviral effect of agnps is due to the particle structure rather than to silver ions present in solution, the antiviral activity of silver sulfadiazine (agsd) and silver nitrate (known antibacterial silver salts) was evaluated. both salts showed a much lower therapeutic index than agnps in vitro, indicating that silver ions themselves are less efficient. these results point out that the antiviral efficacy is not only related to the dose of ag + ions present in solution but is also regulated by different other parameters (e.g., size, charge, and surface functionalization) associated with the nanosize dimension. for instance, in the case of herpesviridae and paramyxoviridae viruses (both enveloped viruses with embedded viral-encoded glycoproteins), agnps can effectively reduce their infectivity, by blocking the interaction between the viral particles and the host cells with an antiviral activity strictly dependent on the size and ζ potential of the agnps. as a general observation, it was reported that smaller nanoparticles have better antiviral effect. this effect was associated with the increase of the surface area, where smaller-sized agnps could bind more efficiently to the viral particles exerting a higher antiviral activity. another study reported the impairment of peste des petits ruminants virus (pprv) replication after incubating infectious viral particles with agnps, which did not exhibit any virucidal effect even up to μg/ml. this result suggested that the anti-pprv activity of the agnps is due to the inhibitory effect alternatively, nanosilver can be combined with viral nucleic acids to change the capsid structure, affect the replication of viral genetic material, and make the virus inactive. for example, tem analyses have shown that nps can cause a change of the structure of the ad virus from a hexahedral shape to an irregular shape, destroying its fibers and capsid proteins, leading to inhibition of the virus from binding to the host cells and destroying the dna structure, preventing adenoviral infection. nanosilver can also bind directly to the doublestranded dna of hepatitis b virus to inhibit its replication. in other studies, it has been demonstrated that silver ions released from nanosilver can directly damage the viruses. based in this property, an interesting application has been proposed. agnps were used as a coating on polyurethane condoms, (hsv) . the hypothesized mechanism is that silver ions are transferred directly from oxidized nps to biological targets, such as viral membrane proteins gp and gp . in addition, a small amount of silver ion is also released from the coated contraceptives to improve the antiviral level. although the studies on naked agnps to reduce viral infectivity have shown their potential as broad-spectrum antiviral agents, the understanding of the specific antiviral action mechanism still needs to be elucidated in depth. many studies have shown that the antiviral performance of naked agnps is related to their size, and smaller nanoparticles have better antiviral activities. in addition to particle size, the antiviral action of agnp morphology has also attracted interest to fight against coronavirus. agnps and two types of silver nanowires were able to significantly cause an inhibitory effect on coronavirus transmissible gastroenteritis (tgev)-induced host cell infection and tgev replication. the mechanism is likely based on a direct interaction of agnps with tgev surface proteins (e.g., tgev glycoproteins) to inhibit the beginning of viral infection. it is possible that agnps and ag nanowires alter the structure of some surface proteins of tgev and then inhibit their recognition and adhesion to the cellular receptor papn. although the potential of agnps as antiviral agents has been commonly recognized, unfortunately, their wide biological applications are limited by the risks of self-aggregation and environmental pollution. silver ions can be released from the surface of agnps and potentially pollute the environment, and their agglomeration into bulkier particles or fibers may change their biological characteristics, diminishing the antiviral effect. in several cases, it has been reported that naked agnps may affect human health. therefore, research and development of agnps whose surface is modified or stabilized by protecting molecular layers is an urgent need to overcome these problems ( table ) . poly(n-vinyl- -pyrrolidone) (pvp) is the most commonly used stabilizer of agnps. the pvp-coated agnps are able to inhibit the activities of hiv- , herpes simplex virus (hsv- ), and respiratory syncytial virus (rsv). , , but compared to foamy carbon, small-sized pvp and bsacoated agnps showed poor antiviral activity to the hiv- virus. for rsv, pvp-coated agnps have a specific binding capacity to the viral surface, evidencing a regular spatial arrangement and a clear interaction with g-protein. in addition, to improve the stability of agnps, their surface modification with antiviral drugs was proved to reduce the drug resistance caused by the drugs administered alone. tannic acid-modified agnps showed good antiviral effects on hsv- infection in vitro and in vivo. the viral infection was inhibited only when these nps directly interacted with hsv- virions. indeed, the pretreatment of host cells with such agnps did inhibit the entry of hsv- . due to the high affinity of tannins to proteins and sugars, tannic acid can bind glycoproteins on the surface of viruses to make them inert, impairing glycoprotein function and preventing viruses from attaching and entering host cells. the surface modification can also exert a synergistic antiviral effect. agnps decorated with polyphosphonium-oligochitosan (pqpoc) exhibited moderate to excellent antiviral activity against hav, nov, and coxb . in addition, agnps could interact with the virion glycoproteins and prevent viral attachment and penetration. pqpoc can also serve as an effective virus inhibitor by blocking the interaction of the targeted virus with the host through the electrostatic interaction between the cationic polymers and the negatively charged binding sites of the virus. surface-modified agnps can also prevent viral infection by competitive adsorption on host cells. the process of infection of cells by herpes simplex virus type (hsv- ) involves the interaction between viral envelope glycoproteins and heparan sulfate (hs) on cell surface. therefore, researchers designed agnps capped with mercaptoethanesulfonate (ag-mes) to compete with the cellular hs through the sulfonate end groups, thereby blocking the virus from entering the cells. a few years ago, it was shown that curcumin could prevent the replication and the budding of rsv, but the disadvantage of poor solubility and low bioavailability limited its clinical application. curcumin was used as a reducing and capping agent to prepare stable curcumin agnps (cagnps) under physiological conditions. cagnps could reduce cytopathic effects induced by rsv and showed efficient antiviral activity against infection by directly inactivating the virus prior to entry into the host cells. its antiviral effect was higher than curcumin alone or unmodified agnps ( figure ). alternatively, zhu et al. prepared agnps surface-modified with oseltamivir, amantadine, and zanamivir (ag@otv, ag@am, and ag@znv ), by chemical methods. the results showed that these nanoparticles can directly interact with the virions, resulting in viral function damages. overall different studies have reported the capacity of agnps to block viral entry. however, there is not a concerted antiviral mechanism, but their activity differs from case to case, based on viral particle adsorption, capsid structure alteration, or surface protein denaturation. for agnps, the antiviral activity can be associated with different parameters including size, shape, surface charge, and functionalization but also to the topical release of silver ions able to disturb the viral cycle replication. as described before, bare agnps can be used as disinfectant agents, however their use in biological media is acs nano www.acsnano.org review limited by their low colloidal stability and potential cytotoxicity. surface functionalization can alleviate cytotoxicity, but it can also mask the nanoparticle surface, reducing their affinity for viral particles, thus reducing agnp antiviral activity. for these reasons, agnps at the moment could find application mainly for surface disinfection and for topical administration. further studies are needed to prepare safer agnp formulations for systemic administration. in particular, the clarification of the antiviral mechanisms and the use of surface functional groups able to stabilize agnps in biological fluids without affecting their prominent antiviral activity are probably the most important challenges to tackle. gold nanoparticles. compared to agnps, aunps exhibit reduced toxicity on healthy cells, making them more attractive for in vivo and clinical applications. indeed, aunps have been successfully tested as inhibitors of viral entry into the host cells. aunps interact with hemagglutinin (ha), where au is able to oxidize the disulfide bond of this glycoprotein causing its inactivation, thus impeding the membrane fusion of the virus with host cells. targeting ha has emerged as an alternative strategy to the actual therapies (e.g., matrix protein and neuramidase), especially to pandemic viruses that show an accelerated mutation speed of their surface proteins, hence a resistance to conventional treatments increasing their infectivity and mortality. this strategy has been applied to influenza (e.g., h n , hcv) and herpes viruses. − the activity of aunps is proportional to the surface area exposed. as a consequence, the size and the morphology of these metal nps play a substantial role in their antiviral activity. recently, kim et al. have reported that porous aunps are able to inhibit influenza a infection more efficiently than nonporous aunps. this effect has been associated with the higher surface area of the porous material that favors their interaction with capsids and thus increases their antiviral activity ( figure ). besides the per se antiviral activity, aunp surface modifications have been developed in order to enhance their overall therapeutic benefits. the engineering of tailored aunps with selected ligands has allowed the preparation of efficient antiviral nanoagents. the target ligands can be introduced directly during the particle synthesis via ligand exchange reactions or ligand modifications. for instance, direct reduction of gold ions in the presence of gallic acid produced homogeneous aunps able to sensibly reduce herpes simplex virus infection in vitro. compared to free ligand nps, functionalized aunps benefit from the multivalency effect and higher circulation times, decreasing the needed therapeutic concentrations. functionalized aunps can present organic groups that mimic host cell surfaces or other specific molecular patterns that selectively target the virus. normally, negative charges are used to mimic cell surfaces and favor the interaction between the particles and the capsid. in particular, sulfonates and organic sulfates have been used for their capacity to attract the virus via capsid protein interaction and block the ha activity. aunps functionalized with sulfonates showed an increasing inhibition of influenza a compared to the nanoparticles capped with succinic acid. this study also demonstrated that there is not a correlation between the negative charge and the antiviral activity, but instead the inhibition depends mainly on the organic groups used. thiolcapped aunps also displayed powerful inactivation of bovine viral diarrhea virus in vitro. multivalency has been exploited in more complex systems using dendrons as capping agents. this strategy allows to generate higher concentrations of the target ligand in close proximity to the aunps and to increase the binding efficiency of the nanoparticles to the capsid. the driving force of the antiviral efficiency relies on the concentration of the targeting agent onto the particles. sulfonated dendrons were grafted to aunps via a sulfide bond and tested for hiv inhibition. the results showed that acs nano www.acsnano.org review the decorated aunps exerted a higher affinity to the virus. additionally, comparing aunps functionalized with different generation dendrons, those with a third generation displayed the highest inhibition performance with an ic below . μmol/ml, thus making them attractive for in vivo translation. it is worth noting that the inhibition efficiency is strictly dependent on the available sulfonate groups present on the surface of the nps, making crucial a thorough characterization of the material. the size of the aunps clearly plays an important role in the concentration of targeting ligands exposed per particle. indeed, too big nps have a limited surface area, while too small would not allow an efficient grafting of the dendrons due to steric hindrance. for instance, it has been shown that dendron-functionalized aunps showed a size-dependent antiviral activity for influenza virus, where nm particles exhibited a higher efficiency than nm aunps. this has been associated with the low functionalization grade of the small nanoparticles and to the inappropriate spatial distribution of the interacting ligand/receptor pairs. the development of viral proteomics has profoundly transformed the antiviral and disinfection strategies. in particular, small molecules and peptides able to target and block the viral biochemical machinery have been developed. however, despite these efforts into the drug design, many of these molecules suffer from poor biological effect, low concentration in the diseased areas, and undesired side effects. in this context, aunps have been coupled to biologically inactive small molecules to create biologically active multivalent aunp therapeutics. a bright example has been reported by bowman et al., where the authors functionalized aunps with sdc- , a small membrane fusion inhibitor of hiv. the results demonstrated that, while pure sdc- has low activity, functionalized aunps are able to inhibit hiv replication at μm concentrations. similar results have been reported using targeting peptides. in particular, it was evidenced that the functionalized aunps can sensibly reduce the ic up to orders of magnitude compared to pure peptides. preliminary results in vivo confirmed the biosafety of the aunps. nanoparticles generating reactive oxygen species. one of the main advantages of using nps compared to oxidized metals relies on the slow release of ions and clusters from these particles, leading to an enhancement of the antiviral activity. additionally, the use of metal nps containing cu or fe in ionic form catalyzes the generation of radicals via fenton and fenton-like reactions oxidizing the capsid proteins and consequently blocking the viral infection at early stage. for instance, copper ions (derived from sulfates or iodide salts) have been widely used as antiviral agents because of their activity on several kinds of enveloped and non-enveloped viruses including influenza virus, − herpes simplex virus − and hepatitis a virus. their mechanism of action relies on the formation of cu + ions (from soluble salts or nanoparticles) that generate hydroxyl radicals. the use of metallic copper nanostructures in the form of particles or sheets has shown only a moderate efficiency due to the low concentration and low release of cu + . for these reasons, cu + salts, where the copper ions are readily present in their active monocationic form, have been favored. in particular, cui nanoparticles (stable at room temperature) have been extensively studied for deactivation of feline calicivirus and h n pandemic influenza virus. however, the use of copper salts at high concentrations can irreversibly alter reactive oxygen species (ros) homeostasis of healthy cells, provoking a general toxicity for the organism, limiting their applications to disinfection. nanostructured cuprous and cupric oxides have been also extensively employed as antiviral agents for in vitro applications. for instance, cuprous oxide nanoparticles (cuonps) were successfully employed against hepatitis c. in particular, it was found that these nps exerted a favorable antiviral activity with no cytotoxic effects. cuonps target the binding and entry step of viral infection to hepatic cells ( figure ). similar results were reported on the use of cuonps against hsv- , however without any profound investigation on the antiviral mechanism. alternatively, zinc salts have been successfully used as antimicrobial agents from research up to clinical trials for viral warts. , more recently, zno nanoparticles (znonps) were developed for the treatment of hsv- . znonps were prepared with a tetrapod morphology. the results showed that they can mimic cell surface interacting with the hs present on the viral capsid. additionally, these particles have been used for photocatalysis showing to efficiently destroy the viral proteins upon uv irradiation. besides all these interesting examples, in vivo applications are still needed to validate this therapeutic modality. due to the generation of high levels of ros, the toxicity of copper nanoparticles has been widely debated. the antiviral activity of copper nanoparticles is generally associated with the release of cu + ions in solution, thus the leakage of cytotoxic cationic species can be modulated by surface functionalization before in vitro and in vivo applications. on the other side, the use of nanomaterials generating ros can find applications in textile and surface coating. the general broad virucidal efficiency of copper oxide nanoparticles shown for h n pandemic influenza should be tested on sars-cov- and might be used for improving mask protection efficiency. carbon nanomaterials. due to their diversity, versatility, and tunable surface chemistry, carbon nanomaterials have been attractive for several types of applications. in particular, the past decade has seen a tremendous raise in the preparation of performant carbon-based nanomaterials in the antiviral field. fullerene and its derivatives are the most studied carbon nanomaterials for their virucidal activity. due to the lack of solubility of pristine fullerene, functionalization strategies have been developed to prepare water-soluble drugs. investigations in the biomedical field evidenced the membranotropic capacity of fullerene derivatives. by modulating shape and functions, fullerene derivatives have been shown to possess antiviral properties through inhibition of viral entry and blockage of viral replication. from these results, the attention has been directed also to other carbon nanomaterials. in particular, functional carbon dots (cds) and graphene oxide (go) have been investigated for their ability to block viral entry into host cells. glycofullerenes. the emerging of mortal viruses, like ebola or zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes. most of these pathogens are rna enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide small spacer between core c and surrounding fullerenes. b large spacer between core c and surrounding fullerenes. www.acsnano.org review spectrum antivirals. the external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells. this strong interaction allows the attachment of the virions to the cells, followed by internalization and infection. blocking lectin receptors is a general strategy used to stop viral infection at an early stage. fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds. fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells. mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells. for example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific icam-grabbing non-integrin (dc-sign). dc-sign receptors mediate the interactions between dcs and t cells. , to exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit ebola, dengue, and other pathogens. for this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from to ) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules. the synthetic route is composed of three steps based on "click chemistry": ( ) assembly of glycodendrons by cu(i)-catalyzed azide−alkyne cycloaddition (cuaac), ( ) synthesis of alkynesubstituted bingel-hirsch hexakis-adducts, and ( ) the coupling between the last two products again by cuaac. to increase the number of mannose moieties up to , the glycodendron core was changed from malonate to trialkynyl pentaerythritol. in order to compare the different derivatives, in vitro studies were performed. jurkat cells (lymphocyte t cd immortalized cells) expressing dc-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of ebola ( figure , route a). the study revealed an ic in the μm range for the mannose fullerene, a lower efficiency with the mannose fullerene with a short spacer (peg, with ethylene oxide units), while a nanomolar ic was achieved with mannose fullerenes with a longer spacer (peg, with ethylene oxide units) ( table ). this first proofof-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers. based on these studies, another class of multivalent fullerene dendrimers was then designed. a fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups. the first study was performed with tridecafullerenes containing mannoses. the molecular structure is composed of hexakis c surrounding a c core ( figure , route b). compared to the previous study, an ic orders of magnitude lower was measured on the inhibition of ebola virus (table ) . , in order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with carbohydrates. in this case, the molecule was synthesized with a c tridecafullerene bearing α( , )mannobioside. the use of this disaccharide was already investigated, showing an increase of affinity with dc-sign receptors by a factor of − . the synthetic strategy exploited also the use of strainpromoted copper-free cycloaddition of azides to alkynes (spaac) for the coupling of the core fullerene to the surrounding fullerenes. spaac allows an easier purification avoiding the removal of cytotoxic copper ions. the inhibition performance of this molecule was studied in vitro with viral pseudoparticles of dengue and zika. the comparison was made between and disaccharides tridecafullerenes and disaccharide monofullerene. the results highlighted a picomolar ic inhibition on both zika and dengue models for the disaccharide glycofullerene ( table ) . the ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs. moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules. following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested. these micelles present a uniform and spherical shape ( figure , route c). the aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation. this self-assembled c functionalized with or mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of ebola virus in the nanomolar range (ic of nm for six mannoses and nm for mannoses, respectively, table ). further in vitro studies evidenced again a good biocompatibility of these glycofullerenes. while there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses. functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells. there has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome. first, glycofullerenes do not have an intrinsic virucidal activity. they can reduce the infectivity, but they are not able to completely inactivate the virus. second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles. thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells. in addition, glycofullerenes can be internalized into host cells losing their viral "shield" activity. on the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry. for instance, in the case of the current sars-cov- pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ace receptors and so inhibiting viral entry. other carbon nanomaterials. alongside fullerenes, other carbon nanomaterials (nms) have been scrutinized for their ability to block viral entry. cds and go are the most known and studied carbon nms with marked antiviral properties. cds are zero-dimensional carbon nanoparticles. they are generally produced via hydrothermal decomposition of carbon containing "low-cost" precursors. the use of cds in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization. pristine cds have shown moderate viral blocking activity for hiv infection in vitro. this has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent acs nano www.acsnano.org review interaction with viral membranes. moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy. therapeutic targeting molecules can be grafted onto a cd surface to enhance their antiviral activity. in this context, the design of multifunctional cd platforms can be obtained through two different strategies. the first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation. target molecules are decomposed with the other precursors, generating the desired functional cds. this protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate. indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect. for these reasons, the reaction conditions must be carefully controlled. the second method is a twostep reaction and implies the postfunctionalization via amide formation on the surface of the cds rich in carboxylic groups. this strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively. different functionalized cds were prepared to hamper host cell viral entry. for instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the cd structure during their preparation (figure ) . the as-prepared cds showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., japanese encephalitis virus, dengue virus, and zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus). these positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the cds to the viral particles amino-functionalized cds were also tested for the treatment of human norovirus. in this study, cds were functionalized with , ′-(ethylenedioxy)bis(ethylamine) (eda) and ethoxypropylamine (epa) via amide bond formation. these nms exerted a good viral blockage. in particular, epafunctionalized cds were able to inhibit % of viral infection at concentration of μg/ml, while in the case of cds prepared with the other amines, % of inhibition was reported. these effects have been associated with the higher positive charge of cd-eda compared to cd-epa. another surface group used for viral targeting is boronic acid (ba), which can bind glycosylated surfaces forming boronic esters. this strategy was successfully adopted to treat hiv where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp receptors on the viral envelope inhibiting the infection. another recent study proposed the use of cd functionalized with phenylboronic acid for prevention of hiv infection. the functional materials showed good inhibition properties compared to nonfunctionalized cds by preventing the binding to the target cell in vitro. overall, the use of cds for stopping host cell viral entrance has shown good results in vitro. however, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks. in addition, most of the in vitro studies foresee first the contact of the cds with the viral particles and then their incubation with host cells. deeper investigations should be performed adding the nms at other time points (for instance in infected cells) to understand if the antiviral activity is maintained. in addition, cds have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment. the same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ros generation under irradiation. graphene materials, and in particular go and reduced go (rgo), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering. go platforms have shown also interesting antimicrobial activity. regarding viral infection, go was used to block the virus entrance in host cells. go and rgo can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins. go showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles. additionally, go can be used as photothermal agent (generation of heat by nir irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively. the use of phototherapies may significantly augment the antiviral properties of the materials. however, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo. photodynamic therapy has been successfully exploited using bacteriophage ms as a model virus. in this study, go was functionalized with an aptamer recognized by the viral surface. the results showed that this functionalization is able to enhance the binding efficiency of the ms capsids onto the go surface compared to nonfunctionalized go. subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized go showed much less activity due to the lack of adsorption. despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose ( w for − min) and the lack of material recovery and reuse make its application for surface disinfection difficult. go can be also used as a platform to link antiviral agents. encouraging results were reported using go with hypericin for the treatment of a recently appeared duck reovirus. more recently, deokar et al. reported an original rgo-based multifunctional platform for hsv- treatment. in this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (fenps). the rgo functionalized with the sulfate is able to mimic the host cell surface and to bind hsv- . subsequently, the viral particles captured onto the rgo-fenp surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy. this approach is highly efficient for disinfection with low energy ( . w/cm for min) and cost effectiveness. hs is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, dengue virus). the use of organic sulfate-functionalized graphene sheets mimicking hs, like go and rgo, has been already explored. however, it is worth noting that these nms are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy. high loadings of sulfate groups were introduced onto rgo using polyglycerol sulfate. , this approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and african swine fever virus in vitro. , graphene has also been used as antiviral material. polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus. this strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy. overall, graphene materials have shown a good capacity to block host cell viral entry. disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments. regarding the go and rgo activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo. so far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged. we take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions). mechanical disruption of the capsid. the most direct way to suppress viruses and stop the spreading of viral infection is to inactivate them before the attachment to the host cells, by binding to the acceptor proteins. one of the most conserved targets of viral attachment ligands is the heparan sulfate proteoglycan (hspg), previously mentioned. hspgs are expressed on the surface of almost all eukaryotic cell types, and many viruses like hiv- , hsv, human papilloma virus (hpv) exploit hspgs as the target of their viral attachment ligands. bearing in mind this behavior, different studies have used hspg-mimicking materials to target this type of virus−cell interaction and to achieve broad spectrum efficacy. for example, in one key study, aunps were functionalized with mercaptoethanesulfonate (mes) based on its mimicry of hs (au-mes nps). au-mes nps were shown to interfere with viral attachment, viral entry, and cell-to-cell spreading. the importance of the polyvalent interactions with the virus makes these nps a good candidate for antiviral therapy. however, au-mes nps presented virustatic activity, meaning that upon dilution of the nps, the virus recovers its infectivity due to the reversibility of the cell-virion interaction. this problem was solved by stellacci and colleagues who developed nps coated with mercapto- -undecanesulfonate (mus) ligands (au-mus nps). the long aliphatic and flexible linkers provide stronger associations with the viral particles compared to au-mes nps, leading to local distortions and eventually inducing a global deformation and breaking of the capsid that inactivates its contagion irreversibly (figure ). these au-mus nps were tested against different hspgdependent viruses, showing a high viricidal activity over hsv, hpv, and rsv ( figure a) . furthermore, the activity of the au-mus nps was studied in vivo using mice infected with rsv, indicating that the material can prevent pulmonary dissemination of the infection and showing potential use as medically relevant virucidal drugs to fight viral infections. more recently, the concept was further extended to cyclodextrins modified with mercapto- -undecanesulfonate, proposing this system as a broad spectrum virucidal macromolecule. besides au-nps, a similar mechanism of disruption was studied with other materials like graphene or go. in a study, in which the toxicity of graphene was evaluated theoretically, it was also shown that graphene nanosheets can interrupt the hydrophobic protein−protein interaction, which is essential to biological functions. this feature was attributed to the hydrophobic nature of graphene. thus, it seems energetically favorable for graphene to slide between the interface of two proteins in contact, due to hydrophobic interactions. in another study inspired by this behavior, the authors performed molecular dynamics simulations of graphene nanosheets in the proximity of the surface of the ebola viral matrix protein vp showing that the nanosheets can break the hydrophobic interactions in vp , a key protein for the replication and stability of ebola virus (figure ). these findings suggest that graphene nanosheets might have potential antiviral activity against ebola; however, there is a lack of experimental evidence that corroborates this mechanism of disruption. on the other hand, go was tested experimentally against pseudorabies virus and porcine epidemic diarrhea virus (pedv), showing significant decrease in the infectivity. it was found that the negatively charged surface of go is important for the adsorption of the virus, whose surface is positively charged, and that go could directly interact with the viral particles and destroy their structures due to the sharp edges of the material (figures and b) . the mechanical disruption of the capsid is a peculiar antiviral mechanism associated with some nms. in particular, the use of specific sulfonates able to mimic heparan sulfate can be also used to target sars-cov- infection. blocking the viral entry, via liquid/surface disinfection or once in the body, is a powerful strategy to hamper early stage viral contagions. on the other hand, when infections have already spread and reached middle and late stages, alternative pharmacological strategies are required. the study of the viral pathogenesis and machinery inside host cells has allowed the preparation of different drugs. due to the present pandemic, such antiviral drugs are now of top interest in the scientific and medical communities. so far, few antiviral drugs are clinically available, and their mechanisms of action consist on the inhibition of reverse transcriptase (hiv, hepatitis), dna inhibition of polymerase (herpes, hiv), inhibition of protease (hiv), blockage of ion channels (influenza), and inhibition of neuramidase (hiv, influenza, hepatitis). however, these drugs suffer from moderate to severe side effects. additionally, rapid mutations in the viral machinery make them resistant to the treatments, making the control and the stop of the infection challenging. the use of hnms in drug delivery has shown several advantages. first, hnms can increase drug solubility and its circulation time. additionally, they can be functionalized with targeting molecules able to direct the drug to the desired organs and so avoiding side effects and reducing the dosage. more recently, new antiviral mechanisms were discovered. in particular, it was found that different types of hnms are able to change the ros homeostasis in infected host cells, stopping the viral replication and preserving the cell survival. in this section both drug delivery materials and hnms with ros modulation properties will be critically presented ( figure ) . nanomaterials for drug delivery. different hnms have been widely tested for drug delivery applications. the advantages of this strategy are several including enhance drug solubility and the possibility of targeting and multivalent effects. as a potential broad spectral antiviral agent, agnps can prevent the virus from adsorbing to the host cell in the early stage of infection and thus show strong antiviral activity. after the cells are infected by the virus, there are ways to inhibit cell apoptosis (figure , right) . for example, lv et al. studied the anti-tgev activity of agnps in swine testicle cells and explored the possible mechanism of agnp inhibition of tgev infection-induced apoptosis. the results showed that these agnps are able to decrease cell apoptosis through the activation of p /mitochondria-caspase- signaling. although the use of agnps has shown good antiviral action to further improve the therapeutic effects and reduce both side effects and drug resistance, the way of binding drugs or genes and other therapeutic agents to agnps has received particular attention. it has been observed that agnps inhibit the activities of neuraminidase and hemagglutinin, preventing the h n influenza virus from attaching to host cells. at the same time, the potential molecular mechanisms revealed that caspase- -mediated apoptosis was inhibited by ros generation. agnps modified with polyethylenimine (pei) can bind sirna. ag@pei@sirna exhibited superior abilities for enhanced cellular uptake and blocking ev virus infection acs nano www.acsnano.org review and significantly decreased the apoptotic cell population, which prevented the spread of ev virus. in addition to drugs and sirna, neutralizing antibodies in combination with agnps were developed. the results demonstrated that there is an additive effect between the antibody and agnps when combined against cell-associated hiv- infection in vitro. the membranotropic properties of fullerenes were widely exploited. for example, pristine c , after accumulation at the cell membrane, can translocate into the cytoplasm by crossing the membrane through multiple energy-dependent pathways despite its hydrophobic character. fullerenes can be made water dispersible using surfactants, sonication or first dissolving them in appropriate organic solvents (dmso). the use of fullerenes as inhibitors of viruses started in with a study focused on hiv infection. this work revealed the interaction between c derivatives and hiv protease through molecular modeling and experimental verification. hiv protease (hiv-pr) is involved in the mechanism of replication in the maturation of hiv virion, cleaving newly synthesized proteins. the active site of hiv protease has a cavity of Å closed to the diameter of c cage. molecular docking and experiments on hiv-pr catalytic activity revealed a blockage of the active site of the enzyme by van der waals interactions leading to an antiviral effect. the following studies were based on a structure−activity relationship between functionalized fullerenes and hiv-pr with the aim to increase the antiviral activity. the introduction of pyrrolidinium salts onto c was tested against the activity of hiv- strain. in a second study, c bearing two ammonium groups was applied against the activity of hiv- and hiv- strains. the results showed the importance of having two moieties in a precise position on the fullerene cage and the influence of the charge of the different salts sensibly increasing its antiviral activity. further investigations using different functional groups (e.g., amino acid derivatives) were explored. c functionalized with aminobutyric acid and aminocaproic acid was able to inhibit hiv viral replication at subnanomolar concentrations. in another work, anionic and cationic pyrrolidinium salts and amino acid functionalized fullerene derivatives were used for the inhibition of hiv reverse transcriptase (hiv-rt). fullerene compounds were compared to nevirapine, an available drug against hiv-rt, revealing a better inhibition compared to the pure drug. the antiviral property of carboxylated fullerenes was confirmed by another study. results obtained in vitro on cem cell line showed low toxicity and a submicromolar ec against hiv- and hiv- strain viral replication. several mechanisms of hiv protease inhibitors have been hypothesized and simulated. some studies investigated the action of fullerene derivatives on viral replication cycle and the virus maturation ( figure ). , for the latter, it was suggested an impairment due to a strong interaction between fullerene and the immature capsid. other rna viruses share ways of viral replication similar to hiv. c functionalized with an amino acid derivative was investigated against hepatitis c rna polymerase (hcv-rp). this essential enzyme for viral replication was inhibited in a submicromolar range, similarly to benzo- , , thiadiazine, a potent specific inhibitor of hcv-rp. another publication highlighted the effect of a c poly(carboxylic acid) derivative on different strains of influenza virus (e.g., a, h n , h n , and b). the inhibition was comparable to tamiflu, rimantadine, ribavirin, and amantadine, but the mechanism of action remains still unclear. these data emphasize that fullerenes c or c can be potent universal antiviral drugs for rna enveloped viruses. however, clear elucidations of the mechanisms of action and in vivo studies are still a missing point. due to their high surface/weight ratio and capacity to pass through cell membranes, carbon nanotubes (cnts) have been extensively explored for drug and gene delivery applications. cnts have been also successfully used for the delivery of antiviral agents. compared to pristine materials, oxidized cnts (ox-cnts) showed an inhibitory activity for hiv viruses per se. this effect has been associated with the oxygenated groups that increase the hydrophilicity and the colloidal stability of the material. the antiviral efficiency has been correlated to the ox-cnt interaction with host cells. however, it is not clear how and what kind of mechanism blocks the viral machinery in host cells. different anchoring strategies have been explored to link antiviral drugs onto cnt surface. for instance, ox-cnts were covalently linked to amino- -nitro- -( , -dimethylbenzyl)-aniline (chi ) and n-( -aminophenyl- -nitro-)- , -dimethylbenzenesulfonamide (chi ), two active non-nucleoside reverse transcriptase inhibitors for hiv treatment. following the covalent conjugation, only a moderate antiviral effect was observed compared to pure drugs, indicating that most probably the nm cell trafficking played a key role on the virucidal activity. in another study, ox-cnts functionalized with cyclodextrin were used for the delivery of acyclovir (a prodrug inhibitor of the viral dna polymerases) for the treatment of hsv- . preliminary results showed that when acyclovir was delivered via the nanotubes, the viral antireplicative effect was higher than the free drug. more recently, a similar approach was applied to herpes virus using cyclodextrin and pei-functionalized cnts for co-delivery of cidofovir and plasmid dna. however, the antiviral effect of the materials was not explained, and the transfection effect was not satisfactory. functionalized cnts have been also used for delivery of ribavirin in vivo using grass carp as an animal model for the study of grass carp reovirus. ox-cnts were first functionalized via amidation with bsa, and then ribavirin was covalently bound to the protein via esterification (figure ) . in vivo tests demonstrated that, when ribovirin was shuttled by ox-cnts, the antiviral efficiency was significantly increased without any evident toxicity and no significant changes in ros-generating enzymatic activities. the use of cnts in drug delivery is however still controversial. graphene-based materials have been limitedly studied as antiviral drug delivery carriers. only a few examples can be found in the literature. graphene quantum dots (gqds) were used for drug delivery of chi and chi and tested in vitro against hiv similarly to cnts. both the prepared gqd-chi and gqd-chi showed a high antiviral activity once into host cells, with low toxicity. go has been also used for the delivery of dnazyme into hepatic cells allowing to block the hepatitis c infection. this specific dna single strand is able to recognize the viral mrna and to silence its expression. overall, carbon nms proved to be interesting carriers for antiviral drugs. however, several questions need to be answered before their safe application as antiviral materials. different reports have demonstrated that pristine cnts display relevant toxicity for healthy cells, but by oxidation of the tubes, the side effects can be sensibly reduced. in addition, more investigations should be performed on cnt toxicity. these nanocarriers indeed are not "innocent delivery agents", but they play a key role in drug internalization pathway and in host cell machinery that might be averse to the expected therapeutic effects. so far, cnt application in biological systems has been studied for years. however, due to their possible toxicity, their real application in clinics seems to be steeper and difficult to achieve. materials tuning reactive oxygen species. ros homeostasis in infected cells has been studied for both rna and dna viruses. for instance, it was shown that infection of mice with influenza a decreased the concentration of lung glutathione and the antioxidant vitamin c, providing evidence that the viral infection was associated with oxidative stress in vitro as well as in vivo. similarly, in hiv infection, induced oxidative stress in host t cells and high concentrations of antioxidants are able to slow down the cell-to-cell viral spreading. the increase of ros concentration is a common process in most of the viral infections. however, the mechanism of radical generation is different from case to case. several proofs suggest that modulating ros homeostasis in infected cells can slow down or block the infection. hnms have been shown to be powerful allies against viral infections. in particular, metal or metal oxide nms, once internalized, can regulate the radical production into infected host cells. in this scenario, the nms can work following two different mechanisms: ( ) enhancing radical activity or ( ) quenching ros inside cell compartments. in the first case, metal oxide nps are able to convert superoxide ions into more reactive hydroxyl radical species via fenton or fenton-like reactions. the excess of superoxide ions is able to oxidize the viral proteins and the genetic material and therefore efficiently block the infection. this approach has been reported using zno nps. , in these studies, zno nps have been successfully applied for the treatment of h n influenza virus and herpes simplex virus. the preliminary results showed that pegylated zno particles were able to efficiently reduce the viral infection with ic similar to acyclovir. more importantly, toxicity of zno was modulated by functionalization with peg, which allowed a higher colloidal stability and a more controlled release of zn + ions to catalyze ros formation. indeed, ros (e.g., superoxide and hydroxyl radicals) produced by the nps should be highly reactive and should not only damage the exogenous biological molecules but also attack different cell compartments. overproduction of ros may reduce virus spread but also induce cell death. interestingly, this approach is applicable only at the early infection stage (after h incubation of the host cells with a virus), but it loses its activity at later time points. these , this specific mechanism of action restricts zno nps to an application only at the early stage of infections. however, the same approach with other metals able to induce fenton or fenton-like reactions (e.g., fe, cu, and mn) has not been reported yet. the choice of proper capping agents may allow to control the metal ion release and thus tune the ros-mediated antiviral activity. we would like to take an advantage here to suggest the growth of the antiviral research in this direction. another successful approach relies on the reduction of ros concentration in host cells. ros scavenging is able to alleviate the toxicity of the infection enhancing cell viability, giving time to start its endogenous antiviral mechanisms. so, this approach may both block infection and ensure host cell survival. in this context, selenium nps (senps) have been extensively studied for their antiviral activity. the mechanism of action of these nps relies on the quenching of the radicals into host cells due to the infection, stopping the mitochondria depolarization and the consequent apoptotic cascade. additionally, senps can also adsorb onto the viral capsid sensibly reducing their infectivity. senps can be prepared via classical mixing of selenium salt precursors in the presence of a reducing agent. more recently, senps have been instead biosynthesized from actinobacteria showing good stability and capacity to inhibit dengue virus in vitro. moreover, senps were used to carry different antiviral drugs including zanamivir, oseltamivir, amantadine, and ribavirin. their functionalization with the desired drug can be easily achieved adding the molecule during their synthesis through the se ion controlled reduction. these nms have been applied for the treatment of h n virus. notably, senps with ribavirin (administered via intranasal absorption every h for days) showed that infected mice had much less alveolar collapse and perivascular and peribronchiolar edema, compared to the group challenged with the virus (figure ). due to their efficacy and low toxicity, senps can be considered a useful material for the treatment of other viral diseases including sars-cov- . as a matter of fact, oxidative stress as well as chronic inflammation may contribute to the aggravation of the covid- symptoms and to the general spread of the infection. the use of senps could eventually alleviate the toxicity of infected patients, giving time for the immune system to react against the contagion. however, the lack of preclinical studies on senps, together the scarce knowledge of their biosafety and long-term toxicity, still remain the main challenges to tackle for clinical translation. the vast majority of the studies show that human survival to viral attack is based on the stimulation and response of our immune system. when a virus enters and starts infecting tissues, the body reacts and triggers a strong immune response to overcome the pathogen invasion and spread. normally, two different immunological reactions take place. the oxidative stress induced by infection causes the activation of the inflammasome through upregulation of pro-inflammatory cytokines such as il- β, pro-il- , and nlrp . excessive upregulation of this mechanism leads to cell damage and eventually to pyroptosis activated by caspases. it was also shown that generation of radicals is able to depolarize mitochondria, hence affecting host cell respiration and inducing ros-mediated apoptosis at the late stage of infection. besides, activation of pro-inflammatory cytokines can alert the immune system blocking the infection (the innate acs nano www.acsnano.org review immune response). the second immune response is specific (the adaptive immune response). immune cells are trained to attack the virus (cellular response), while specific antibodies are produced by b cells (humoral response). in the last years, hnms were proved to tune the immune responses, demonstrating to be a possible alternative against viral infection. in this section the most relevant strategies for the activation of innate and adaptive immune responses using nms will be described ( figure ) . innate immune response. innate immune response is the first response that takes place in the presence of any type of infection. during this early stage, interferon stimulating genes (isgs) are upregulated in the infected cells. this selfdefense mechanism slows down the viral replication and alerts sentinel immune cells that start producing proinflammatory cytokines and trigger inflammation. however, many viruses are able to escape this complex mechanism, retarding the immune response and spreading the infection. the interaction of hnms with the immune system has been more and more studied. in the case of antiviral hnms, many examples can be found in the literature where the nms not only slow down the infection but also tune the innate immune response. certain hmns can display an intrinsic immune stimulation. we have already mentioned above that in the early stage of infection, agnps mainly prevent the virus from entering the host cell through the interaction with the external capsid, but in vitro cellular experiments lack to understand the complex interaction with primary immune cells. recent studies have shown that agnps can potentially induce the expression of genes involved in innate and adaptive immunity-associated pathways, which are known to play crucial role in immune regulation. for example, toll-like receptor can be upregulated by agnps after h, by recognizing the singlestranded rna of the viruses and regulating the antiviral immune response. another study showed that in rsvinfected mice treated with agnps, the particles reduced the production of pro-inflammatory tnf-α and il- cytokines, but potentiated the anti-rsv activity of neutrophils in an experimental mouse model. however, activation of agnps in the reduction of rsv has been noted only when the nm was intranasally inoculated together with the virus, and no results have been reported on the use of agnps administrated on infected mice. in the case of influenza virus infection of lung epithelial cells, it was found that agnps targeted infected lung epithelial cells and reduced viral replication, by preventing autophagy. however, the blockage of the autophagic flux by agnps does not inhibit viral replication in already infected cells. therefore, agnps are more suitable as viral preventive agents due to their pro-inflammatory response rather than drugs. more recently, agnps were combined with graphene materials and exploited as antiviral material for the treatment of porcine reproductive and respiratory syndrome virus (prrsv). go-agnps were able to clump the virus diminishing its fusion with cell membrane. additionally, once go-agnps were internalized in host cells, they stimulated the isgs that blocked viral budding and its diffusion to other cells in vitro (figure ). similarly, cds used for the treatment of rsv and prrsv were able to activate the innate immune response via an upregulation of isgs in vitro. gold nanorods were applied to boost the innate immune response against rsv in vivo. interestingly, it was shown that these nanorods (when intranasally administered with rsv) were not only able to activate the isgs but also to tune the production of pro-and anti-inflammatory cytokines, resulting in the blocking of the infection with a reduced pulmonary inflammation. as drug carriers, hnms can also affect the internalization pathways, modulate drug efficacy, and the immune cell responses. for instance, it was found that the isoprinosine immunomodulatory antiviral drug displays a much higher antiviral efficacy in vivo when delivered with cnts than as a pure drug (in acs nano www.acsnano.org review zebrafish larvae food administration), probably due to a better cellular uptake and the anti-inflammatory properties of the nanotubes. fullerenes also exhibit immunomodulation properties through the release of cytokines by bovine alveolar macrophages. a study explored the influence of functionalization of c with molecules presenting different surface charges like hydroxyl groups and amino acids. the study concluded that negative charges upregulated tnf-α up-secretion in raw . macrophages, but highly positively or negatively charged surfaces increased cell toxicity. the upregulation of the cytokine tnf-α is a proof that fullerene can promote cellular immune response. despite these preliminary results, the actual mechanisms of interaction between hnms and the immune system are still at early stage of understanding. we must consider that the immune response needs to be proportional to the infection grade. if on one side nanosized immuno-boosters can alert more efficiently the sentinel cells, the use of hnms that trigger an exaggerated immune response can promote excessive inflammation, damaging healthy cells and promoting uncontrolled side effects. in the particular context of sars-cov- , the use of agnps, fullerenes, or other pro-inflammatory hnms at the middle and late infection stage may cause an aggravation of the symptoms due to already diffused inflammation. in particular, most of the studies showed the ability to reduce infection when hnms were first incubated with the pathogenic virus, thus limiting their potential use in the early stage infection. the formulation of hnms able to both alert the immune system (e.g., upregulating cytokine) and control lung inflammation (e.g., ros scavenger) even after the early stage infection may be a possible strategy for treatments against sars viruses. adaptive immune response. adaptive immune response is the specific response that the immune system exerts against pathogens. this mechanism is particularly active toward viral infections where the immune system produces specialized lymphocytes (to fight the virus), called memory b cells (to be effective in case of new infections) and antibodies (corresponding to the humoral response). the stimulation of adaptive responses in case of specific infections can be induced artificially through the introduction of attenuated pathogens, stimulating the production of specific antibodies. this is the principle of vaccination, which is the most common procedure for immunization of large areas of population against many kinds of lethal viruses. besides, the use of viral proteins as antigens in the vaccine formulation leads to neutralizing antibodies, but, due to the low immunogenicity of isolated proteins, does not always stimulate sufficiently the immune system to reach total protection. more recently, nanotechnology has been applied to develop more efficient vaccines (e.g., nanovaccines). the use of nanostructures with a size similar to virus (virus-like nanoparticles) sensibly enhances the response helping to reach immunity. hnms can adsorb viral particles and present them to the immune system. , this method of vaccination has been successfully applied in vivo for the challenge of herpes simplex virus. hsv- starts its spreading in vaginal tissues and then diffuses to the neurons causing death in mice. zno nps (teardrop morphology), after vaginal inoculation with hsv- , are able not only to prevent viral cell adhesion but also to expose viral antigens to t cells and dcs, leading to immunization. the preclinical trials acs nano www.acsnano.org review against hsv- showed a survival to infection higher than %. this approach highlights the possibility to couple cell mimicking nms to other co-adjuvants for the formulation of large spectrum nanovaccines ( figure ). , hnms have been also applied to the delivery of antigens, exposing them to the immune system. fullerenes were found as suitable carriers for the delivery of drugs or nucleic acids. functionalized fullerene can also self-assemble into virus-sized nps. investigated as vaccines in cancer immune therapy, polyhydroxy fullerenes (called fullerenols) display interesting properties for antiviral therapy, based on their capacity to selfassemble into virus-like particles (vlps) and so to enhance the immunogenicity of the antigens. the great advantage of this strategy relies on the easy encapsulation process during the self-assembly making fullerenols versatile for the formulation of different kinds of vaccines. these vlps were investigated against hiv- and hepatitis c viruses. compared to conventional protein-or peptide-based vaccines intended to induce antigen-specific adaptive immune responses, dna vaccines are more stable, cost-effective, easy to manufacture, and safe in handling. however, dna vaccines have the disadvantage of being poorly immunogenic. fullerenol vlps allow to avoid the use of other adjuvants. in the case of a vaccine against hiv- , fullerenol vlps penetrated easily into the cells resulting in an enhancement of dna transfection. this was proved in a study using fullerenol encapsulating dna encoding the hiv- envelope protein gp ( figure ). in vitro assays were performed in human embryonic kidney cells line (hek ) showing good transfection ability. following various immunization routes (e.g., activation of toll-like receptor signaling or effector memory t cell immune response), fullerenol vlps can induce an innate and a cellular immunity. a similar study was performed for hepatitis c using the hcv recombinant protein as antigen, confirming the potential efficacy of using fullerenols as antiviral vaccines. nevertheless, these results require further mechanistic investigations. indeed, in vitro studies also evidenced a suppressive effect of acquired immune response of c pyrrolidine tris-acid and fullerenol c (oh) . the fullerenol had a dose-dependent effect on t cell receptormediated activation and antibody production by b cells under anti-cd /il- stimulation. however, the molecular mechanism is still unknown. other hnms including aunps (two subcutaneous injections in guinea pigs) and nanodiamonds (three subcutaneous injections in balb/c mice) were explored as carriers of viral proteins for immunization of swine transmissible gastroenteritis virus and h n influenza, respectively, with good preliminary results. , in these cases, the vaccine formulation relies on the adsorption of the viral antigen onto the surface of the nanoparticles. however, an effective vaccination depends on several factors. first of all, the size of the nps plays a key role on the immune system. for instance, size-dependent vaccination efficacy has been reported in mice immunization against the foot-and-mouth disease virus (intraperitoneal and subcutaneous injection, every days for weeks) using aunps as antigen carriers. in this study, the most effective activity to stimulate the immune system was exerted by particles with a diameter in the range of nm. both smaller or bigger particles evidenced a drop-off of the immunization effect. this aspect cannot be ascribed to the antigen concentration, but must be associated only to the acs nano www.acsnano.org review nanoparticle size; however, the mechanism of interaction remains unknown. the selected antigen plays also a crucial role in the preparation of wide spectrum vaccines. for instance, in the case of influenza, two major membrane glycoproteins, hemagglutinin and neuraminidase, are generally used as antigens. however, the antibodies produced by this vaccination strategy are selective to the dominant epitope which has a low effectiveness or is totally ineffective against other epitopes or other kinds of influenza viruses. m (a viral protein responsible for the budding and scission of the influenza virus) is commonly expressed in different types of influenza viruses with a high rate of conservation but with low antigenicity. it has been shown that aunps functionalized with m e protein have a high immunization capacity in comparison to the antigen alone. mice immunized with aunps (two intranasal injections), and then challenged, showed a survival rate higher than % to california-h n pdm, victoria-h n , and vietnam-h n infections. this strategy shows that hnms can be used to boost the immune response of low immunogenic molecules, providing a wide spectrum vaccination potential. unfortunately, it has not been determined if the budding process in sars-cov- is mediated by viral proteins or via the host cell's endosomal sorting complex, thus more research on the sars-cov- viral machinery is highly desirable. all these approaches are based on the capacity of the nanoparticles to adsorb the antigens and expose them to the immune system in the appropriate conformation to produce the neutralizing and protective antibodies. however, the adsorption is not an easy process to control. for instance, aunps have been ineffective in the immunization against sars-cov, when s viral proteins were used as antigens. in particular, the immunization with the protein alone was more efficient than when it was adsorbed onto the aunp surface. this failure was associated with a conformational change or denaturation of the antigen, which did not lead to the production of the specific antibody. covalent chemistry strategies offer a higher control on the antigen quantification with higher reproducibility and possibility to bind different groups onto the surface of hnms. for example, calcium phosphate nanoparticles (capnps) were successfully covalently functionalized with hen egg lysozyme as model antigen showing an immunization times higher in vivo compared to antigens alone using (one subdermal injection in mice). a similar approach was used with iron oxide nps using mannose (to target dcs) and hepatitis b antigen showing good immunological activity in vitro (two subdermal injections in mice at days distance). more recently, other types of vaccination strategies have been applied using hnms. a smart example has been reported using multifunctional capnps on herpes virus. in this study, capnps have been covalently functionalized with alum/mpl as the adjuvant and two peptides as antigens selected via reverse vaccination. the nm stimulated the immune system generating highly efficient antibodies able to block cell-to-cell infection of herpes virus in vivo (three intramuscular injections in mice every days), increasing the survival rate of immunized mice to % against the controls ( % survival, figure ). the recent history has shown the spread of different viral pandemics such as h n flu, hiv, and sars. nowadays, the sars-cov- pandemic global lockdown has profoundly changed the daily life of most humans, causing uncertainty in short and middle time perspectives. in this context, the scientific community has responded to protect the population by studying new vaccines and disinfection methods to be applied in the near future. despite this tough work, a sars-cov- vaccination will hopefully be available in − years, making this epidemic transient period gloomy with increased instability. the study of more effective vaccines and the production of a wide range antiviral agents is nowadays an extremely hot topic. hnms comprise a family of materials that share a nanostructured hard core and a tunable surface chemistry. in this contribution, we have methodically reviewed different hnms for antiviral properties. hnms can have antiviral properties per se, blocking the viral replication and diffusion, or their antiviral properties can be tailored, playing with surface chemistry. hnms can be used to block viral entry and arrest infection at the early stage. the mechanisms rely on different actions including breaking of capsid disulfide bonds (e.g., noble metal nanoparticles), capsid oxidation (e.g., cuonps), mimicking of cell surface (e.g., carbon nms), or mechanical disruption (e.g., aunps or graphene). surface functionalization additionally confers a higher specificity and pharmacological activity toward the targeted virus. in particular, the high local concentration of ligands on functionalized hnm surface imparts a high multivalent effect, enhancing the viral trapping efficiency of nms. interestingly, hnms that show an intrinsic antiviral activity can further enhance their antiviral efficacy via surface functionalization. some antiviral hnms are good photosensitizers (e.g., cuonps) or exert photothermal activity (e.g., carbon nms), thus their antiviral activity can be trigged by light stimulation. more importantly, most of the settled strategies target common viral entry mechanisms and can be adopted to fight a wide viral spectrum. hnms have been also applied as antiviral agents by their interaction with host cells. hnms are able to block viral replication machinery in host cells (e.g., cnts and fullerenes), inhibiting endogenous enzyme activity. additionally, hnms can be explored for the delivery of antiviral molecules, showing a better antiviral activity and reducing side effects in vitro and in vivo. some hnms can also regulate the ros homeostasis of host cells, reduce apoptosis, and enhance host cell survival during the infection. finally, we have reviewed the role of hnms on immunity. in particular, hnms can stimulate the innate immune response, mainly inducing overexpression of interferon and cytokines. this effect can alert sentinel cells and generally warn the immune system of the infection. hnms can be also used for the activation of the adaptive immune response, foreseeing vaccination. due to the similar size of a virus, functionalized hnms with antiviral molecules (virus-like particles) can enhance their immunogenicity. certainly a huge effort should be done for translation of the research into clinics. indeed, hnm applications as antivirals are still in the early phase of research. several challenges still need to be tackled before their safe use. at the current stage, some hnms have been approved only for surface disinfection. for instance, cunps have been used in filters for the preparation of highly efficient broad spectrum antiviral masks. some hnms have been already clinically approved. for example, fenps were approved for imaging and as a drug to treat iron deficiency anemia in adult patients with chronic kidney disease, while aunps are in clinical trials for the treatment of prostate cancer (photothermal therapy). , however, at the moment no clinical trials are running for the use of hnms as antiviral agents. in fact, there are still several concerns on the applications of hnms in drug formulations. compared to molecules, where mainly concentration and exposure routes are concerned, solving hnm toxicity issues is much more complicated. composition, size, shape, and surface functionalization must be considered to respond to the requirement for safety regulations. additionally, interaction on hnms with the immune system must be better elucidated. in particular, the activation of the immune system and complement activation-related pseudoallergy must be taken into account. for instance, ferumoxytol (a fenp-based drug) has been reported to generate severe anaphylactic reactions in humans, of which were fatal. this is due to the possible interaction of hnms with mast cells, provoking their degranulation/activation and release of histamine even at the first exposure (pseudoallergic-mediated hypersensitivity). besides, the mechanism of activation of these cells is still unknown, although it was found that it depends on the hnm composition, size, and surface chemistry and on the corona formation. on the other hand, it has been demonstrated that hnms can travel to the draining lymph nodes, targeting resident dendritic cells and macrophages. therefore, they are able to interact with antigen presenting cells to stimulate innate and adaptive immune responses. we believe that a joint venture of different chemists, materials scientists, virologists, toxicologists, and medical doctors can push forward the preparation and safe application of hnms in this field, hoping to prevent and eventually block the rise of new viral pandemics. alberto bianco − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france; orcid.org/ - - - x; email: a.bianco@ibmc-cnrs.unistra.fr giacomo reina − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france; orcid.org/ - - - ; email: g.reina@ibmc-cnrs.unistra.fr hard nanomaterial, nonpolymeric organic or inorganic materials with sizes comprised between and nm; antiviral, medical term used for any agent or drug altering virus integrity or process involved in viral infection disease; viral infection, process by which viruses invade the body through multiple pathways and multiply in susceptible host cells; viral pathogenesis, approach in biomedical research to understand the process by which a viral infection leads to disease, including mechanism of infection into the host (e.g., viral entry, viral replication) and factors that affect this mechanism (e.g., virus susceptibility to host defenses); membranotropism, the ability of an organism or an agent to interact with biological barriers; immunogenicity, capacity of an exogenous substance or material to trigger an immune 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for controlled genetic immunization potential suppressive effects of two c fullerene derivatives on acquired immunity prospects for the use of spherical gold nanoparticles in immunization nanodiamond enhances immune responses in mice against recombinant ha/ h n assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide consensus m e peptide conjugated to gold nanoparticles confers protection against h n , h n and h n influenza a viruses gold nanoparticle-adjuvanted s protein induces a strong antigen-specific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs nanoparticle-based b-cell targeting vaccines: tailoring of humoral immune responses by functionalization with different tlr-ligands hbs antigen and mannose loading on the surface of iron oxide nanoparticles in order to immuno-targeting: fabrication, characterization, cellular and humoral immunoassay induction of herpes simplex virus type cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine progress in nanomedicine: approved and investigational nanodrugs gold nanoshell-localized photothermal ablation of prostate tumors in a clinical pilot device study engineered nanomaterials and type i allergic hypersensitivity reactions shiyuan peng − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france lucas jacquemin − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france andreś felipe andrade − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france complete contact information is available at: https://pubs.acs.org/ . /acsnano. c the authors declare no competing financial interest. the authors gratefully acknowledge the financial support from the eu graphene flagship project (no. ). this work was partly supported the agence nationale de la recherche (anr) through the labex project chemistry of complex systems (anr- -labx- _csc). we wish to acknowledge the centre national de la recherche scientifique (cnrs) and the international center for frontier research in chemistry (icfrc). s.p. is indebted to the chinese scholarship council for supporting her ph.d. internship as a visiting ph.d. student. a.f.a. wish to thanks the eur csc graduate school (strasbourg, france) for supporting his master studies. key: cord- -b c aye authors: roberts, lisa o.; jopling, catherine l.; jackson, richard j.; willis, anne e. title: chapter viral strategies to subvert the mammalian translation machinery date: - - journal: prog mol biol transl sci doi: . /s - ( ) - sha: doc_id: cord_uid: b c aye viruses do not carry their own protein biosynthesis machinery and the translation of viral proteins therefore requires that the virus usurps the machinery of the host cell. to allow optimal translation of viral proteins at the expense of cellular proteins, virus families have evolved a variety of methods to repress the host translation machinery, while allowing effective viral protein synthesis. many viruses use noncanonical mechanisms that permit translation of their own rnas under these conditions. viruses have also developed mechanisms to evade host innate immune responses that would repress translation under conditions of viral infection, in particular pkr activation in response to double-stranded rna (dsrna). importantly, the study of viral translation mechanisms has enormously enhanced our understanding of many aspects of the cellular protein biosynthesis pathway and its components. a number of unusual mechanisms of translation initiation that were first discovered in viruses have since been observed in cellular mrnas, and it has become apparent that a diverse range of translation mechanisms operates in eukaryotes, allowing subtle regulation of this essential process. viruses do not carry their own protein biosynthesis machinery and the translation of viral proteins therefore requires that the virus usurps the machinery of the host cell. to allow optimal translation of viral proteins at the expense of cellular proteins, virus families have evolved a variety of methods to repress the host translation machinery, while allowing effective viral protein synthesis. many viruses use noncanonical mechanisms that permit translation of their own rnas under these conditions. viruses have also developed mechanisms to evade host innate immune responses that would repress translation under conditions of viral infection, in particular pkr activation in response to double-stranded rna (dsrna). importantly, the study of viral translation mechanisms has enormously enhanced our understanding of many aspects of the cellular protein biosynthesis pathway and its components. a number of unusual mechanisms of translation initiation that were first discovered in viruses have since been observed in cellular mrnas, and it has become apparent that a diverse range of translation mechanisms operates in eukaryotes, allowing subtle regulation of this essential process. viruses are obligate intracellular parasites and therefore depend on host cells for their replication. viruses have evolved a number of ways in which to modify the translation apparatus of the host cell to ensure preferential translation of virus mrnas, and use alternative and novel mechanisms to initiate translation of viral mrnas under such conditions. the study of viral translation mechanisms has been a rich source of information about cellular protein synthesis and its regulation. viruses are diverse in the ways they interact with the host and replicate. for example, their genomes can be made up of dna or rna, and within the rna viruses the genomes may be positive-sense rna, negative-sense rna, or dsrna. production of mrna transcripts may take place in the nucleus and therefore the virus can make use of host cell enzymes. alternatively, this process may occur in the cytoplasm, in which case the virus must encode, or bring with it, its own transcriptional system. viral mrnas also differ in their structure; some viral mrnas are capped and polyadenylated, and these viruses often adopt novel ways in which to ensure the viral mrnas are preferentially translated over cellular mrnas, either through modification of the host cell translational machinery and/or adoption of novel translational mechanisms. there are also a large number of viruses that do not produce capped mrnas and have evolved novel strategies to direct initiation of protein synthesis. no virus has been discovered that encodes its own translation system, and in fact this is one of the main distinguishing features between a virus and a living cell. however, the recent discovery of the ''giant'' virus, acanthamoeba polyphaga mimivirus , has challenged this long-held belief. trnas have been discovered in a number of such giant viruses and this new mimivirus has also been shown to encode a number of translation factors and four aminoacyl-trna synthetases. the virus encodes homologs of eukaryotic initiation factor (eif) e, eif a, and eif and also possesses a homolog of the release factor erf . although the virus therefore encodes proteins involved in all stages of translation, it does not encode any ribosomal components. it has been speculated that these viral components probably represent the remains of a more complex translational system that has been gradually lost over time, rather than an acquisition of cellular components. in summary, to enable efficient synthesis of viral proteins a virus needs to be able to do one or more of the following: ( ) modify the host translation machinery to favor the translation of viral rather than host encoded mrnas. many viral rnas are uncapped and/or contain highly structured -untranslated regions (utrs) that would inhibit the scanning ribosome, so the viral rna would compete poorly with host encoded mrnas for the translation machinery. ( ) use novel mechanisms that allow the selective synthesis of viral proteins. ( ) circumvent the host defense mechanisms that function to inhibit translation following viral infection. a detailed molecular understanding of these three processes has the potential to provide unique insights into viral replication strategies and therefore highlight potential new targets for antiviral therapies. translation in mammalian cells is a multistep, highly regulated process (see chapter by fraser, this volume). it can be considered as three phases; initiation, elongation, and termination. all three phases are regulated, although initiation is thought to be the rate-limiting step for the whole process. for initiation to occur the eif f complex, comprised of the cap-binding protein eif e, the helicase eif a, and the bridging protein eif g, binds to the mrna. the s ribosomal subunit is recruited via an eif g-eif - s interaction together with the ternary complex, which contains eif , gtp, and initiator met-trnai. the resulting complex is known as the s complex. the scanning model of translation initiation predicts that this complex then scans along the mrna until the start codon is reached. the poly(a)-binding protein (pabp) interacts with both the polya tail and the n-terminal half of eif g to circularize the mrna (refs. , ; fig. ). an interaction between eif b, which binds to eif a, and pabp further stabilizes this circularization. the rate of translation initiation in mammalian cells is also controlled by sequence elements within the -and -utrs of mrnas which regulate this process by providing sites for interaction of regulatory proteins and rnas. these include upstream open reading frames (uorfs), microrna (mirna) target sites, and polyadenylation elements. , transcription of mrnas from mammalian dna virus genomes such as herpesviruses and adenoviruses occur in the nucleus and results in the production of capped viral mrnas. these viruses need to establish conditions to permit selective translation of viral mrnas over cellular transcripts. it is therefore desirable to stimulate cap-dependent translation pathways and at the same time inhibit or downregulate the translation of host mrnas, which otherwise would be similarly stimulated, to provide the virus with a selective advantage. a major mechanism of regulation of cap-dependent translation is mediated by the eif e-binding proteins ( e-bps), which regulate the formation of the eif f complex ( the scanning model of translation initiation predicts that the s preinitiation complex moves along the mrna in a - direction until it encounters an aug codon that is in a good context. many viruses produce proteases that cleave protein components of this complex to inhibit cap-dependent scanning. thus the cleavage of eif g dissociates the ribosome binding ability of the complex from cap recognition. the cleavage of pabp by viral proteases will prevent the interaction of the and ends of the mrna and also reduce the stability of the complex. three e-bps in mammals, all of which act by binding to eif e and inhibiting its interaction with eif g, leading to an inhibition of cap-dependent translation initiation. hyperphosphorylation of e-bp releases eif e and allows it to interact with eif g in the eif f complex. this hyperphosphorylation is stimulated by growth factors via the mammalian target of rapamycin (mtor) signaling pathway, and is a target of regulation for a number of viruses. how such regulation contributes to viral replication is described below. the herpesviruses are a large family of dsdna viruses and are responsible for a number of different diseases of vertebrates, such as cold sores caused by herpes simplex virus- (hsv- ) and chicken pox caused by varicella zoster virus (vzv) in humans. viruses of the herpesvirus family have adapted to colonize a variety of terminally differentiated cells in which translation rates are generally low. these viruses establish latent infections during which a restricted subset of viral mrnas is expressed at a low level, so that the virus evades the host immune system. the virus replication may undergo periodic reactivation to a productive lytic infectious fig. . regulation of eif e by e-bps. the availability of eif e (the cap-binding protein) for eif f complex formation (which also contains eif g (the bridging protein)) and eif a (a deadbox helicase), is controlled by interaction with its binding partners the e-bps which bind to and sequester eif e. the interaction of eif e with e-bp is regulated by phosphorylation and viral infection controls, either positively or negatively, the phosphorylation status of this protein. when bound to eif g, eif e can be phosphorylated by mnk and it has been suggested that this may increase the affinity of eif e for the cap. the k protein from adenovirus displaces mnk from eif g and so prevents the phosphorylation of eif e. viral strategies to subvert the mammalian translation machinery cycle, accompanied by major changes in viral gene expression. this switch from latent to lytic infection requires the induction of protein synthesis, and e-bp modification appears to play an essential role in this process. the switch from latent to lytic infection in hsv- -infected sensory neurons is accompanied by induction of e-bp phosphorylation. inactivation of e-bp in hsv- -infected cells is increased further by proteasome-dependent degradation, and a decrease in the level of this protein accompanies the increase in phosphorylation. the viral icp protein, an important master regulator of lytic reactivation, is required for both these processes. e-bp phosphorylation during hsv- infection is sensitive to the effects of rapamycin, suggesting that virus-induced signaling through mtor (see chapter by blenis and mahoney, this volume) is required for inactivation of this protein. direct regulation of the mtor pathway occurs in cells infected with epstein-barr virus (ebv). the protein product of the lmp- a gene activates mtor via pi kinase/akt signaling and it has been suggested that the resulting phosphorylation and inactivation of e-bp may be required to increase translation in the transformed cells. for other viruses in this family, such as human cytomegalovirus (hcmv), it would appear that additional mechanisms are also required, as rapamycin is not sufficient to abolish the e-bp phosphorylation induced in the virus infection. , infection of b cells with kaposi's sarcoma associated herpesvirus (kshv) also stimulates hyperphosphorylation of e-bp , although complete release of eif e from e-bp is not observed in this case. the best known poxviruses are smallpox virus and the smallpox vaccine virus, vaccinia virus. recently, the effects of poxvirus infection on the eif f complex have been studied for the first time. poxviruses replicate in the cytoplasm of infected cells and manufacture capped viral mrnas using a viral methyltransferase complex and therefore must effectively compete with host mrnas for the eif f complex. it has now been shown that in vaccinia virus-infected cells e-bp is inactivated through its hyperphosphorylation. in addition, the overall amount of e-bp decreases following vaccinia virus infection, so the virus is able to inactivate this protein through two different mechanisms. adenoviruses (adv) are also dna viruses and are widespread in humans and birds. these viruses have been shown to increase the phosphorylation of e-bp , allowing eif e to bind to eif g and stimulate formation of the eif f complex. this suggests that the regulation of e-bp by multiple mechanisms is a common mechanism of regulation of translation initiation used by most dna viruses. the picornaviruses are a large family of positive-sense rna viruses including important animal and human pathogens such as foot-and mouth-disease virus (fmdv) and poliovirus (pv). the picornaviruses have uncapped mrnas that are translated by the cap-independent mechanism of internal ribosome entry. encephalomyocarditis virus (emcv) and, to a lesser extent, pv, affect e-bp activity by increasing the ability of this protein to bind to and sequester eif e in order to silence cap-dependent translation of host mrnas and permit selective translation of emcv and pv mrnas. upon infection with emcv, e-bp becomes dephosphorylated, and this coincides with the shutoff of protein synthesis that occurs. dephosphorylation of e-bp in pvinfected cells lags behind the shutoff of cellular protein synthesis, and it appears that in this situation protein synthesis inhibition is initiated by the cleavage of eif g. further evidence of a role for dephosphorylation of e-bp in inhibition of protein synthesis during emcv and pv infections was demonstrated by addition of rapamycin, an inhibitor of e-bp phosphorylation, to virus-infected cells. this results in enhanced synthesis of emcv and pv viral proteins. rhabdoviruses are negative-stranded rna viruses. inactivation of e-bp by dephosphorylation and downregulation of host protein synthesis is also observed in cells infected with vesicular stomatitis virus (vsv), despite the fact that vsv mrnas are capped. however, multiple other factors control the translation of viral mrna including a relocalization of certain hnrnps from the nucleus to the cytoplasm. c. other regulation of eif f assembly in addition to their regulation of e-bp , some herpesviruses stimulate the assembly of eif f complexes in cells directly using a range of mechanisms. , , for example, it has been shown that the icp protein produced in hsv- lytic infection is required for increased eif f complex formation and interacts directly with eif g, suggesting that it has a chaperone function. hcmv infection leads to an increase in the abundance of eif e, eif g, and pabp, and to enhanced eif f assembly. reactivation from latency in kshvinfected cells also leads to a stimulation of eif f assembly. interestingly, no viral strategies to subvert the mammalian translation machinery corresponding increase in the level of pabp association with the eif f complex was observed, and pabp was seen to redistribute from the cytoplasm to the nucleus. this is perhaps surprising given the role of pabp in stimulating translation. it was suggested that alternative eif f complexes lacking pabp could selectively promote the synthesis of viral, but not host, proteins, so that kshv-encoded mrnas would compete more effectively for host translation machinery in infected cells. poxvirus infection results in the reorganization of discrete cytoplasmic regions into replication factories. the poxvirus vaccinia virus induces the redistribution of eif e, eif g, and pabp to these replication compartments. it is not fully understood how redistribution of initiation factors occurs although it has been proposed that this may be important in selectively promoting translation of viral mrnas. eif e is phosphorylated on residue serine by the map-kinase signalintegrating kinases mnk and mnk (reviewed in ref. and the chapter by blenis and mahoney, this volume). the mnks bind to eif g, bringing the kinase into close proximity to eif e. [ ] [ ] [ ] the exact role of eif e phosphorylation in translational regulation is still unresolved, and although mnk and mnk are essential for constitutive and inducible phosphorylation of eif e they are not required for cell growth or development. however, it is thought that phosphorylation of eif e leads to stimulation of cap-dependent translation and this is associated with tumorigenesis. changes in the phosphorylation state of eif e are often seen in virus-infected cells and these have been shown to affect virus replication. adenovirus infection results in changes in eif e phosphorylation that are important for virus replication. adenovirus mrnas are capped, but they are selectively translated during late viral infection. during this stage, the first protein to be synthesized is the k protein, encoded by the l transcription unit, and this is produced in very large amounts. the k protein then binds to the carboxyl-terminus of eif g at, or near, the site that is normally occupied by the eif e kinase mnk . by competing with mnk for binding, the k protein acts as a direct inhibitor of mnk and displaces this protein from eif g. , the removal of mnk from eif g results in the dephosphorylation of eif e, which is thought to be associated with the inhibition of cellular capdependent translation, although the precise mechanism by which this occurs is not understood. late adv mrnas are capped but are still translated when cellular protein synthesis is inhibited as they possess a common -noncoding region ( -ncr) known as the tripartite leader. the tripartite leader allows the late mrnas to be selectively translated by an alternate mechanism of initiation known as ribosome shunting. during late infection, the k protein enhances the binding of eif g and eif a to the tripartite leader complex at the end of the adenovirus mrna. the activity of the k protein is stimulated by tyrosine phosphorylation, and this phosphorylation event is necessary to promote viral translation following shunting and does not affect the binding of this protein to eif g. similar decreases in eif e phosphorylation are observed in vsv and influenza virus infections. , in contrast, hsv- induces the phosphorylation of eif e, which promotes its association with eif g and may enhance capdependent (and therefore viral) protein synthesis, although it is not fully understood how selective translation of viral protein synthesis is achieved (reviewed in ref. ). eif g is the central component of the eif f cap-binding complex and is frequently targeted during virus infection. there are a number of functional homologs of eif g including eif gi and ii. many picornavirus infections induce a rapid inhibition of host cell translation. in the case of the entero and rhinoviruses and fmdv, this shutoff is associated with the cleavage of eif g. the entero-and rhinovirus a proteases cleave eif gi such that the protein is separated into an n-terminal one-third, containing the eif e-binding site, and a c-terminal two-thirds, to which eif and eif a bind. [ ] [ ] [ ] the bridging function of eif gi between the capbinding activity of eif e and the helicase and s recruitment roles of eif a and eif is therefore lost. the fmdv l-protease similarly cleaves eif gi at a site close to, but distinct from, the a protease cleavage site. a secondary cleavage event is mediated by a second fmdv protease, c, in a species-specific manner. both cleavage events result in separation of the eif e-binding domain from the c-terminal portion of the protein, similar to the effects of the entero rhinovirus a protease (fig. ) . the effects of eif gi cleavage on host translation are more complex than was originally thought. experiments conducted in the presence of inhibitors of viral rna synthesis indicated that, although eif gi is still cleaved under these conditions, host translational shutoff is minimal. it was subsequently shown viral strategies to subvert the mammalian translation machinery that a protease also cleaves eif gii. cleavage of both proteins is required for the virus to inhibit host translation. in the case of pv, eif gii is cleaved with slower kinetics than eif gi, and eif gii cleavage is therefore the ratelimiting step for induction of host translational shutoff. fmdv l-protease also cleaves both eif gi and ii, but with similar kinetics for each protein. the significance of eif gii cleavage is not certain, as it is much less abundant than eif gi in cells and is no more active in supporting translation initiation. moreover, the central domain of eif g, which lacks the eif ebinding domain, can support translation initiation on capped mrnas. this eif g p domain is fourfold less effective than intact eif f in mediating translation initiation on capped mrnas, but is more active than intact eif f for initiation on pv rna. it is likely that, when viral rna synthesis increases the pool of pv rna in the cell, the p fragment of eif g is redirected to pv rna at the expense of host translation. other effects of picornavirus infection, such as pabp cleavage, may also be involved in mediating the inhibition of host translation that occurs during picornavirus infection. picornavirus translation is directed by internal ribosome entry sites (iress) within the -utrs of the viral rnas. the central one-third of eif g, containing the eif and one eif a-binding domain, is sufficient to support translation initiation from these iress. this allows picornavirus rnas to compete effectively for the host translation machinery following infection, although the situation appears to be more complicated than this (see section iii). an exception to this is hepatitis a virus (hav), which does require full-length eif g and eif e for translation initiation, and hence does not cleave eif g or induce shutoff. the caliciviruses are an important family of viruses, being the main cause of outbreaks of viral gastroenteritis in man (noroviruses) and the causative agents of a number of animal diseases. infection with feline calicivirus (fcv) induces cleavage of eif gi and ii, somewhat closer to the n-terminus than the picornavirus a protease cleavage site. this cleavage occurs late in infection and correlates with host translational shutdown. despite this induction of cleavage, fcv requires intact eif g to mediate translation of viral mrnas. it may be that in this case, cleavage of eif gi results in a cleavage product that retains the eif e-binding site, but removes the pabp-binding site. this would make sense as fcv mrna translation requires eif e, although the pabp requirement is currently unknown. however, translation of mrna from the related calicivirus murine norovirus (mnv) is insensitive to fmdv l-protease treatment and it therefore seems that intact eif g is not required for translation of mnv mrnas. it appears that even within the same family of viruses, different requirements for specific initiation factors in viral translation exist. retroviruses have rna genomes that undergo reverse transcription in infected cells to give a full-length dsdna copy, which then integrates into the host genome. human immunodeficiency virus (hiv) is a lentivirus responsible for acquired immunodeficiency syndrome (aids). proteases encoded by hiv- and - cleave eif gi, but not eif gii, in infected cells and in vitro. , unlike the picornavirus proteases, this cleavage occurs at multiple sites and results in inhibition of hiv ires-driven translation, in addition to host translation. several other retrovirus proteases cleave eif gi and eif gii at sites in a similar location when the protease is expressed in cells or introduced into cell-free systems. the significance of this result is not clear, as most retroviruses other than hiv do not inhibit host translation. it is now well accepted that pabp plays a central role in stimulation of translation. by binding to poly(a) tails on capped mrnas, pabp can mediate the circularization of mrnas by simultaneously binding to eif g at the end of the mrna, thus promoting the recycling of ribosomes; this has been termed the ''closed loop model.'' , a number of viruses have been shown to target pabp as a mechanism of inhibiting host cell translation. it has been shown that infection of cells with the picornaviruses pv and coxsackie virus b (cvb ) results in the cleavage of pabp. , furthermore, it was demonstrated that the viral a proteases directly cleaved pabp between m -g . , pabp contains four rna recognition motifs (rrms) that participate in eif g-and rna-binding and a conserved c-terminal domain that interacts with other factors such as eif b. , cleavage of pabp by the picornavirus a proteases separates the rrms from the c-terminus and results in inhibition of protein synthesis, although pabp cleavage does not fully correlate with shutoff. in pv-infected cells pabp is cleaved by the viral c protease at different sites to the a proteases. recent work has provided new information on the role of pabp cleavage in picornavirus infections. it is known that pabp also stimulates picornavirus ires-directed translation through its interaction with poly(a) tails and eif g. , one question that has been the focus of interest for picornavirologists for many years is the mechanism of switching from translation to replication of the viral rnas. as these two processes are occurring in opposite directions on the same rna, it is believed that something must stall translation to allow replication to occur. it has also been shown recently that hav c protease cleaves pabp in vivo and in vitro. the resulting n-terminal cleavage product binds viral strategies to subvert the mammalian translation machinery to the hav -utr (to the py region upstream of the ires) and suppresses translation of the hav mrna. hav does not induce host cell shutoff and infection does not result in cleavage of eif g. a model has been proposed in which pabp binds to the poly(a) tail on the hav rna early in infection and stimulates translation. once enough viral proteins have accumulated, the c protease cleaves pabp and the n-terminal cleavage product binds to the py region of the -utr of hav rna, inhibiting translation. the rna is then cleared of ribosomes to allow replication to occur in the opposite direction. this model is also likely to apply to other picornaviruses, as it has now been shown that pabp cleavage by pv c protease also inhibits translation directed by the pv ires, both on rnas with and without poly(a) tails. it was also demonstrated that expression of a pabp that is resistant to cleavage by c protease within cells resulted in reduced production of viral rna and reduced virus production. this suggests that pabp cleavage may be important in promoting the switch from translation to replication in picornavirus infections. it is not only in picornavirus infections that pabp cleavage is seen. the caliciviruses norovirus (nv) and fcv also induce pabp cleavage and this results in inhibition of translation of polyadenylated rnas in vitro. the c-like protease is responsible for this cleavage. pabp cleavage does not occur until relatively late in fcv infection. as calicivirus rnas are also polyadenylated it is possible that pabp also stimulates translation of viral rnas and that pabp cleavage would inhibit viral translation, but this has not yet been demonstrated. in line with the model described above, it is tempting to speculate that pabp cleavage in calicivirus infections may also modulate the switch from translation to replication of the viral rnas. a different mechanism of targeting pabp that does not involve its cleavage has recently been described in rubella virus infection. the rubella virus capsid protein binds to pabp and there is an increase in pabp levels during infection. addition of the rubella virus capsid protein to in vitro translation reactions inhibited translation of viral rnas, but this inhibition could be rescued by the addition of pabp. an inhibition of host protein synthesis was also observed, although this was not complete. the authors suggested that the binding of the capsid protein to pabp may mediate the switch between translation and packaging of the new genomes, in a similar manner to the model described for picornaviruses and caliciviruses above. rotaviruses belong to the reoviridae family and have segmented dsrna genomes. rotaviruses cause gastroenteritis and infect many animal species; in humans, they are responsible for severe diarrheal disease in infants which is a cause of high mortality in the developing world. these viruses replicate in the cytoplasm of infected cells where capped, nonpolyadenylated viral mrnas are made. the rotavirus nonstructural protein nsp binds to the n-terminal region of eif gi, both in vitro and in infected cells and also binds to the terminal sequence common to all rotavirus mrnas, similar to pabp binding to cellular mrnas. rotavirus-infected cells also undergo inhibition of cellular protein synthesis and this has been attributed to the novel action of the nsp protein. the nsp protein specifically evicts pabp from the eif f complex by competing for binding to eif gi. in spite of this similar function, there is no sequence homology between pabp and nsp . it is therefore believed that nsp binding to a consensus sequence in the end of rotavirus mrnas recruits the eif f complex to the viral mrnas via nsp binding to eif gi, effectively circularizing the viral mrnas. it has been proposed that nsp functions in a similar way to pabp binding to polyadenylated eukaryotic mrnas. however, more recent data have questioned this model as it has been demonstrated that nsp (and its interaction with eif gi) is not required for rotavirus mrna translation. in this study, rnai-induced silencing of nsp in infected cells had no effect on viral protein production (except nsp ) or virus replication, although it did result in a less severe shutoff of host cell protein synthesis. in fact, viral progeny production was enhanced in the nsp -silenced cells. similarly, these authors suggested that eif gi is also not required for viral protein synthesis, as silencing of this factor also had no effect on virus production. a new model on the role of nsp was put forward that proposes that binding of nsp to the viral mrnas either protects them from degradation or prevents binding of the virus polymerase, thereby ensuring they are utilized for translation. the exact role remains to be determined. bunyaviruses possess tripartite, negative-sense rna genomes and are responsible for a febrile illness in humans that is mosquito-borne. bunyavirus mrnas, like rotavirus mrnas, are capped but not polyadenylated. infection of cells with these viruses induces shutoff of host cell protein synthesis, possibly through the inhibition of transcription. it has been shown recently that a translational enhancer element (tee) within the -utr of bunyamwera virus s segment mrna is able to substitute for a poly(a) tail. a similar element has been shown to exist in dengue virus mrna. translation of the bunyavirus mrnas requires eif gi but does not require pabp, although the exact role of eif gi is currently unknown. furthermore, bunyavirus infection of cells in culture resulted in a redistribution of pabp localization from the cytoplasm to the nucleus, and it was suggested that this may contribute to the inhibition of translation of host (polyadenylated) mrnas. the viral n protein binds to pabp in the cytoplasm but it is not yet known if this protein is directly involved in the nuclear relocalization. an alternative mechanism of translation initiation that is used in mammalian cells is termed internal ribosome entry. in this case, a complex, highly structured rna element (an internal ribosome entry site or ires) is formed in the -utr of the mrna and the ribosome is recruited via the ires to an aug start codon that may be a considerable distance from the end of the mrna. accessory proteins termed ires trans-acting factors (itafs) are usually required by cellular iress and the data suggest that these act as rna chaperones that permit the ires to attain the correct structure to recruit the s ribosomal subunit. in general, ires-mediated translation is used under conditions of pathophysiological cell stress which include genotoxic shock, temperature shock, hypoxia, and viral infection. members of several different families of rna viruses are able to bypass the canonical, cap-dependent, translation initiation process by employing this strategy of internal initiation of protein synthesis. the translation initiation factors required, and mechanisms used, by different viral iress vary considerably. the picornavirus rnas are not capped, but are covalently linked to a small peptide known as vpg at the terminus; this peptide is rapidly lost once the virus enters the cell, leaving an uncapped rna. picornavirus -utrs tend to be long and structured, with many upstream aug codons that are not used for translation initiation. this suggested that a cap-dependent scanning mechanism of translation initiation was unlikely to be utilized, and led to the discovery of the first iress in the -utrs of emcv and pv rna. these were identified by construction of dicistronic reporter rnas, in which the viral -utr was placed between two cistrons and was able to promote translation of the downstream cistron. iress were subsequently identified in many different picornavirus rnas and divided into two major categories, within which there are common secondary structural and mechanistic features. type i iress are found in enteroand rhinoviruses, such as pv, and recruit ribosomes to an aug codon at the end of the ires. a ribosomal scanning process then transports the s subunit and associated factors to the next aug codon further downstream, where translation initiation occurs. type ii iress, located in cardio-and aphthovirus mrnas (e.g., emcv), are similar in length to type i iress, at about nucleotides (nt). they also recruit the s ribosomal subunit directly to an aug codon at the end of the ires, but in this case this aug is the initiation codon (fig. ) . the fmdv ires is structurally related to the type ii iress, but only a minority of translation initiation occurs at the site of ribosome recruitment. the remaining ribosomes initiate translation at the next aug downstream following a scanning process, so this ires uses a hybrid of type i and type ii mechanisms. both type i and type ii iress require the entire canonical translation initiation machinery, with the exception of the cap-binding protein eif e, and the eif e-binding domain of eif g. in vitro reconstruction of initiation complexes using purified and recombinant initiation factors and s subunits indicated that eif g binds directly to the j-k domain of type ii iress, that this binding is stimulated by eif a, and that this induces a conformational change in the region surrounding the initiation codon that is likely to promote s complex recruitment. recently, similar experiments on the type i pv ires have indicated that an analogous mechanism is used to recruit eif g- a to domain v of the ires and to induce structural changes at the border of the ires. other structural features of both classes of ires are also required for initiation, and therefore eif g/ a recruitment alone is not sufficient for ires activity. in addition to their requirement for components of the canonical translation initiation machinery, many picornavirus iress need to recruit noncanonical ires itafs to achieve optimal activity. a number of itafs that interact with specific iress have been identified, although in some cases the physiological role of these factors is questionable. well-characterized examples include the polypyrimidine tract-binding protein (ptb), which was first shown to interact with the human rhinovirus (hrv), and was subsequently found to be required for efficient pv and emcv translation in cells. a role for the autoantigen la in stimulation of pv ires activity has also been demonstrated in vitro and in cell culture. itafs are thought to act by modulating the secondary structure of the ires such that canonical initiation factors are more effectively recruited, and such a role was demonstrated for ptb and itaf binding to the fmdv ires and more recently in cells. picornavirus infection is frequently associated with rapid shutoff of host translation, providing a rationale for the use of iress to maintain viral translation under these conditions. the enterovirus a and fmdv l-proteases, for example, cleave eif gi and ii such that the n-terminal eif e-binding domain is separated from the remainder of the protein. although picornavirus iress show unaffected or even enhanced activity in the presence of a protease, some of this stimulation is thought to be independent of inhibition of host translation or expression of the c-terminal fragment of eif g. stimulation of ires activity can still occur when a protease with a mutant active site is expressed, or when eif g is resistant to a cleavage. the effects of this protease on ires activity are therefore more complex than a simple competition between full-length and truncated eif g mediating host and viral translation, and it is probable that other factors are regulated by the protease and have an effect on picornavirus iress. two further categories of picornavirus ires have also been identified. the hav ires forms a minor class of its own, and requires the full canonical initiation machinery including eif e and full-length eif g, although the viral rna is not capped. it has been suggested, although not yet experimentally proven, that the requirement for eif e may be due to its conformational effects on eif g. a fourth, and very distinct, class of picornavirus ires elements was recently identified in porcine teschovirus- (ptv- ), and subsequently in several other picornaviruses such as avian encephalomyelitis virus. these iress are distinct from other picornavirus iress in their initiation factor requirements and mechanism of action, and instead are very similar to the hcv and pestivirus iress. the ptv- and aev ires elements show sequence and structural homology to the hepatitis c virus (hcv) ires and act similarly to directly recruit the s complex in the absence of the eif factors. the ptv- ires has also been shown to interact directly with the s ribosomal subunit and eif . this suggests that exchange of genetic information between picornaviruses and flaviviruses has occurred at some point. the mechanism of initiation used by the ptv- ires and its relatives was initially identified in hcv and pestivirus rnas. , hcv belongs to a subgroup of the flaviviridae family and is a major cause of human disease, causing blood-borne hepatitis that can result in the development of hepatocellular carcinoma. the flavi-and pestiviruses are positive-sense rna viruses with uncapped structured -utrs that direct synthesis of the viral polyprotein. the hcv and pestivirus -utrs are somewhat shorter than those of the picornaviruses, and almost the entire utr, approximately nt, is required for ires activity. hcv and related iress can bind directly to the s subunit (in the absence of any initiation factors) such that the start codon is positioned close to the ribosomal p site. the iress then bind directly to eif and require this factor and the ternary eif /gtp/met-trnai complex to form correctly positioned s* complexes. the ires can then recruit the s subunit and assemble functional s ribosomes. these iress do not require any components of the eif f complex, and it was recently demonstrated that at high magnesium concentrations the hcv ires is able to initiate translation independently of the eif /gtp/met-trnai ternary complex. eif -and eif -independent hcv ires activity was mediated by eif b in a manner analogous to prokaryotic translation initiation. this ability to function in an eif -independent manner displays an intriguing similarity to the dicistrovirus intergenic region (igr) iress and is likely to allow the hcv ires to function under conditions of cell stress that induce eif phosphorylation. the minimal factor requirements for hcv ires activity have allowed close study in vitro and it has been possible to use cryo-electron microscopy (cryo-em) to determine the tertiary structure of the ires in complex with the s ribosomal subunit. the hcv ires was shown to induce conformational changes in the s subunit, some of which are very similar to those induced by eif and eif a binding to the s subunit. this suggests that the hcv ires may function in a similar manner to these factors to promote initiation. a pathway for recruitment of hcv ires rna to the s subunit has been determined using directed hydroxyl radical probing, providing a further indication of similar, but distinct, effects of the hcv ires and eif / a binding to the s subunit. the secondary structure of the hcv and pestivirus iress has been clearly defined (fig. ) . domain iii is necessary for s binding and for subsequent eif recruitment, but domain ii is not required to recruit these components. however, deletion of domain ii results in severely impaired ires activity, and this region of the ires was shown to be important for s formation. domain ii folds independently of the remainder of the ires, and is responsible for the conformational changes induced in the s subunit by hcv ires binding. an analysis of the role of this domain in subunit joining indicated that it promotes eif -induced gtp hydrolysis and release of eif /gdp. despite its ability to initiate translation by binding directly to the ribosome, there is some evidence for a role for several different itafs in hcv ires activity. several proteins have been shown to bind to the hcv ires, but the most convincing evidence is for a role for the autoantigen la. this factor interacts directly with the hcv ires at a site close to the start codon, and stimulates s binding in vitro. depletion of la in cultured cells led to a loss of ires activity. the most recent class of ires to be identified was found in the igr of the cricket paralysis virus (crpv) genome and other members of the dicistroviridae family , of insect-infecting viruses that belong to the picornavirus superfamily. these viruses have a naturally discistronic genome and iress have been discovered in both the -utr and the igr. the crpv igr ires is approximately nt long, and adopts a high degree of secondary structure, with three pseudoknots (pki-iii) that are involved in different aspects of the ires activity. the crpv igr ires and its relatives recruit the s and s ribosomal subunits independently of any host initiation factors or met-trnai. the igr ires binds to the s subunit via the pkii-pkiii domains, and is positioned correctly for elongation by pki, which occupies the p site of the ribosome. a gcu codon is positioned in the a site and recruits its cognate trna such that the first amino acid is alanine (fig. ) . the mechanism of initiation by the crpv igr ires has been analyzed in detail by in vitro reconstitution experiments. the ires mediates peptide synthesis following incubation with the s and s ribosomal subunits, the elongation factors eef a and eef , and aminoacylated trnas. , the first translocation step on the ires occurs without peptide bond formation. the ires therefore mimics both the initiator and elongator trnas by its interactions with the ribosomal p site. the structure of the igr ires in complex with s and s ribosomes has been revealed by cryo-em and crystallography. , these structures demonstrate the ability of the ires to mimic a trna in the ribosomal p site, and to form contacts with the a, p, and e sites. this binding pattern is very different to that of the hcv ires, which interacts predominantly with the solvent side of the s subunit, although there is some overlap in the e site. despite this difference in binding, the crpv and hcv iress induce similar conformational changes in the s subunit, suggesting that these changes may be intrinsic to translation initiation. the ability of the crpv igr ires to initiate translation in the absence of initiation factors allows it to function effectively under conditions in which host translation is inhibited. the ires is relatively inactive in wild-type yeast, but is activated by eif phosphorylation, or by depletion of various other canonical initiation factors. , this implies that the ires competes with host mrnas for ribosomes, and is able to do so effectively under conditions of cell stress. the ires found in the -utr of the dicistroviruses is very different in both structure and mode of action to that of the igr ires. it has been demonstrated that the -utr of the dicistrovirus rhopalosiphum padi virus (rhpv) is mostly unstructured and requires only a minimal set of factors for function, eif , eif , and eif , although the addition of eif f did stimulate s complex formation on the ires. it may be that this simplified mode of action is responsible for the ability of the ires to direct translation initiation in mammalian, insect and plant systems. two iress have been identified in hiv- rna. the first to be identified is located in the coding region of the gag gene and directs synthesis of an nterminally truncated gag protein. the second ires is located in the -utr and directs protein synthesis during the g /m phase of the cell cycle. an unusual form of internal ribosome entry has been described in hiv- . hiv- viral particles contain gag p (the translation of which initiates at the first codon, aug ), and two shorter isoforms of p (which initiates at aug ) and p (which initiates at aug ). an ires that directs translation of all three isoforms of the gag protein is located in a highly structured region which is downstream of the authentic aug start codon and spans to aug . this ires is therefore required to deliver the preinitiation complex upstream to produce gag p polyprotein from the first aug codon. h. mirnas mirnas have recently emerged as major regulators of gene expression (discussed in detail in the chapter by sarnow, this volume). metazoan mirnas function predominantly by binding via imperfect complementarity to sites in the -utr of mrnas and repressing gene expression, both at the level of translation and by mrna degradation. this important mode of gene regulation has been found to affect the life cycles of a number of viruses in a variety of different ways (fig. ). the first observation that viruses utilize the mirna pathway came from the herpesviruses. these large dna viruses encode their own mirnas within their genomes and express these mirnas during both latent and lytic infection. cloning and sequencing of small rnas derived from b cells infected with the -herpesvirus ebv led to the identification of the first five viral mirnas. mirnas expressed by a number of other , , and -herpesviruses that infect a range of species have subsequently been identified by cloning and computational methods, implying that this is a general mechanism used by this viral family. conservation of sequence or genomic location between mirnas derived from different viruses was generally not observed. mirnas are also expressed by the small dna virus simian virus (sv ) and several other polyomaviruses. [ ] [ ] [ ] two mirnas, derived from opposite strands of a single pre-mirna, are expressed by these viruses. a number of small rnas derived from adenovirus va rnai, and predominantly from va rnaii, were identified and shown to associate with the rnai-induced silencing complex (risc), although a functional role has not yet been ascribed to these small rnas. it is likely that viral mirna expression is limited to dna viruses, as drosha and dicer-dependent processing of mirna precursors results in the destruction of the parent rna, and would therefore be undesirable for a virus with an rna genome. cloning of small rnas from cells infected with hcv, yellow fever virus (yfv), or hiv- did not yield any mirnas derived from these rnas and viruses. mirnas derived from both strands of the hiv- tar rna have since been detected in infected cell lines and primary cells. however, hiv- -derived mirnas were undetectable in another recent study, so it is questionable whether any expression that does occur is at a sufficiently high level to have functional consequences. viral mirnas are similar to those of the host, as they are derived from longer hairpin transcripts expressed by rna pol ii or iii and undergo processing to yield mature, cytoplasmic mirnas. identification of novel viral mirnas is therefore amenable to computational analysis. , ebv is now known to encode at least mirnas, located in two genomic clusters, within introns of the bart and bhrf genes. mirnas derived from each cluster show differential expression patterns across different stages of viral latency. the temporal regulation of herpesviral mirna expression in general is not yet well understood, as most viral mirnas have been cloned exclusively from cells at a particular stage of the viral life cycle. however, hsv- mirnas show distinct expression profiles, with four mirnas expressed in latency, one during productive replication, and one throughout infection. sv mirnas are only expressed in late infection. the first identified target for a viral mirna was the sv large t antigen (tag). the viral mir-s is expressed antisense to the tag mrna and acts in an sirna-like manner to cleave and degrade the tag transcript. this mechanism appears to function to mediate downregulation of antigen levels late in the viral life cycle and thus allows the virus to evade a cytotoxic t cell response. the location and function of this mirna appear to be conserved in other polyomaviruses. , regulation of a viral transcript by an antisense viral mirna has also been observed or predicted for some herpesvirus mirnas. mir-h - p, which is expressed during latent hsv- infection, is antisense to the viral icp transcript. surprisingly, despite its exact complementarity to its target, mir-h - p does not significantly affect icp mrna levels, but reduces the expression of the encoded protein at the level of translation. this regulation is likely to be important for establishment and maintenance of viral latency, as the icp protein is important for productive replication and is thought to be involved in reactivation from latency. a search for imperfect targets for viral mirnas within viral -utrs yielded a number of attractive candidates, suggesting that viruses also use this mechanism to regulate their own gene expression. this was confirmed experimentally for hcmv mir-ul - , which downregulates expression of the major immediate early gene ie by binding to its -utr. ie protein is important for the switch from latent to lytic infection, as are some of the other predicted viral targets of herpesvirus mirnas, so viral mirnas may play a general role in regulation of herpesvirus latency. an important function of herpesvirus mirnas appears to be in the regulation of host gene expression. various host mrna targets of herpesvirus mir-nas have been detected, and the viral mirnas appear to function in a similar manner to cellular mirnas, by binding to the -utr with imperfect complementarity and negatively regulating translation and rna stability. ebv mir-bhrf - downregulates the chemokine cxcl- , a t cell attractant, and may thus allow infected cells to escape the t cell response, whereas mir-bart- targets the proapoptotic factor puma and protects infected cells from apoptosis. several kshv mirnas repress expression of bclaf , a protein involved in apoptosis ( ), and others downregulate thbs , a multifunctional protein which has a role in recruitment of monocytes and t cells to sites of infection. despite a lack of sequence homology, mirnas from several different herpesviruses bind to adjacent sites in the -utr of micb mrna and inhibit expression of micb protein, which is involved in immune surveillance. the herpesvirus mirna targets identified to date appear to allow these viruses to regulate the host immune response or apoptosis, and thus are likely to be important in allowing effective infection. a particularly intriguing viral mirna is kshv mir-k , which has an identical seed sequence to cellular mir- , and has been shown to regulate many of the same targets. mir- levels are upregulated in cells infected with ebv, and a similar viral orthologue is encoded by another oncolytic herpesvirus, marek's disease virus type (mdv- ). mir- overexpression is linked to various cancers, so these results suggest that viral mirna expression, or virus-induced modulation of host mirna expression, may contribute to oncogenesis. viral infection can affect both the cellular mirna machinery as a whole, and the expression of individual mirnas. in addition to its effects on mir- expression, discussed above, ebv modulates the levels of multiple host mir-nas, with different effects in latent and lytic infections. , hcmv also regulates the expression of specific host mirnas, some of which affect viral replication. hiv- -dependent downregulation of the mir- - cluster had a positive effect on viral replication, mediated by the tat cofactor pcaf. inhibition of the mirna and rnai pathways by viruses is well established as a mechanism of evasion of the host immune response in lower eukaryotes, but few examples exist in mammalian systems. adenovirus infection results in a global inhibition of host mirna expression and activity. expression of va rnai led to inhibition of processing of ectopically expressed pre-mir , and to inhibition of rnai induced by short hairpin rna (shrna) precursors that require dicer cleavage for activity. this activity was mediated by va rnai competition with pre-mirnas and shrnas for binding to both exportin- , required for pre-mirna export from the nucleus, and to the cytoplasmic processing enzyme dicer. va-derived small rnas also compete with endogenous mirnas for incorporation into the risc. va rnai is expressed at very high levels in infected cells, so it is possible that its interference with the mirna and rnai pathways may be a consequence of this expression and have little functional relevance for the virus. although some studies have suggested repression of the mirna pathway by retroviral transcription factors, , a recent analysis did not identify any such activity. a direct effect of a cellular mirna on virus replication has been observed in the case of the liver-specific mir- . mir- binds to the -utr of hcv rna and is required to maintain viral rna abundance. the mechanism by which this occurs has not yet been resolved. no effects of mir- on hcv translation were initially observed, suggesting that the mirna acts at the level of viral rna replication, whereas translational stimulation mediated by mir- binding to the hcv -utr was observed in a subsequent study. it is possible that multiple mechanisms may operate. it is not yet known whether this mechanism is unique to hcv, or whether other viruses may use similar strategies. various mirnas, including several induced by interferon stimulation, have been shown to negatively regulate hcv, and mirna-dependent repression of other viruses, including primate foamy virus (pfv- ) has also been observed. however, the extent to which these mirnas regulate virus replication in naturally infected tissues remains unclear. the strong requirement for conservation of complementary sequences to an mirna seed for regulation to occur, coupled with rapid viral evolution, would suggest that any mirna that has a detrimental effect on virus replication would quickly be evaded. tissue specificity is an important feature of the expression of many cellular mirnas. it is possible that viruses may have evolved to selectively avoid targeting by host mirnas that are expressed in the tissue they infect, and therefore results obtained outside normal target cells should be interpreted with caution. attempts to target viruses by introduction of artificial-binding sites for tissue-specific host mirnas were effective in restricting viral tropism, but some viral escape mutants evolved. , in conclusion, the interplay between viruses and the mirna pathway is complex and varied, and has many important consequences for viral infection. viruses have evolved a number of unconventional mechanisms that allow the translation of distal orfs, contributing to the complexity of gene expression from compact viral genomes. these include (i) leaky scanning, where the aug of the -most orf is poorly recognized and the ribosomes scan and initiate at a downstream orf, (ii) reinitiation, where a posttermination complex remains associated with the rna and reinitiates at a downstream orf, and (iii) frameshifting. although few of these mechanisms of translation initiation have been described in host mrnas thus far, it is interesting to speculate that, as in the case of iress, viruses could use or adapt a preexisting system to initiate synthesis of viral proteins. a number of rna viruses have a protein known as vpg covalently linked to the end of the viral rna. in picornaviruses, the vpg protein is small and is removed from the rna following viral entry, such that an uncapped viral rna serves as a substrate for translation, which occurs by internal initiation. many of these novel mechanisms have been attributed to the presence of rna structures within the virus genome that are involved in translation initiation, however, recent work has described the presence of a proteinaceous ''capsubstitute'' on calicivirus rnas that directs translation of the viral mrnas. the vpg on calicivirus rnas is much larger than its picornaviral counterpart and has a different function, as it is retained on the rna and serves as a cap substitute that directs translation of the viral mrnas. the calicivirus vpg protein binds to eif e at a site distinct from both the cap and e-bp -binding sites. in the case of fcv, the vpg:eif e interaction is required for translation initiation (at least in vitro) on the viral mrna, but in the case of mnv this interaction is not required for efficient translation, although the presence of vpg on the mrna is necessary. it has also been reported that human norovirus vpg binds to eif , suggesting that vpg has multiple interactions with key components of the translational apparatus. the interaction of calicivirus vpg with eif e is unique amongst mammalian rna viruses but a similar interaction occurs on plant potyvirus mrnas (reviewed in ref. ) . in this case, plants that do not express eif(iso) e are resistant to infection with turnip mosaic virus. potyvirus vpg is thought to bind eif e in competition with the cap structure. hence, even though the principles of vpg-directed translation are common, the specific mechanisms by which the vpg proteins interact with eif e are distinct. influenza virus is a major human health problem with worldwide prevalence. influenza virus is well known for causing pandemic outbreaks and is a zoonotic disease, infecting pigs, avian species and horses, as well as humans. influenza a viruses belong to the orthomyxoviridae family and have a singlestranded, negative-sense rna genome which is made up of eight segments. in influenza virus-infected cells there is a dramatic inhibition of host cell translation while the viral mrnas are selectively translated. it is well established that influenza virus mrnas acquire their caps through a process of ''cap snatching'' or ''cap stealing'' during which the virus polymerase complex ''snatches'' the - nt from host nuclear mrnas which then prime the synthesis of viral mrnas. the viral mrnas therefore contain host cell sequences at their ends, which are followed by conserved viral sequences that are known to bind the viral polymerase. it is believed that polymerase binding to these conserved sequences prevents the snatching of caps from the viral mrnas and contributes to the selective translation of viral mrnas during infection; the cellular mrnas being degraded once decapped. the polymerase itself is a complex of three subunits, pa, pb , and pb . the pb subunit is involved in binding to the caps on cellular mrnas that are then viral strategies to subvert the mammalian translation machinery thought to be cleaved by the endonuclease activity of the polymerase. the crystal structure of the pa subunit has recently been solved and the endonuclease active site was shown to reside in the amino-terminal end of the protein. , as described above, influenza virus infection results in changes to the eif f complex as eif e is dephophorylated and eif g is hyperphosphoryated. a study to understand the components of the eif f complex that are required for translation of viral mrnas has recently demonstrated that influenza virus translation has no requirement for eif e, as viral mrnas are translated in cells depleted of eif e and in cells treated with rapamycin. the authors suggested that the polymerase is able to substitute for eif e by binding to the conserved sequences in the -utr of the viral mrnas and recruiting initiation factors. this seems to be another example of how some capped viral mrnas display a reduced requirement for eif e in a similar manner to the adenoviruses. whereas influenza virus substitutes the eif e component of the eif f cap-binding complex with a viral protein, a recent report has demonstrated the unique ability of hantaviruses to replace the entire eif f complex with just one viral protein. the hantaviruses are rodent-borne viruses of the bunyaviridae family and the genome is made up of three negative sense, single-stranded rna molecules. hantaviruses include sin nombre virus and hantaan virus, viruses associated with high-mortality rates and symptoms including hantavirus pulmonary syndrome and haemorrhagic fever, respectively. the nucleocapsid (n) protein of sin nombre virus has been shown to uniquely possess activities that mimic all three components of the eif f complex, eif e (as n binds to the end of capped mrnas), eif g (as n recruits the s complex to the cap), and eif a (as n replaces the rna helicase). it was shown that n enhances translation of capped mrnas as a whole, but preferentially stimulates translation of capped mrnas that contained nt of noncoding sequence from the virus. following inhibition of translation by the addition of a picornavirus a protease to rrl such that eif g is cleaved, n rescues translation of capped mrnas. finally, it was shown that n increased the rate of recruitment of the s complex to mrnas. in summary, n protein increases the translation of both viral and cellular mrnas, although the enhancement is greater on viral mrnas. hantaviruses do not induce shutoff of cellular protein synthesis but the binding of n to viral mrnas is likely to permit their preferential translation over host mrnas. there are many examples of important mammalian virus pathogens including retroviruses (e.g., hiv- ) and coronaviruses (sars) that employ frameshifting during translation. in most of the systems examined to date, frameshifting is required for the expression of viral replicases. in retroviruses, frameshifting is necessary for the synthesis of gag-pol and gag-pro-pol polyproteins and the production of reverse transcriptases, whereas for the majority of other viruses it is essential to permit the synthesis of rna-dependent rna polymerases. ribosomal frameshifting is a process that alters the triplet decoding of the mrna by the elongating ribosome. a specific signal in the mrna causes the ribosome to change reading frame from the to the À frame and translation then continues in the new frame. in eukaryotes frameshift signals require two elements, a heptanucleotide ''slippery sequence,'' where the ribosome changes reading frame, and a stimulatory element that is located a few nucleotides downstream in the form of an rna pseudoknot or a stemloop. a spacer region of - nt between the slippery sequence and the stimulatory rna element is also required, and frameshifting efficiency is dependent upon the length of this sequence (fig. ; refs. , ) . several models for how frameshifting occurs have been proposed (reviewed in refs. , ) and the model that is most consistent with experimental data suggests that ribosomal pausing at the stimulatory rna element increases the time that the ribosome is held over the slippery sequence and this permits the trna to realign in the À frame. , . coronaviruses coronaviruses are a family of animal viruses with a large positive stranded rna genome. in this family of viruses the replicase gene is composed of two partially overlapping orfs, a and b, and the fused polyprotein a/ b (pp ab) is synthesized by programmed À ribosomal frameshifting. , the first characterized frameshift signal described for pp a/pp b in coronaviruses was in avian infectious bronchitis virus (ibv; ref. ); subsequently, very similar mechanisms were shown to achieve this mode of translation elongation in other coronaviruses. it was shown by mutational analysis that the slippery sequence in ibv is uuuaaac and that the downstream frameshift stimulatory element - nt away from this sequence is a hairpin (h)-type mrna pseudoknot ( ref. ; fig. ). to carry out detailed structural analysis of ribosomes paused on frameshift sequences, a variant of the frameshift sequence of ibv was generated in which the slippery sequence was changed to cgaggca and ribosomes, stalled in the act of decoding the frameshift signal, were purified and viral strategies to subvert the mammalian translation machinery subjected to cryo-em. this allowed images to be generated of s ribosomes stalled over the a/ b ibv pseudoknot frameshift signal and important mechanistic details of the frameshift process have been derived from these basic mechanism of action of frameshifting. two elements are required in the viral rna for frameshifting to take place; a heptanucleotide slippery sequence and a structured downstream stimulatory element which is present in the form of either a rna pseudoknot or a hairpin-stem. ribosome pausing, due to the presence of the rna structure, over the slippery sequence permits the trna to realign in the À frame. fig. . structures of the stimulatory sequences that are found in ibv and hiv. the structure of the stimulatory sequence found in ibv is an rna pseudoknot whereas the structured element in hiv forms a complex stem-loop. reconstructions. importantly, these data indicate that translocation is the point in the elongation cycle at which frameshifting occurs. the rna pseudoknot interacts with the ribosome in close association with the putative s helicase at the entrance to the mrna channel as well as with the s rrna helix , rps , rps , and the ubiquitous eukaryotic ribosomal regulatory protein rack . eukaryotic elongation factor (eef ) was shown to be trapped in the a site of the ribosome and this would prevent binding of trna to this site until the frameshift has been completed. in the presence of the pseudoknot the p site trna is distorted and bent toward the a site that contains eef . taken together these data allowed the following three step model for frameshifting to be proposed. (i) the helicase at the entrance of the mrna tunnel on the elongating ribosome is unable to unwind the pseudoknot structure in the mrna, pausing the ribosome. (ii) the blockage imposed by the pseudoknot partially inhibits the movement of the trna, bound to the mrna by the codon-anticodon pairing, during translocation. the trna is unable to return to the a site due to the presence of eef , and the resulting strain on the trna causes it to bend in a (þ) sense direction such that it now moves to the roof of the p site. (iii) due to the strain of these opposing forces the codon-anticodon interactions breaks. the trna then relaxes and moves in the (À) sense direction and repairs with the mrna in the À position. this elegant model agrees well with earlier data and supports a number of other models that have been proposed. , , severe acute respiratory syndrome (sars) in humans is caused by a novel coronavirus and the frameshift element in this virus is highly related to those described previously although with some interesting differences. in sars-cov the site of frameshifting signal that allows the production of pp ab is also a u_uua_aac stretch and this is found bases upstream of the a stop codon. the frameshift is very efficient; using reporter-based systems, frameshift frequencies of between % and % were measured in vitro and in vivo. , as with other coronaviruses there is a downstream pseudoknot, and disruption of base pairing in this element substantially reduces the efficiency of frameshifting. , however, there are notable differences in this pseudoknot when compared to other coronaviruses. the pseudoknot conforms to the h type structure found in ibv, but there is extensive base pairing in loop . most of loop can be deleted without great effect. however, there appears to be an essential conformation that needs to be maintained to achieve maximum frameshifting efficiency. frameshifting is essential to hiv replication and related lentiviruses (e.g., siv, hiv- ) as it controls both the expression of gag-pol polyproteins and importantly the precise ratio of the gag:gag-pol polyproteins. even small variations in the gag and gag:pol ratio can have adverse effects on the virus in terms of infectivity. the site of frameshifting for hiv is a u_uuu-uua stretch located within the gag/pol overlap nt upstream of the gag termination codon. , the downstream stimulatory element found at the gag-pol junction is a simple but very stable rna stem-loop. , [ ] [ ] [ ] [ ] for hiv- the nmr data suggest that the stimulatory rna is comprised of a two stem structure (fig. ). there is an bp helical stem and a highly ordered hairpin loop, the top of which contains an acaa tetraloop (fig. ) . a less stable stem is also present which is separated from the upper loop. , it has been suggested that the lower stem acts as a ''positioning element'' that permits the stem-loop to induce the ribosomal pausing required to perturb ribosome translocation. in the longer term a full understanding of frameshift regions is likely to be of considerable importance in the development of antiviral drugs. for example, hiv has an absolute requirement for a À ribosome frameshift during translation and this would provide an attractive new target to interfere with the viral lifecycle. it may be feasible to disrupt this process using small molecules, peptides, or oligonucleotides. eukaryotic translation initiation generally occurs close to the end of the mrna. in some mrnas, the -proximal aug is followed by a short uorf, and if this is fewer than codons a significant percentage of the ribosomes that have completed uorf translation may resume scanning (as s subunits) and reinitiate translation at a downstream aug codon. since not all ribosomes resume scanning after termination, the presence of the uorf results in a decrease in translation of the major orf. the uorf must be translated rapidly for reinitiation to occur, [ ] [ ] [ ] suggesting that rescanning occurs only if some of the initiation factors that promoted initiation at the uorf aug remain ribosome-associated during uorf translation. although the mechanism of reinitiation is not fully understood, it has been shown that in mammalian systems efficient reinitiation following uorf translation only occurs if the complete eif f complex, or eif a, b, and the central domain of eif g participated in the original initiation event. some viral mrnas are able to mediate translation reinitiation following translation of a long orf, and special mechanisms have been developed to promote this event. the best studied case of translation reinitiation occurring following translation of a long orf is found in caliciviruses. the subgenomic mrna encoding the structural proteins of fcv is bicistronic with two overlapping cistrons. the first orf of the rna encodes the viral major capsid protein (vp ) and the second cistron encodes the minor capsid protein vp . the two uorfs overlap by nt in fcv (auga), one in norovirus (uaaug) and eight in rabbit hemorrhagic disease virus (augucuga). , the expression of the downstream orf requires a termination-reinitiation event which is different from those identified in mammalian systems studied to date since it is independent of eif g or the eif f complex. instead reinitiation requires an interaction of eif and the s ribosomal subunit with a sequence element that is present in the -terminal nt of the upstream orf, denoted the termination upstream ribosomal-binding site (turbs). , two short sequence motifs present in turbs are required for reinitiation, the first of which is a pentameric uggga sequence that is complementary to the apical loop of helix in the mammalian s rrna. evidence for a direct interaction between fcv mrna and s rrna was obtained using a yeast model system where mutations were introduced into both rnas. thus when the yeast s rrna was mutated such that it was adapted to the fcv sequence or vice versa there was a dramatic increase in the translation of the downstream frame. this ugga motif is conserved in caliciviruses. the second motif is not conserved among caliciviruses but is located at an equivalent position in the turbs of fcv and rhdv, and it is the secondary structure of these sequences, and not the primary sequence, that is important for function. it has been proposed that the binding of posttermination eif / s complexes to turbs retains them in a position suitable for reinitiation once they have acquired the eif /gtp/met trnai ternary complex. , in agreement with this hypothesis it has been shown that eif is involved in termination and recycling of ribosomal complexes, thus providing in part an explanation for the interaction of eif with the s ribosomal subunit (ref. and discussed in the chapter by fraser, this volume). in influenza b virus the genes encoding the m (matrix protein ) and the bm proteins, both of which are important for virus viability, are located on segment of the viral genome. the termination codon of m overlaps the start codon of bm (uaaug) and the bm polypeptide is expressed by termination-dependent reinitiation. , the mrna sequence requirements for reinitiation are similar to those identified for the caliciviruses and are dependent on a nt stretch of rna that is immediately upstream of the uaaug pentanucleotide and includes the uggga motif. the process of reinitiation and termination is thought to involve the interaction of a stemloop structure in this region that has complementarity with the apical loop of helix in s rrna. there are two genera of pneumovirinae: the pneumoviruses, including human respiratory syncytial virus (rsv) and the metapneumoviruses including avian pneumovirus (apv) and human mpv (hmpv). all these viruses cause acute respiratory infections in their hosts. the pneumovirinae direct the synthesis of eight mrna transcripts, encoding nine primary translation products and the m transcripts all contain two uorfs, m - and m - , which are overlapping. expression of the rsv m - orf occurs via an unusual coupled translation event in which termination of translation of the m - orf is required before translation of m - can be initiated. [ ] [ ] [ ] a number of regions in the rsv m - orf have been shown to play a role in coupled translation. the most important region is not between the overlapping cistrons, but is located nts upstream in the m - orf and contains stable structural elements. , a similar mechanism is used to initiate the translation of m - transcripts of apv and hmpv, although there are differences in the efficiencies of the process, which appear to be due to lack of stimulatory sequences in the m - orf. the reinitiation mechanism is likely to be quite different from that found in influenza bm and calicivirus subgenomic mrnas, but may well involve the same principle of capturing some of the posttermination s subunits and restraining them in a position suitable for reinitiation. although translation initiation generally occurs at the proximal aug codon that is reached after scanning of the -utr, alternative sites of translation initiation also exist which contribute to the complexity of viral genomes. it is known that the sequences flanking the aug codon contribute to the efficiency of translation initiation. a purine, particularly adenosine, at À , and guanosine at þ , when the a of aug is designated þ , gives the greatest enhancement to the initiation event. in many viral mrnas the first aug codon is in a suboptimal context, so the scanning ribosome may migrate past this first aug codon and initiate at an alternative downstream aug. this process is termed ''leaky scanning.'' the process of shunting is occasionally observed following leaky scanning and examples of such a mechanism are provided by studies of adenovirus, sendai virus, and avian reoviruses. adenoviruses contain a double-stranded linear dna genome and the timing of the expression of mrna from this genome is dependent upon the stage of infection. during early stages of infection there is a production of proteins for viral dna replication whereas in late infection this switches to proteins that are required for the assembly of viral capsids. most late adenovirus transcription is initiated from the major late promoter (mlp). mrnas derived from this promoter all possess a common -ncr of nt in length called the tripartite leader which arises from splicing of three small exons. , there is an inhibition of host protein synthesis following adenovirus infection. the continued selective translation of viral mrnas under these conditions is due to the presence of the tripartite leader which is able to direct ribosome shunting. , shunting involves the loading of the s subunit at the end of the capped tripartite leader-containing mrnas, followed by linear scanning over a short distance and then a direct translocation of the s subunits via ''shunting elements'' to the start codon. within the tripartite leader, the elements that are required for translation initiation during late viral infection are an unstructured end to the mrna and hairpin structures that have complementarity to the s rrna at the end. the s mrnas transcribed by the fusogenic avian (arv) and nelson bat (nbv) reoviruses encode three unrelated proteins from sequential partially overlapping orfs. the -orf encodes the p fusion-associated small transmembrane protein that is responsible for the syncytium-inducing phenotype of these reoviruses. the second orf encodes a nonstructural nucleocytoplasmic protein (p ) that has no known function. the terminal orf encodes the sigma c protein. this is similar to the mammalian reovirus sigma protein that has a function in cell adhesion. analysis of the start codons of these three orfs revealed that the first aug codon is in a suboptimal context, whereas the sequence surrounding the aug of the second uorf provides a good context for translation initiation with a highly conserved a at the À position. the start codon of sigma c is in a strong context but it is a considerable distance from the -terminal cap structure. by altering the sequences surround the augs of the uorfs that encoded the p and p proteins to optimal contexts it was shown that the coordinated expression of these proteins occurred by leaky scanning. however, the translation initiation of sigma c was shown to be independent of leaking scanning, reinitation, and internal ribosome entry and it was proposed that sigma c translation is mediated by an atypical shunting mechanism. viral strategies to subvert the mammalian translation machinery sendai virus belongs to the parainfluenza virus family, is a pathogen of mice, and has a negative-sense rna genome. in sendai virus p/c mrna there are five start codons that are located - nt from the end of the mrna. a sixth start codon is located more than nt from the end, and together these generate eight protein products, as alternative c-termini are also used. initiation from the first three start codons, which are acg, aug, and aug, can be explained by the leaky scanning model. the sequence that surrounds the first unusual acg codon is in an otherwise optimal context (gccacggat) with a purine at þ and À . if the acg sequence is mutated to aug there is increased expression of the encoded c protein, but the expression of the p and c proteins initiated from the second and third start codons is ablated, presumably because there is no leaky scanning. the expression of the proteins from the downstream start codons is not affected under these circumstances, suggesting that the initiation of these proteins is independent of scanning. , it was shown that in vitro translation of these downstream y proteins was initiated by ribosomal shunting, but in vivo this occurred by both shunting and proteolytic processing of the c and/or c proteins. all proteins encoded by picornaviruses are present in a long single orf which must be ''cleaved'' to give the functional polypeptides. the fmdv a peptide is encoded between sequences that specify capsid and replicative functions of the virus and this is the major site for the processing of this polyprotein. it has been shown that the a region of fmdv (and other picornaviruses) mediates ''cleavage'' of its own c-terminus to release it from the b region. interestingly, the a peptide is active when placed between reporter proteins and the cleavage reaction is not therefore dependent on viral (protease) sequences outside of this region. instead the data suggest that the a peptide is able to interact with the ribosome and direct translational recoding. a detailed analysis of this recoding reaction shows that the ribosomes pause over the final amino acid of the a peptide. the recruitment of release factors to this peptide catalyzes termination and releases the peptide, the sequence of which includes the penultimate amino acid encoded by this region (glycine), while the codon of the terminal amino acid (proline) remains in the a site of the ribosome. these a like peptides have been termed chysel (cis-acting hydrolase element) peptides and their ability to act independently of any proteolytic activity in the ''host'' cell has lead to a number of biotechnological uses. a. inhibition of pkr in mammalian cells there are four proteins that control the formation of ternary complex that is required to bring the initiator trna imet to the ribosome by inducing phosphorylation of eif (fig. ) . these are the gcn , perk, hri, and pkr kinases. these proteins are activated by different external stimuli, generally under conditions of cell stress, and the particular kinase that is activated is dependent upon the stress induced. pkr activation represents one of the major host responses to viral infection. exposure of cells to interferon stimulates the production of pkr which then is activated in response to the presence of dsrna in cells. this often occurs as a result of viral infection by rna viruses, either because the virus has dsrna elements within its genome, or because viral replication induces the temporary formation of dsrna intermediates which could originate from bidirectional transcription. the interaction of pkr with dsrna initiates dimerization, autophosphorylation, and the subsequent activation of this kinase (fig. ) . active pkr dimers then phosphorylate the alpha subunit of eif at serine and this has the net effect of preventing further ternary complex formation (fig. ) such that the translation of host and viral rnas is inhibited. viruses have developed a number of mechanisms to counteract the effects of pkr and there are viral proteins that inhibit pkr activation, sequester dsrna, inhibit pkr dimerisation, activate antagonist phosphatases, or degrade pkr (fig. ). many viruses employ mechanisms to inhibit pkr dimerization and thereby prevent pkr activation in the presence of dsrna. for example, the kshv- (hhv- ) protein virf- binds directly to pkr and prevents activation by inhibiting the autophosphorylation of the protein. hcv encodes two pkr inhibitors, the e envelope and the ns a protein. the cytosolic form of the e protein is unglycosylated, and has been shown to directly bind to and inhibit pkr function. similarly, ns a interacts with pkr and inactivates its kinase function. , this interaction occurs via the interferon sensitivity determining region (isdr) and there is a correlation between a negative response to interferon (ifn) of some hcv-chronically infected patients and mutations in a region of ns a, although the relevance of the ns a/pkr interaction in this regard is still unclear. [ ] [ ] [ ] interestingly, it has also been shown that the ns a/pkr interaction may be involved in the development of liver carcinoma. . mechanisms of inactivation of pkr by viral proteins. viruses have developed a wide range of mechanisms to prevent activation of pkr. these include the production of viral proteins that are able to prevent pkr activation by inhibiting pkr dimerisation and dsrna-binding proteins that sequester dsrna. activity. , in addition, tat directly contributes to the downregulation of pkr as this protein is a substrate for pkr and acts as a competitor of eif . , the phosphorylation of tat by pkr leads to more robust viral transcription since this phosphorylation increases the interaction of tat with tar. the vaccinia virus k l gene encodes an amino acid protein that is % identical to the n-terminus of eif . the k l protein acts as a competitive inhibitor of pkr and a pentapeptide motif is important for this effect. k l is produced early in vaccina virus replication, suggesting that this serves to ensure continued translation once dsrna is produced by the virus. ebv produces two small noncoding rnas eber- and eber- . the eber rnas play an important role in downregulating pkr activity and preventing virusinduced apoptosis. eber- inhibits the protein kinase activity of pkr directly in vitro by competing with dsrna activators for binding to the enzyme. moreover, expression of eber- in ebv negative cells protects against ifn-induced apoptosis. similarly, the adenovirus va i rna which is synthesized in large quantities following viral infection binds to pkr. this rna blocks the activation of pkr in the presence of dsrna and as a consequence both the autophosphorylation of pkr and the subsequent phosphorylation of eif a are inhibited. many viruses synthesize dsrna-binding proteins that prevent activation of pkr by interacting with and sequestering any free dsrna molecules. , the first such dsrna-binding protein identified in this regard was reovirus sigma . the reovirus sigma protein does not have catalytic activity and it was shown that inhibition of pkr by this protein could be overcome by the addition of excess dsrna, suggesting that the protein acts as a competitive inhibitor by directly binding to dsrna. influenza ns protein is a multifunctional protein involved in both protein-protein and protein-rna interactions. this protein dimerizes upon binding to poly(a), and ns -rna complexes block pkr activation. similarly, the e l protein from vaccinia virus contains a dsrna-binding domain which binds to and sequesters dsrnas produced during virus infection and so inhibits pkr activation. both ebv and hsv- encode proteins, sm protein and us , respectively, that bind to dsrna and interact directly with pkr. , both of these proteins contain an rxp domain comprised of multiple copies of the amino acid sequence rxp. this domain has been demonstrated to be a dsrna recognition motif and is involved in the interaction of these proteins with pkr. [ ] [ ] [ ] in pv-infected cells pkr is activated by the presence of dsrna, resulting in an increase in the phosphorylation of eif that leads to a decrease in protein synthesis rates. to circumvent this, pv infection also initiates a series of events that lead to the degradation of pkr. the detailed mechanism of this degradation is not fully understood, although it has been suggested that cellular rather than viral proteases are required. finally, it has been shown recently that the nss protein of rift valley fever virus (rvfv) induces specific degradation of pkr. following infection with rvfv there is a dramatic decrease in pkr protein levels with little change in the levels of the mrna. in the presence of proteasome inhibitors there is a decrease in rvfv growth and an increase in pkr, strongly suggesting that the degradation is mediated via the proteasome pathway. since other related viruses do not have this activity it was suggested that the extraordinary pathogenicity of rvfv is due to the acquired pkr degradation function of nss. . additional mechanisms to overcome pkr activation although many viruses have devised novel ways in which to circumvent the effects of pkr, it is interesting to note that viruses belonging to the dicistroviridae family which infect insects use a unique mechanism to initiate their translation which is independent of ternary complex. [ ] [ ] [ ] in this case viral protein synthesis is stimulated when eif is phosphorylated. an alternative mechanism is adopted by the alphavirus sindbis virus (sv) where activation of pkr by viral infection causes almost complete phosphorylation of eif . however, under these conditions there is still efficient translation of sv s mrna. downstream of the initiation codon on the subgenomic rna there is a stable stem-loop, that is able to stall ribosomes on the correct site to initiate translation of sv s mrna. the data suggest that eif a delivers the met-trnai to the stalled s ribosome, bypassing the requirement for a functional eif . perk is a type i transmembrane protein that attenuates protein synthesis during endoplasmic reticulum (er) stress (see chapter by kedersha and anderson, this volume), including viral infection. virus infection has a profound effect on the er and this can result in activation of the unfolded protein response (upr) leading to perk-mediated eif phosphorylation [ ] [ ] [ ] [ ] [ ] and transient translational arrest (reviewed in ref. ) . viruses have developed a unique set of mechanisms to overcome the effects of perk activation and alternatively use the upr to selectively enhance the synthesis of viral proteins. the herpesvirus human cytomegalovirus (hcmv) is a widespread opportunistic pathogen that can cause disease and death of newborn infants or individuals that are immunocompromised. infection of cells with hcmv results in the production of large amounts of viral glycoproteins and an increase of ca þ release from the er to the cytosol. this induces cell stress, which benefits the virus, as chaperone induction by the upr extends the protein folding capacity of the er. , however, virus infection also induces the phosphorylation of perk and the translational upregulation of the transcription factor atf , yet the virus prevents the activation of the atf -dependent pathway that would lead to cell apoptosis. , this is mediated by viral protein pul which promotes perk activation and atf production but suppresses the persistent c-jun n-terminal kinase (jnk) phoshorylation and er-stress-induced cell death. it has also been shown that the lmp protein from ebv works in a similar manner. again this protein induces the phosphorylation of perk and this translationally upregulates the production of atf . atf then trans-activates the promoter of lmp leading to increased transcription and expression of this protein and enhanced proliferation of the infected b cells. in hsv- infected cells perk is inactivated and not affected by the acute er stress that occurs as a result of the viral infection. this is mediated by the viral glycoprotein (b) (gb) of hsv- that specifically associates with the luminal domain of perk and suppresses its activation. interestingly, gb appears also to directly regulate viral protein accumulation in a perk-dependent manner. similarly the hcv envelope protein e , an er-bound protein, has been shown to directly bind to perk and inhibit its activation. mammalian cells that stably express e are resistant to the effects of er-stress inducers, and e can relieve the translational repression induced by perk. as viruses do not possess their own translational machinery they are completely dependent on the host cell for this critical step in their replication cycle. it is clear that viruses have adopted a number of elegant mechanisms to inhibit host cell translation, or at least modify translation factors, in order to effectively compete with cellular mrnas (fig. ) . viruses, especially rna viruses with their high rna replication error rates, are the best example we have of natural selection (as opposed to directed selection in agricultural and laboratory practice) operating in real time before our very eyes. the most ''successful'' viral strains or species are, by definition, those which are most abundant in the environment, and by this criterion h n swine flu is proving a more successful virus than sars was, notwithstanding the much higher mortality rate (almost %) in sars infection. of course, such success may be short lived. for example, we expect swine flu to peak in the next couple of years and then decline, while other virus strains or species may be considered more successful because they maintain a more enduring high abundance, even though they never reach such extreme peaks. the drivers behind virus evolution seem relatively straightforward: (i) efficient transmission between host organisms, (ii) efficient redirection of host cell mechanisms, especially translation, toward viral multiplication at the expense of host cell functions, and (iii) an ability to evade the host cell defense mechanisms, both extracellular (e.g., immune system) and intracellular. against these, there is evolutionary pressure on the host to elaborate antiviral defense mechanisms, which in turn puts pressure on the viruses to develop mechanisms of negating or bypassing these host cell defenses. although this picture is almost certainly oversimplified, there is little doubt that virus evolution is relatively simple as compared with higher eukaryote whole organism evolution. viruses have contributed enormously to our understanding of eukaryotic protein synthesis mechanisms, and mrna structure and function. this is in part due to the fact that, irrespective of whether the viral genome is dna or rna, positive or negative strand, double or single stranded, all viruses are dependent on the cellular protein synthesis machinery, whereas they vary in their dependence on other facets of host-cell gene expression. the contribution of virus research to our understanding of mrna structure and function, and the mechanism of protein biosynthesis is highlighted by the following list of discoveries (in approximate chronological order) all made through exploiting viruses: eubacterial translation initiation sites (rna bacteriophages), poly(a) tails (vaccinia), caps (reovirus), pkr (pv), translation of only the first cistron of polycistronic mrnas (tobacco mosaic virus), leaky termination (plant rna viruses), the scanning ribosome mechanism (reovirus), eif g (pv), iress (picornaviruses), programmed- frame-shifting (retroviruses and coronaviruses), ribosome shunting (adenovirus), and reinitiation after translation of a long orf (pneumoviruses, caliciviruses, influenza b viruses). moreover, this list has not closed because there are unusual viral rna translation mechanisms that remain to be fully elucidated, for example, the extraordinary translational stop-restart mechanism promoted by the fmdv a peptide. some of the earlier discoveries in the above list exploited the fact that in the era before recombinant dna techniques and transcription vectors had been developed, viruses were often the best source of a single mrna species (e.g., pv, tobacco mosaic virus) or a very limited number of mrnas (reovirus), with the added advantage of being able to synthesize defined radiolabeled mrnas in some cases (e.g., reovirus). viruses also provide excellent tools with which to study cellular protein synthesis or novel viral mechanisms of initiation. for example, the picornavirus proteases that cleave eif g are widely used to study capindependent mechanisms of initiation, and a range of viral ires elements that require different factors for function are routinely used to dissect translational control processes, such as mirna-mediated regulation of gene expression. however, the more recent discoveries are due to the fact that viruses, particularly positive-strand rna viruses with small genomes, have evolved to exploit the extremes of behavior (or what might be thought the weaknesses) in the host cell translation machinery (e.g., programmed frameshifting, leaky termination, shunting, reinitiation after a long orf). some of these viral ''tricks'' (e.g., iress, and perhaps shunting) have evolved to enable efficient viral protein synthesis to proceed despite the virus-induced shutdown of host cell protein synthesis, and, certainly in the case of iress, the discovery has proved highly relevant to mrna translation in the uninfected host cell. many other viral ''tricks'' have evolved to allow more than one protein to be expressed from a single rna: frameshifting and leaky termination give two proteins with the same n-termini, while reinitiation after a long orf gives two unrelated proteins as an alternative to relying entirely on proteolytic processing of a polyprotein by virus-encoded proteases, or synthesis of monocistronic subgenomic mrna(s). it is not yet clear whether these events also occur in the translation of some cellular mrnas. if they do, they certainly concern only a very limited number of cellular mrnas, and without the clues provided by the viral examples as to the sequence and structure of the required rna motifs, the task of identifying such cellular mrnas would be like trying to find the proverbial needle in a field of haystacks. even if they do not occur in any cellular mrna, the viral mechanisms are always instructive, because they provide insights into issues such as why does reinitiation after a long orf not normally occur with the vast majority of cellular mrnas. moreover, if the viral mrna motifs directing leaky termination, frameshifting, or reinitiation after a long orf, are absent from all essential cellular mrnas, they provide potential targets for the development of novel antiviral agents targeted against the intracellular phase of the infectious process (although the high mutation rate of rna viruses may well pose problems with this approach). thus, while the ''tricks'' are essential for viral multiplication, they are also a potential achilles heel. viruses not only interfere with host translational processes to improve competition for factors, etc., but they must also respond to a number of host antiviral ''attacks''-this means that viruses have evolved ways in which to manipulate the host to ensure their replication is not inhibited. viral countermeasures against host attack, and the delicate balance between the host and virus, are only beginning to be understood and it is likely that we will learn much more about this in the future. there is no doubt that a greater understanding of how viruses regulate host translation, and identification of viral strategies, will aid in the development of novel antiviral therapies; the diversity and ingenuity of viruses is also likely to further surprise us. a giant virus in amoebae the . -megabase genome sequence of mimivirus regulation of translation initiation in eukaryotes: mechanisms and biological targets translational control by viral proteinases dissecting eif e action in tumorigenesis disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor b with the poly(a)-binding protein by caspase-and viral protease-mediated cleavages molecular mechanisms of translational control how do micrornas regulate gene expression? oncogenic gamma-herpesviruses: comparison of viral proteins involved in tumorigenesis phosphorylation and dephosphorylation events that regulate viral mrna translation regulation of the translation initiation factor eif f by multiple mechanisms in human cytomegalovirus-infected cells epstein-barr virus: inhibition of apoptosis as a mechanism of cell transformation human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mtor kinase human cytomegalovirus infection alters the expression of cellular microrna species that affect its replication activation of host translational control pathways by a viral developmental switch modification of rna by messenger-rna guanylyltransferase and messenger-rna (guanine- -)methyltransferase from vaccinia virions phosphorylation of eif e by mnk- enhances hsv- translation and replication in quiescent cells adenovirus overrides cellular checkpoints for protein translation activation of the translational suppressor e-bp following infection with encephalomyocarditis virus and poliovirus rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection vesicular stomatitis virus infection alters the eif f translation initiation complex and causes dephosphorylation of the eif e binding protein e-bp hnrnps relocalize to the cytoplasm following infection with vesicular stomatitis virus eif initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation mnk , a new map kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates mitogen-activated protein kinases activate the serine/threonine kinases mnk and mnk circularization of mrna by eukaryotic translation initiation factors micrornas expressed by herpes simplex virus during latent infection regulate viral mrnas does phosphorylation of the cap-binding protein eif e play a role in translation initiation? cleavage of eukaryotic initiation factor eif g and inhibition of host-cell protein synthesis during feline calicivirus infection rna-binding properties of a translational activator, the adenovirus l -kilodalton protein adenovirus-specific translation by displacement of kinase mnk from cap-initiation complex elf f a functional microrna- ortholog encoded by the oncogenic marek's disease virus ebv micrornas in primary lymphomas and targeting of cxcl- by ebv-mir-bhrf - modification of eukaryotic initiation-factor f during infection by influenza-virus inhibition of hela cell protein synthesis following poliovirus infection correlates with the proteolysis of a , -dalton polypeptide associated with eucaryotic initiation factor and a cap binding protein complex mapping of functional domains in eukaryotic protein synthesis initiation factor g (eif g) with picornaviral proteases. implications for cap-dependent and cap-independent translational initiation mapping the cleavage site in protein synthesis initiation factor eif- gamma of the a proteases from human coxsackievirus and rhinovirus cleavage of eukaryotic translation initiation factor gii within foot-and-mouth disease virus-infected cells: identification of the l-protease cleavage site in vitro sv -encoded micrornas regulate viral gene expression and reduce susceptibility to cytotoxic t cells lack of direct correlation between p cleavage and the shut-off of host translation after poliovirus infection proteolysis of human eukaryotic translation initiation factor eif gii, but not eif gi, coincides with the shutoff of host protein synthesis after poliovirus infection activity of the hepatitis a virus ires requires association between the cap-binding translation initiation factor (eif e) and eif g functional dissection of eukaryotic initiation factor f: the a subunit and the central domain of the g subunit are sufficient to mediate internal entry of s preinitiation complexes initiation of protein synthesis from the a site of the ribosome calicivirus translation initiation requires an interaction between vpg and eif e caliciviruses differ in their functional requirements for eif f components in vitro cleavage of eif gi but not eif gii by hiv- protease and its effects on translation in the rabbit reticulocyte lysate system translational resistance of late alphavirus mrna to eif alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase pkr the eukaryotic translation initiation factor gi is cleaved by different retroviral proteases cleavage of poly(a)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro cleavage of poly(a)-binding protein by coxsackievirus a protease in vitro and in vivo: another mechanism for host protein synthesis shutoff ? poly(a)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression interaction of rat poly(a)-binding protein with poly(a)-and non-poly(a) sequences is preferentially mediated by rna recognition motifs þ calicivirus c-like proteinase inhibits cellular translation by cleavage of poly(a)-binding protein picornavirus ireses and the poly(a) tail jointly promote cap-independent translation in a mammalian cell-free system eukaryotic initiation factor g-poly(a) binding protein interaction is required for poly(a) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for x-mediated stimulation of hepatitis c virus translation a late adenovirus factor induces eif- e dephosphorylation and inhibition of cell protein-synthesis cleavage of poly(a)-binding protein by poliovirus c proteinase inhibits viral internal ribosome entry site-mediated translation rubella virus capsid protein interacts with poly(a)-binding protein and inhibits translation capped and conserved terminal structures in human rotavirus genome double-stranded-rna segments rotavirus rna-binding protein nsp interacts with eif gi and evicts the poly(a) binding protein from eif f rotavirus nonstructural protein nsp is not required for viral protein synthesis bunyamwera orthobunyavirus s-segment untranslated regions mediate poly(a) tail-independent translation control of translation by the -and -terminal regions of the dengue virus genome a segment of the nontranslated region of encephalomyocarditis virus rna directs internal entry of ribosomes during in vitro translation internal initiation of translation of eukaryotic mrna directed by a sequence derived from poliovirus rna internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond eukaryotic initiation factors g and a mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site direct functional interaction of initiation factor eif g with type internal ribosomal entry sites polypyrimidine-tract binding protein (ptb) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus- rna the polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation la autoantigen is necessary for optimal function of the poliovirus and hepatitis c virus internal ribosome entry site in vivo and in vitro a cell cycle-dependent protein serves as a template-specific translation initiation factor structural insights into the transcriptional and translational roles of ebp recognition of picornavirus internal ribosome entry sites within cells; influence of cellular and viral proteins competitive translation efficiency at the picornavirus type internal ribosome entry site facilitated by viral cis and trans factors unique characteristics of a picornavirus internal ribosome entry site from the porcine teschovirus- talfan the picornavirus avian encephalomyelitis virus possesses a hepatitis c virus-like internal ribosome entry site element functional and structural similarities between the internal ribosome entry sites of hepatitis c virus and porcine teschovirus, a picornavirus secondary structure of the nontranslated regions of hepatitis c virus and pestivirus genomic rnas mnk and mnk are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor e but not for cell growth or development a prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis c and classical swine fever virus rnas initiation factor-independent translation mediated by the hepatitis c virus internal ribosome entry site internal initiation in saccharomyces cerevisiae mediated by an initiator trna/eif -independent internal ribosome entry site element hepatitis c virus ires rna-induced changes in the conformation of the s ribosomal subunit the eukaryotic translation initiation factors eif and eif a induce an open conformation of the s ribosome the pathway of hepatitis c virus mrna recruitment to the human ribosome the pathway of hcv ires-mediated translation initiation hcv and csfv ires domain ii mediate eif release during s ribosome assembly translation initiation at the cuu codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro the untranslated region of rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus divergent trna-like element supports initiation, elongation, and termination of protein biosynthesis translation elongation after assembly of ribosomes on the cricket paralysis virus internal ribosomal entry site without initiation factors or initiator trna trna-mrna mimicry drives translation initiation from a viral ires cryo-em visualization of a viral internal ribosome entry site bound to human ribosomes: the ires functions as an rnabased translation factor translation initiation factors are not required for dicistroviridae ires function in vivo suppression of microrna-silencing pathway by hiv- during virus replication eukaryotic translation initiation machinery can operate in a bacterial-like mode without eif tethering of eif g to adenoviral mrnas by viral k protein drives ribosome shunting the human immunodeficiency virus type gag gene encodes an internal ribosome entry site the leader of human immunodeficiency virus type genomic rna harbors an internal ribosome entry segment that is active during the g /m phase of the cell cycle hiv- genomic rna contains a novel type of ires located downstream of its initiation codon identification of virusencoded micrornas identification of micrornas of the herpesvirus family evolutionarily conserved function of a viral microrna murine polyomavirus encodes a microrna that cleaves early rna transcripts but is not essential for experimental infection mrna helicase activity of the ribosome human cytomegalovirus protein pul induces atf expression, inhibits persistent jnk phosphorylation, and suppresses endoplasmic reticulum stress-induced cell death identification of functional micrornas released through asymmetrical processing of hiv- tar element analysis of the interaction of primate retroviruses with the human rna interference machinery a combined computational and microarray-based approach identifies novel micrornas encoded by human gamma-herpesviruses epstein-barr virus micrornas are evolutionarily conserved and differentially expressed hiv- protease cleaves eukaryotic initiation factor g and inhibits cap-dependent translation merkel cell polyomavirus encodes a microrna with the ability to autoregulate viral gene expression suppression of immediate-early viral gene expression by herpesvirus-coded micrornas: implications for latency adenovirus virus-associated rnaii-derived small rnas are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes an epstein-barr virus-encoded microrna targets puma to promote host cell survival identification of cellular genes targeted by kshv-encoded micrornas diverse herpesvirus micrornas target the stress-induced immune ligand micb to escape recognition by natural killer cells a viral microrna functions as an orthologue of cellular mir- generation of a conditionally replicating adenovirus based on targeted destruction of e a mrna by a cell type-specific microrna tandem array-based expression screens identify host mrna targets of virus-encoded micrornas epstein-barr virus growth/ latency iii program alters cellular microrna expression epstein-barr virus-mediated dysregulation of human microrna expression the phosphorylation of eukaryotic initiation factor eif e in response to phorbol esters, cell stresses, and cytokines is mediated by distinct map kinase pathways internal ribosome entry site within hepatitis c virus rna adenovirus va noncoding rna can inhibit small interfering rna and microrna biogenesis evidence that hiv- encodes an sirna and a suppressor of rna silencing a cellular microrna mediates antiviral defense in human cells modulation of hepatitis c virus rna abundance by a liver-specific microrna microrna- stimulates translation of hepatitis c virus rna interferon modulation of cellular micrornas as an antiviral mechanism engineering microrna responsiveness to decrease virus pathogenicity crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site the genome-linked protein vpg of the norwalk virus binds eif , suggesting its role in translation initiation complex recruitment translational control in positive strand rna plant viruses complex formation between potyvirus vpg and translation eukaryotic initiation factor e correlates with virus infectivity how does influenza virus regulate gene expression at the level of mrna translation? let us count the ways surprising function of the three influenza viral polymerase proteins: selective protection of viral mrnas against the cap-snatching reaction catalyzed by the same polymerase proteins the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit double-stranded rna-dependent protein kinase (pkr) is regulated by reovirus structural proteins influenza virus mrna translation revisited: is the eif e cap-binding factor required for viral mrna translation? a protein that replaces the entire cellular eif f complex characterization of the rna chaperone activity of hantavirus nucleocapsid protein programmed ribosomal frameshifting in hiv- and the sars-cov characterization of an efficient coronavirus ribosomal frameshifting signal-requirement for an rna pseudoknot structure of the rna signal essential for translational frameshifting in hiv- the sequences of and distance between cisacting signals determine the efficiency of ribosomal frameshifting in human-immunodeficiency-virus type- and human t-cell leukemia-virus type-ii in-vivo frameshifting rna pseudoknots: structure and mechanism signals for ribosomal frameshifting in the rous-sarcoma virus gag-pol region a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting mutational analysis of the rna pseudoknot component of a coronavirus ribosomal frameshifting signal mutational analysis of the slippery-sequence component of a coronavirus ribosomal frameshifting signal a crosskingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry presented at the th asia pacific congress of medical virology programmed ribosomal frameshifting in decoding the sars-cov genome a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal rna pseudoknots and the regulation of protein synthesis incorporation of pol into human immunodeficiency virus type gag virus-like particles occurs independently of the upstream gag domain in gag-pol maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type rna dimerization and viral infectivity the human immunodeficiency virus type ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy programmed ribosomal frameshifting is siv is induced by a highly structured rna stem-loop pseudoknots: rna structures with diverse functions solution structure and thermodynamic investigation of the hiv- frameshift inducing element the murine doublestranded rna-dependent protein kinase pkr is required for resistance to vesicular stomatitis virus ribosomal frameshifting: an emerging drug target for hiv effects of intercistronic length on the efficiency of reinitiation by eukaryotic ribosomes ribosomal pausing at a frameshifter rna pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency constraints on reinitiation of translation in mammals ribosomal pausing during translation of an rna pseudoknot what determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? characterization of the sequence element directing translation reinitiation in rna of the calicivirus, rabbit hemorrhagic disease virus translation of the minor capsid protein of a calicivirus is initiated by a novel termination-dependent reinitiation mechanism the importance of inter-and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mrna the mechanism of an exceptional case of reinitiation after translation of a long orf reveals why such events do not generally occur in mammalian mrna translation recycling of eukaryotic posttermination ribosomal complexes reduced incorporation of the influenza b virus bm protein in virus particles decreases infectivity eukaryotic coupled translation of tandem cistronsidentification of the influenza-b virus bm polypeptide characterization of the termination-reinitiation strategy employed in the expression of influenza b virus bm protein translational termination-re-initiation in viral systems animal pneumoviruses: molecular genetics and pathogenesis expression of the orf- protein of the human respiratory syncytial virus m gene is initiated by a ribosomal termination-dependent reinitiation mechanism coupled translation of the respiratory syncytial virus m open reading frames requires upstream sequences coupled translation of the second open reading frame of m mrna is sequence dependent and differs significantly within the subfamily pneumovirinae adenoviruses: update on structure and function spliced segments at terminus of adenovirus late messenger-rna analysis of rna cleavage at the adenovirus- l polyadenylation site translation by ribosome shunting on adenovirus and hsp mrnas facilitated by complementarity to s rrna rna interaction and cleavage of poly(c)-binding protein by hepatitis a virus protease unshackling the links between reovirus oncolysis, ras signaling, translational control and cancer the second open reading frame of the avian reovirus s gene encodes a transcription-dependent and crm -independent nucleocytoplasmic shuttling protein oligomerization and cell-binding properties of the avian reovirus cell-attachment protein sigma c leaky scanning and scanningindependent ribosome migration on the tricistronic s mrna of avian reovirus* sendai virus y proteins are initiated by a ribosomal shunt replication of paramyxoviruses scanning independent ribosomal initiation of the sendai virus-y proteins in vitro and in vivo scanning independent ribosomal initiation of the sendai virus x-protein intracellular processing of the sendai virus c protein leads to the generation of a y protein module: structure-functional implications dissection of a co-translational nascent chain separation event site-specific release of nascent chains from ribosomes at a sense codon molecular cloning and characterisation of the human double-stranded rna-dependent protein kinase induced by interferon essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection autophosphorylation of the protein-kinase dependent on doublestranded-rna sequential modification of translation initiation factor elf gi by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the c(pro) cleavage site latently expressed human herpesvirus -encoded interferon regulatory factor inhibits double-stranded rna-activated protein kinase detection of a novel unglycosylated form of hepatitis c virus e envelope protein that is located in the cytosol and interacts with pkr interaction of the interferon-induced pkr protein kinase with inhibitory proteins p (ipk) and vaccinia virus k l is mediated by unique domains: implications for kinase regulation evidence that hepatitis c virus resistance to interferon is mediated through repression of the pkr protein kinase by the nonstructural a protein comparison of full-length sequences of interferon-sensitive and resistant hepatitis-c virus b-sensitivity to interferon is conferred by amino-acid substitutions in the ns a region mutations in the nonstructural protein a gene and response to interferon in patients with chronic hepatitis c virus b infection mutations within protein kinase r-binding domain of ns a protein of hepatitis c virus (hcv) and specificity of hcv antibodies in pretreatment sera of hcv-chronically infected patients and their effect on the result of treatment evolution of hepatitis c virus non-structural a gene in the progression of liver disease to hepatocellular carcinoma prevalence of wildtype in ns a-pkr protein kinase binding domain in hcv-related hepatocellular carcinoma the oncogenic potential of hepatitis c virus ns a sequence variants is associated with pkr regulation regulation of messenger-rna accumulation by a human-immunodeficiency-virus transactivator protein the integrity of the stem structure of human-immunodeficiency-virus type- tat-responsive sequence rna is required for interaction with the interferon-induced , -mr protein-kinase tat-responsive region rna of human immunodeficiency virus- can prevent activation of the double-stranded-rna-activated protein-kinase control of the interferon-induced -kilodalton protein-kinase by the hiv- tat gene-product the tat protein of human immunodeficiency virus type is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, pkr hiv- tat directly interacts with the interferon-induced, double-stranded rna-dependent kinase phosphorylation of hiv tat by pkr increases interaction with tar rna and enhances transcription recombinant vaccinia virus k l gene-product prevents activation of double-stranded rna-dependent, initiation factor- -alpha-specific protein-kinase distinct patterns of ifn sensitivity observed in cells infected with vaccinia k l(-) and e l(-) mutant viruses comparative-analysis of the regulation of the interferon-inducible protein-kinase pkr by epstein-barr-virus rnas eber- and eber- and adenovirus va(i) rna epstein-barr virus-encoded poly(a)(-) rna confers resistance to apoptosis mediated through fas by blocking the pkr pathway in human epithelial intestine cells adenovirus vai rna complexes with the , mr protein kinase to regulate its autophosphorylation and activity the e l and k l vaccinia virus geneproducts stimulate translation through inhibition of the double-stranded rna-dependent protein-kinase by different mechanisms selective translation initiation by ribosome jumping in adenovirusinfected and heat-shocked cells inhibition of pkr by rna and dna viruses binding of the influenza-virus ns protein to double-stranded-rna inhibits the activation of the protein-kinase that phosphorylates the eif- translation initiation-factor complementation of vaccinia virus deleted of the e l gene by mutants of e l characterization of rna determinants recognized by the arginineand proline-rich region of us , a herpes simplex virus type -encoded double-stranded rna binding protein that prevents pkr activation identification of a lytic-cycle epstein-barr virus gene product that can regulate pkr activation the herpes simplex virus type u(s) protein interacts with protein kinase r in infected cells and requires a -amino-acid sequence adjacent to a kinase substrate domain the cellular , -mr protein-kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection-implications for translational regulation degradation of the interferon-induced , -mr proteinkinase by poliovirus requires rna nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase eukaryotic translation initiation factor f architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells the lmp oncogene of ebv activates perk and the unfolded protein response to chive its own synthesis maintenance of endoplasmic reticulum (er) homeostasis in herpes simplex virus type -infected cells through the association of a viral glycoprotein with perk, a cellular er stress sensor resistance of mrna translation to acute endoplasmic reticulum stress-inducing agents in herpes simplex virus type -infected cells requires multiple virusencoded functions protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e through the eukaryotic initiation factor alpha kinase perk microrna- is an epstein-barr virus-induced gene that modulates epstein-barr virus-regulated gene expression pathways transcriptional and translational control in the mammalian unfolded protein response production of infectious human cytomegalovirus virions is inhibited by drugs that disrupt calcium homeostasis in the endoplasmic reticulum human cytomegalovirus infection activates and regulates the unfolded protein response key: cord- -gxu refq authors: banerjee, nilotpal; mukhopadhyay, sumi title: viral glycoproteins: biological role and application in diagnosis date: - - journal: virusdisease doi: . /s - - - sha: doc_id: cord_uid: gxu refq the viruses that infect humans cause a huge global disease burden and produce immense challenge towards healthcare system. glycoproteins are one of the major components of human pathogenic viruses. they have been demonstrated to have important role(s) in infection and immunity. concomitantly high titres of antibodies against these antigenic viral glycoproteins have paved the way for development of novel diagnostics. availability of appropriate biomarkers is necessary for advance diagnosis of infectious diseases especially in case of outbreaks. as human mobilization has increased manifold nowadays, dissemination of infectious agents became quicker that paves the need of rapid diagnostic system. in case of viral infection it is an emergency as virus spreads and mutates very fast. this review encircles the vast arena of viral glycoproteins, their importance in health and disease and their diagnostic applications. being an obligate intracellular parasite [ ] , virus is the most deadly microbe to be dealt with. globally it accounts for extremely high morbidity and mortality throughout the age groups of people [ , ] . thousands of new viral strains are discovered till date affecting people producing a huge global burden of viral infections resulting immense challenge towards healthcare system [ , , ] . with the capability of fast mutation, viruses affect the host cells with new and newer mechanisms. so to detect them at the earliest, there is an extreme need of dynamic diagnostic system. glycans are major components of the outermost surface of viruses. thus, majority of the interactions of viral pathogens with their hosts are influenced by the pattern of glycans and glycan-binding receptors that each expresses. [ , , ] glycans are most complex biomolecules due to extensive branching of carbohydrates, and a variety of glycoproteins have been identified in human viral pathogens. these pathogenic glycans either virus encoded or host derived usually elicit high humoral responses in human body [ ] . these virus specific high levels of glycan specific antibodies have been exploited to develop novel diagnostic assays. viral diagnostic tests can be broadly classified into three categories in general. those are direct detection, indirect examination (virus isolation), and by serology. in case of direct detection, the clinical sample is examined directly to identify any presence of virus particles, virus antigen or viral nucleic acids. in case of indirect examination, the sample has to be added into cell culture, eggs or animals to grow the virus in vitro. this is known as virus isolation. serology always constitutes the bulk of the work of any virology laboratory, especially in overpopulated third world countries. serological diagnosis is generally made by detecting titres of antibody in infection [ ] . generally, the majority of common viral infections are diagnosed by serology [ ] . viruses can be directly detected through electron microscopy. it can also be enumerated by molecular biological techniques like pcr/rtpcr by detecting viral genomes. these techniques are extremely useful but are technically demanding, costly and require skilled personnel. on the other hand, indirect detection by virus isolation is dependent on cell culture techniques. the major problem of cell culture is it takes a long time (up to weeks). also, the sensitivity is poor and depends on many factors, such as the specimen condition and the condition of the cell line. cell cultures are also very susceptible to microbial contamination and toxins present in the specimen. also, many viruses do not grow at all in cell culture e.g. hepatitis b and c, viruses causing diarrhea, parvovirus etc. serology is the mainstream of viral diagnosis [ , ] . with increase in the growth of sophisticated immunoassay techniques, effective viral immunodiagnostic assays are now available in the market [ , , ] . the detection of structural glycoproteins of viruses or early glycoprotein antigen formation in the host due to viral infection or the quantification of titres of antibodies against viral antigenic glycoprotein is an emerging discipline in viral immunodiagnostics [ ] . the detection of these structural glycoproteins of viruses is done by lectins or monoclonal antibodies acting as probe or by measuring the titres of host antibodies against antigenic glycoprotein. there are several good review works on viral glycoproteins. namely, the work of kazuya i.p.j. hidari and takashi suzuki on glycan receptor in influenza virus [ ] . yuan et al. [ ] worked on receptor glycoprotein interaction in zaire ebola virus (zev). this review attempts to conglomerate the importance of glycoprotein in widely studied viral infection and their application in diagnosis. a fully assembled infectious virus is known as virion. the simplest virions consist of two basic components, namely nucleic acid (single-or double-stranded rna or dna) and a capsid, which is a protein coat, functions as a shell to protect the viral genome from nucleases. this capsid comes into play during infection to attach the virion to specific receptors exposed on the prospective host cell. capsid proteins are coded by the viral genome. due to its limited size, the genome codes for only a few structural proteins (besides non-structural regulatory proteins involved in virus replication). capsids are formed as single or double protein shells and consist of only one or a few structural protein species. therefore, multiple protein copies must self assemble to form the continuous three-dimensional capsid structure [ ] . the structural viral proteins are extremely important to the virus, so as to facilitate the transfer of the viral nucleic acid from one host cell to another. the proteins determine the antigenicity of the virus. host's primary immune response is directed against the antigenic determinants of these proteins rather glycoprotein in major cases. there are enveloped viruses and these envelopes are made up of either lipid or glycoprotein. viral envelopes mainly consist of envelope proteins (e), membrane proteins (m) and spike proteins (s) [ ] . lipid envelopes are derived from the host cell. whereas the envelope glycoproteins are virus encoded. however, there are sugars attached to the viral glycoproteins which often reflect the host cell that harboured the virus. the surface glycoproteins of an enveloped virus attach the virion to a target host cell by properly interacting with a cellular receptor [ ] . structural biological analysis of viral envelope glycoproteins reveals that viruses have wide range of folds to facilitate their attachment with proper host receptors. bowden et al. [ ] stated that arenaviridae group of viruses have a/b fold, whereas filoviridae possess 'chalice' of gp . similarly, paramyxoviridae shows six bladed b propeller and large trimeric haemaglutinin is shown by orthomyxoviridae. glycosylated gp trimer is observed in the lentiviruses of retroviridae. viruses exhibit 'semaphorins' which are family of cell surface signalling glycoproteins [ ] . these semaphorins binds with cell surface receptors to initiate important physiological processes. these observations are made by recent study of viral glycoproteins by employing macromolecular crystallography [ ] . the m and s proteins of the virus are usually rich in n glycosylated proteins, which have been demonstrated as important virulent factor of viruses [ ] . thus, e, m and s viral glycoproteins are involved in viral host binding and subsequent virus-host membrane fusion to establish the pathogenesis of the virus. two envelope glycoproteins, namely e and e develop the viral spike of the virions of flaviviridae family [ ] are involved in the engagement with host receptor and conformational change required for membrane fusion (fig. c) . studies show that e can express independently but e is dependent upon e in case of hcv. sars coronavirus possess a spike(s) glycoprotein [ ] , which itself performs the membrane fusion for the entry of the virion and its fusion with host cell [ ] . in case of chikungunya virus, attachment is facilitated by the e glycoprotein [ ] and fusion mainly by the e glycoprotein, thus both the processes are mutually exclusive, whereas in dengue virus, it is carried by the same e protein (http://www.uniprot.org/ uniprot/q jux ). interestingly, the dengue virus apart from synthesizing the basic capsid, membrane and envelope proteins also produces seven non-structural secretory glycoproteins ns , a, b, , a, b, [ ] . these proteins are not integrated in the virus but secreted in the host. studies have found heterogeneity in the e glycoprotein of dengue virus [ ] . five different glycans are present in this glycoconjugate including mannose, galnac and glcnac, fucose and sialic acid. b cell and t-cell epitopes are predicted in a study by analysing this e glycoprotein [ ] . the dengue viral envelope is more ordered than the inner viral core, as the envelope is composed of glycoprotein e dimer icosahedral scaffold [ ] . computational studies are there to develop vaccines against dengue virus [ , ] . there are three glycoproteins present in hiv [ ] ; namely gp , gp and gp [ ] . all these are encoded by the env gene [ ] . the hiv envelope glycoprotein gp contains nine disulphide bridges and is highly glycosylated, carrying on average n-linked glycans ( fig. d ) [ ] . experiments proved that the glycan part of the gp protein has important role in the efficient intracellular transport of another glycoprotein gp . those gp proteins lack gp are arrested in golgi complex after their biosynthesis [ ] . zaire ebola virus is the member of filoviridae group, and the glycoproteins (gp) have found to be major pathogenic determinants [ - , , , ] . in the ebola virion gp gene is the th gene among total seven genes in the linear gene order. this synthesizes several proteins. among them two are predominant. those are sgp and d-peptide (delta peptide). these two proteins are produced due to a furin cleavage of a precursor pre-sgp protein. the gp is actually a spike protein which is composed of two subunits joined by disulphide linkage gp -gp [ ] . the chikungunya virus on the other hand are known to produce non structural glycoproteins (nsp - ) [ ] these nsps have been demonstrated to have important role in keeping the replicase complex of the virus intact in the host as well as to circumvent important host immune responses. chikungunya virus has two envelope proteins, namely e and e [ ] . thus, viral glycoproteins have diverse structure and function. taken together, glycoproteins are important components of the virus structure and each have unique role to establish pathogenesis. [ ] . viral glycoproteins have a definite role in their pathogenesis. the primary goal of viral infection is to identify a receptor on the host cell surface and binding with it. subsequently this will pave the way of viral entry into the host cell. in most cases, the first attachment site of the virus is a glycan, either a glycoprotein or a glycolipid. so, glycoproteins play a crucial role in viral pathogenesis. the study of glycoproteins in viral infection is most important to know the disease process as well as to develop antiviral treatments. glycoprotein-receptor interactions also play important roles in pathogen pattern recognition and in the regulatory signals that control the activities of cells of the immune system. the most important cause behind viral infection is that it has evolved to present its own sugars and receptors in a manner that mimics or interferes with host glycan-based immune functions. glycomic studies are ongoing in several viruses. several advanced technologies are there to decipher structural and functional aspects of glycans like glycan microarray [ ] , mass spectrometry and nano lc. glycan array represents the actual in vivo interaction in silico. the arrayed multivalent demonstration of polysaccharides mimics the cell surface display. there are two types of carbohydrate microarray. those are polysaccharide and oligosaccharide microarray [ ] . natural polysaccharides are randomly immobilized on solid matrices exploiting hydrophobic physical absorption or charge-based interaction. polysaccharide microarrays are useful for comparative antigenicity analyses. being hydrophilic in nature, oligosaccharides need chemical derivatization before arraying. through oligosaccharide microarray we can study structure-activity relationships [ ] . microarrays were developed on maleimide-functionalized surfaces using seven thiol-containing synthetic highmannose oligosaccharides for the identification of human immunodeficiency virus (hiv) vaccine candidate antigens [ ] . the binding profile reveals that several proteins which interact with gp of hiv, like the receptor of the innate immune system known as dc-sign (cd ). in case of influenza glycans, there are protocols for fluorescent labeling of virus, coupling of virus to a glycan microarray, analysis of a glycan microarray slide experiment, and data interpretation. studies have shown that there are a , -linked sialic acid motif (sa , gal) in avian, equine, and canine species. whereas a , -linked sialic acid motif (sa , gal) is present in humans. saa , gal and saa , gal are present in swine, these are causing corresponding host tropism. zhao et al. [ ] showed that, association mining results of glycan microarray [ ] data with influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds [ ] . the study suggest that besides neu aca - galb, human-origin viruses could bind glycans with neu aca - neu aca - neu ac and neu gca - galb - glcnac substructures; galb and glcnacb terminal substructures, without sialic acid branches these were linked with the binding of human, swine, and avian origin viruses. sulfated neu aca - substructures were associated with the binding of human-and swine-origin viruses. finally, through three-dimensional structure characterization, it has been revealed that the role of glycan chain shapes is more important than that of torsion angles [ ] . though characterization of glycoproteins is tough but, through mass spectrometry, it is now easier to identify structural details of complex glycoproteins. mass spectrometry derived glycoproteomics [ ] helps us to precisely identify viral and cellular proteins that are functionally, structurally, and dynamically altered during virus infection, but enables us to identify important proteins having active role in the infection pathway. additionally, isolation and purification techniques along with quantitative strategies in conjunction with ms significantly improve its sensitivity to detect low-abundant proteins. with time, more virus and host genomes are being sequenced and ms-based glycoproteomics is becoming a very important tool for virology. a work by barrientos et al. [ ] revealed that post translational modification of secretory glycoprotein of zaire ebola virus can be characterized by mass spectrometry. maldi-tof ms (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry) also enables to identify regions susceptible to limited proteolysis in sgp of zev. another work by anastassia et al. [ ] shows that . microgram of viral glycoprotein can be purified by nano-liquid chromatography. after nano-lc the sample is analysed through mass spectrometry. one more work showed that they purified heat shock protein by nanolc-ms of respiratory syncitial virus which have important role in virus particle assembly [ , ] . so it is evident that using these modern techniques, the biological roles of glycoproteins can be studied more conveniently. virus is a nucleic acid surrounded by proteins. this infective particle is called a virion. in most cases this virion is covered with a fascinating coat composed of glycoproteins through which the virus communicates with its host. the co-evolution of host and virus leads the way of making the glycoprotein coat so fascinating [ ] . it is evident that the infectivity of a virus rather of its nucleic acid is fully dependent on its glycoproteins. enveloped viruses generally encode membrane proteins and these special proteins are necessary to mediate the specific binding against host cell ligands. this also directs initial events of membrane fusion and viral internalization. these fascinating envelope proteins are generally glycosylated [ ] . the process of glycosylation takes place in the endoplasmic reticulum (er)-golgi complex secretory pathway. the host cell encoded glycosyl-transferase enzyme catalyses the glycosylation. this glycosylation is necessary to make the virion host compatible, which is needed by the virus for its pathogenesis. so glycans present at the envelop proteins acts as immunological barriers to resist evasion by the host immune system [ ] . several viruses exhibit different glycoproteins on their surface (table ). hepatitis c virus has two envelope glycoproteins namely e and e [ , ] . these two proteins play an important role in viral infectivity and can be used as candidate subunit vaccine [ ] . on the other hand, in case of ebola virus there are glycoproteins gp and gp which causes cell attachment and cell fusion and therefore the main target of the host antibodies. the ebola virus has an rna editing mechanism to regulate these gp and genes which when expressed at high level, disrupts normal cell physiology [ ] . in case of hiv- , the gp- protein initiates viral entry to the cd cells [ ] . recent studies proved that several types of glycans in hiv- produce different levels in infectivity. those viruses have more oligomannose and less structural complexity, infects more efficiently. this ultimately proves that mature oligosaccharide structure of the envelope glycans play a pivotal role in the infection process of hiv- . due to n-linked glycosylation of gp- protein in the endoplasmic reticulum, and further folding and cleaving in the golgi complex; the gp- protein of the envelope is produced [ ] . there are two popular glycoproteins present on the surface of influenza virus namely, haemaglutinin (ha) and neuraminidase (na) [ , ] . these are the key molecules for the viral infection which binds with sialic acid. initially, ha binds with sialic acid during the initiation of infection. after viral replication na degrades its substrate sialic acid to accelerate release of new viruses [ ] . so it is evident from the examples of viruses of diverse family that glycoproteins are the key molecules for a virus to establish an infection within the host and to survive further within the host system. in the study of viral pathogenesis, a special type of glycoprotein, called semaphorin have been established [ ] . semaphorins are family of cell surface signaling glycoproteins which binds to the family of plexin glycoprotein cell surface receptors. semaphorins also activate repulsive guidance pathways having active part in axon guidance, immune regulation and activation, and vascular development [ , ] . semaphorins have eight known classes. among them two are found in invertebrates, five in vertebrates, and the eighth class in viruses which are known as 'viral semaphorins' [ ] . the ectodomains of cellular semaphorins contain c-terminal domain elaborations like psi (plexin, semaphorin and integrin) domains, immunoglobulin (ig)-like domains, thrombospondin domains and pdz-domain-binding sites which occassionally attach to the cell-surface. whereas the n-terminal having a plexin-binding sema-domain, is conserved in all cases of virus host cell attachment. the sema-domain is the [ , ] fusion with host cell membrane sialic acid and attachment [ ] - million cases worldwide [ , ] sars-cov spike(s) glycoprotein [ , ] membrane fusion [ ] within the duration of st november to th august occurring worldwide [ , ] hepatitis c virus e and e [ , ] binding to host receptor and conformational change necessary for membrane fusion [ ] to million people globally [ , ] human immunodeficiency virus gp , gp , gp [ ] intracellular transport [ ] million globally up to [ , , , ] zaire ebola virus spike protein gp -gp [ ] primary host cell activation [ ] up to th june total , cases [ , , ] dengue virus e (dimer) [ ] host cell fusion and attachment [ ] who reported recently that there are million dengue infections per year globally [ ] . presently dengue is endemic in countries [ ] . chikungunya virus e and e [ , ] host cell binding according to who, this disease occurs mainly in africa, asia and indian sub-continent [ ] . [ ] have revealed that human sema a and mouse sema d semadomains comprises of structurally conserved homodimer of seven-bladed b-propellers [ , , , , ] . the immune-regulatory semaphorins like sema a, a, d, and a helps in b cell mediated immunity (sema d), t cell activation as well as differentiation (sema a, sema a, and sema d), and inflammation (sema a) [ ] . these semaphorins provide a molecular basis for how viruses can optimize their own proteins to override normal physiological interactions. a work by shirato h as 'norovirus and histo-blood group antigens' in the journal jpn j infect dis. ( ; ( ): - ) describes that norovirus (nov) causes viral gastroenteritis and interestingly bind to histoblood group antigens (hbgas), like abh antigens and lewis antigens. it has been shown epidemiologically that persons with different abh phenotypes are infected with nov strains in a genotype-dependant fashion. an in vitro binding assay using nov virus-like particles (vlps) showed a uniform recognition pattern for type and core structures of histo blood group antigens. nov vlps bind more tightly to type carbohydrates than to type . type carbohydrates are found to be expressed at the surface of the small intestine and targeted by nov. this property speaks about nov tissue specificity. so it is evident that glycoproteins perform a major and active role in viral pathogenesis and disease progression. glycoproteins provide tissue tropism to the virus. some virues used to infect the respiratory system whereas some affects the liver. the cause is the type of glycoprotein with which the virus binds to accelerate its invasion. in a study by raska et al. [ ] , it has been proved that there are differential glycosylation in viruses like hiv depending upon the cells which produce the virus. n-glycosylation of reconbinant gp of hiv is varied and affected the recognition by serum antibodies. glycosylation of gp protein of hiv affects its recognition by neutralizing and non neutralizing monoclonal antibodies. this study also says that this glycosylation is cell specific. another study by lin et al. [ ] stated that there are c-type lectins expressed on the dendritic cell surface known as dc-sign(dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin) or cd and dc-signr which binds to hiv and transmit to t cells through the viral envelope env glycoprotein. but interestingly other highly glycosylated viruses failed to interact with dc-signr [ ] . lin et al. showed that dc-sign (r) or cd selectively binds with hiv env and zaire ebola virus glycoproteins containing more highmannose. by modulating n-glycans on env or glycoprotein during virus production in different primary cells or in the presence of the mannosidase i inhibitor deoxymannojirimycin affected dc-sign(r) infectivity enhancement. they also predict that viruses containing glycoproteins with a high amount of high-mannose n-glycans effectively interact with dc-sign(r), but those viruses having only complex n-glycans cannot effectively react with dc-sign(r). so it is evident that virus-producing cell type is a crucial factor in depicting both n-glycan status and virus interactions with dc-sign(r), which establishes virus tropism and infection within the human body [ ] . liu et al. [ ] described in their study that sialic acid present on cell surface is essential for human enterovirus d (ev-d ) entry. crystallographic studies showed that ev-d with sialylated glycan receptor analogues binds on the viral surface. sialic acid receptor induces a cascade of conformational changes within the virus to secrete a fattyacid-like molecule which regulates the stability of the virus. so, it is evident that binding of virus to a sialic acid receptor and to immunoglobulin-like receptors facilitates viral entry in enteroviruses. glycan based viral immunodiagnostics usually have high sensitivity and specificity. glycoprotein based igm serology was developed for the diagnosis of recent primary rubella virus infections and significant sensitivity and specificity was obtained. similarly, glycoprotein based serology tests to detect antibodies to herpes simplex virus glycoproteins g- and g- , which evoke a type-specific antibody response have also been developed. these tests are used to confirm a diagnosis of genital herpes, and also to establish diagnosis of hsv infection in patients with atypical complaints, to identify asymptomatic carriers, and identify persons at risk for acquiring hsv. glycan based immunodiagnostics have also been developed for the rapid identification of different strains of influenza virus. a novel peptide based elisa which has sensitivity and specificity of . and . % respectively is very promising. on the other hand, the main diagnostic challenge related to sars is to diagnose it differently with atypical pneumonia [ , ] . the key diagnostic tools are immunofluorescent staining, elisa and rt-pcr [ ] . but all these techniques are extremely sophisticated and of little use in case of epidemics; especially in the developing world. sars coronavirus possess a spike(s) glycoprotein, which itself performs the membrane fusion for the entry of the virion and its fusion with host cell (fig. b) [ ] . igg based diagnostics against this s protein has been developed [ ] . indirect elisa test has been developed by using recombinant sars 's' protein and the n (nucleoprotein) protein. the sensitivity of sars 's' and 'n' proteins are and . % respectively whereas the specificity of sars 's' and 'n' are . and . % [ ] . similarly, as acute hepatitis c infection is asymptomatic, so it is difficult to diagnose early. generally patients come to the clinic with damaged liver. initially patients are screened by anti-hcv antibody test and further confirmed by testing viral rna. there are two glycoproteins e and e in hcv. standardised pcr system is available in hcv diagnostics but a core antigen test is also in the market. in tanaka et al. [ ] suggested that the hcv core proteins can be used as antigens for the chronic stage. studies for developing algorithms confirms . % sensitivity in case of rt-pcr whereas . % for the core antigen test [ ] . another novel glycoproteomic serum biomarker has been identified which can diagnose hcv along with progressive liver cirrhosis is wisteria floribunda agglutinin positive mac- -binding protein (wfa?-m bp) [ ] . the diagnostic threshold for cut-off index values of this protein is . and . in hcv negative and hcv positive patients showing progressive liver cirrhosis [ ] . in case of hiv , elisa and pcr are two gold standard tests. type specific conformational epitopes of the gp and gp glycoproteins of the hiv envelope are used for the recognition of 'early hiv antibodies' [ ] . enzyme immuno assay (eia) and rt-pcr are used in these assays. these tests are very specific but they fail to diagnose early infection [ ] . the most challenging part in hiv diagnosis is to diagnose the acute stage and differentiate ''window phase'' patients from the serologically positive patients [ ] . popular serological tests fail to diagnose all patients of hiv as they exploit the gag proteins which literally decrease after disease progression [ , ] . there are three glycoproteins present in hiv; namely gp , gp and gp . all these are encoded by the env gene [ ] . the hiv envelope glycoprotein gp contains nine disulphide bridges and is highly glycosylated, carrying on average n-linked glycans (fig. d) [ ] . experiments proved that the glycan part of the gp protein has important role in the efficient intracellular transport of another glycoprotein gp . those gp proteins lack gp are arrested in golgi complex after their biosynthesis [ ] . as stated above, there are limitations of the popular gag antigen based serological tests which cannot diagnose hiv patients of different clinical stage. but antibodies against precursor gp env protein and final env proteins gp and gp can detect all clinical stages of hiv [ ] . the sensitivity of current available diagnostic system is % at \ days, % at - days and % at - days [ ] the specificity is almost % [ ] . in case of zaire ebola virus, initially there was controversy about the role of glycoproteins in the pathogenesis of ebv. but later on, scientific researches proved that the primary host cell activation by the ebv is mediated by gp - [ ] . an antigen capture elisa has been developed in zaire ebov using mabs [ ] . it has been reported that these tests have both high sensitivity and specificity [ ] . similarly, dengue (denv) ns is a highly conserved glycoprotein, expressed as both membrane-associated and secreted forms [ , , ] . secreted ns has been detected ranging from - . lg/ml in the serum of dengue-infected patients during the early stages of the disease. a high ns level has been demonstrated to circulate as early as day after onset of symptoms up to early convalescences thus provides an alternative to virus culture or pcr for early dengue diagnosis when igm or igg antibodies are not present yet in dengue infected patients [ , ] . circulating dengue ns in sera can be detected either using elisa assay or lateral flow based rdts [ ] . thus, glycan based viral immunodiagnostics or glyco-immunodiagnostics are helpful in early diagnosis of patients with viral infection [ ] . current diagnosis scenario in chikungunya is igm and igg based elisa and nucleic acid detection by rt-pcr [ , ] . but there is no antigen based elisa. this makes the condition crucial as the primary health care providers in the virus affected countries do not have rt-pcr facilities. it is not recommended to maintain rt-pcr facilities in primary health care centre by policy. it is extremely costly and demands expertise. it is not possible to provide such facility in the densely populated tropical countries. as chikungunya causes short duration fever, often the patients are not diagnosed properly. the joint pain generally persists for some days but can be present for a year [ ] . there is a study which shows even multi organ failure in chikungunya infected patients [ ] . chikv has two envelope glycoproteins, namely e and e (fig. g) [ ] . recombinant chikv e and e glycoprotein based elisa showed a sensitivity of . and % respectively whereas the specificity for both cases was % [ , ] highlighting the potential for these two glycoproteins in the diagnosis. viral glycoproteins are integral parts of enveloped viruses and they actively take part in their pathogenesis. exploting glycoproteins, viruses enter into their host and combat with host immune system. recent advances in technology deciphers different role of glycoproteins which are dependent on their structures. different viruses have different mode of pathogenesis and glycoproteins directly takes part in the host binding and entry. during maturation from host cell viruses have viral glycoproteins: biological role and application in diagnosis host glycoproteins on their surface to avoid the immunity of the host. so, to detect viruses and to decide for developing vaccines [ ] , glycoproteins always play a key role. antibody production is a prominent feature of the immune response in patients with viral infection, and particular isotypes correlate with resistance or susceptibility to infection [ ] . a substantial proportion of the antibodies detected in patients with acute or chronic infections is directed against viral glycan epitopes. as levels of anti glycan antibody are high and specific for each viral infection this permits diagnostic discrimination between the different viral infections. taken together, viral glycoproteins have important functions in pathogenesis and can be exploited to develop viral diagnostics. oligosaccharide and glycoprotein microarrays as tools in hiv glycobiology; glycan-dependent gp / protein interactions comparative dynamics, morbidity and mortality burden of pediatric viral respiratory infections in an equatorial 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large-scale glycan microarray data reveal novel hostspecific substructures in influenza a virus binding glycans current approaches on viral infection: proteomics and functional validations viral glycoproteins: biological role and application in diagnosis key: cord- -xigv u f authors: benotmane, i.; gautier-vargas, g.; wendling, m.-j.; perrin, p.; velay, a.; bassand, x.; bedo, d.; baldacini, c.; sagnard, m.; bozman, d.-f.; della-chiesa, m.; solis, m.; gallais, f.; cognard, n.; olagne, j.; delagreverie, h.; gontard, l.; panaget, b.; marx, d.; heibel, f.; braun-parvez, l.; moulin, b.; caillard, s.; fafi-kremer, s. title: in-depth virological assessment of kidney transplant recipients with covid- date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: xigv u f severe acute respiratory syndrome coronavirus (sars-cov- ) has spread widely, causing coronavirus disease (covid- ) and significant mortality. however, data on viral loads and antibody kinetics in immunocompromised populations are lacking. we aimed to determine nasopharyngeal and plasma viral loads via rt-pcr and sars-cov- serology via elisa and study their association with severe forms of covid- and death in kidney transplant recipients. in this study we examined hospitalized kidney transplant recipients with non-severe (n = ) and severe (n = ) covid- . sars-cov- nasopharyngeal and plasma viral load and serological response were evaluated based on outcomes and disease severity. ten recipients ( %) displayed persistent viral shedding days after symptom onset. the sars-cov- viral load of the upper respiratory tract was not associated with severe covid- , whereas the plasma viral load was associated with covid- severity (p= . ) and mortality (p= . ). all patients harbored antibodies the second week after symptom onset that persisted for two months. we conclude that plasma viral load is associated with covid- morbidity and mortality, whereas nasopharyngeal viral load is not. sars-cov- shedding is prolonged in kidney transplant recipients and the humoral response to sars-cov- does not show significant impairment in this series of transplant recipients. in december , severe acute respiratory syndrome coronavirus (sars-cov- ) emerged in china, and it has since spread widely across the world. the resulting coronavirus disease (covid- ) has led to a high death toll. scientific knowledge on sars-cov- has evolved rapidly since the outbreak, but little is known about responses to the virus in immunocompromised populations. infection with respiratory viruses has been shown to be particularly concerning in transplant recipients due to prolonged viral shedding and a higher risk of complications. , however, there have been no reports indicating whether sars-cov- infection presents the same risks for transplant recipients as other respiratory viruses. determining viral loads and antibody kinetics in immunocompromised individuals is necessary to protect this highly vulnerable population. because prolonged viral shedding and/or a lack of protective immunity could lead to significant viral spread in patients' environments, protective measures may need to be increased. we thus conducted a retrospective cohort study in kidney transplant recipients (ktr) in alsace, grand-est france, to determine the dynamics of nasopharyngeal and plasma viral loads and sars-cov- serology and to study their association with mortality and severe forms of covid- . continuous data are presented as medians and interquartile ranges (iqr) and were analyzed using the non-parametric mann-whitney u test. categorical variables are expressed as counts and percentages and were compared using the fisher's exact test. the associations between maximum nasopharyngeal sars-cov- viral load and clinical, demographic, and laboratory variables were determined using spearman's correlation coefficient (ρ) values. receiver operating characteristic (roc) curves were generated to investigate the viral loads in the upper respiratory tract and plasma with respect to disease severity and mortality. survival plots of severe covid- -free survival and covid- -specific survival were graphically represented with kaplan-meier curves according to rnaaemia (i.e., positive plasma viral load), using the log-rank test to compare differences in survival. severe covid- was defined as one or more of the following: an oxygen requirement of > l/min, the need for icu admission, and patient death. patients were censored at the time of last follow-up. statistical analyses were performed using graphpad prism . (graphpad inc., san diego, ca, usa). a p value < . (two-tailed) was considered statistically significant. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint (mpa) was being taken by patients ( %), mtor inhibitors were being administered to ( %) patients, and steroids were taken by ( . %) patients. the median interval between the onset of symptoms and covid- diagnosis was days (iqr: − days). all but two patients had a fever. respiratory (n= , %) and gastrointestinal (n= , . %) symptoms were the most common clinical manifestations. twenty-one patients had a non-severe clinical presentation, whereas had a severe clinical course. during follow-up, the mortality rate was found to be . % ( / ). a total of upper respiratory specimens were analyzed (median of three swabs per patient [iqr: − swabs]). the median viral load was . log copies/reaction (iqr: . − . ) at diagnosis (between day and day after symptom onset). a total of patients ( . %) had their peak viral load at admission, and two patients hospitalized at day and day after symptom onset had negative rt-pcr results at admission and during the follow-up. the viral load at admission was not statistically different between severe and non-severe patients ( . log copies/reaction versus . log copies/reaction, respectively p= . ) and was not predictive of disease severity (area under the roc curve = . , p= . ) at admission, at the peak of viral load (figure a and b) , or during the course of the disease ( figure ). recipient age (ρ= . , p= . , figure ) and sex (p= . ) were marginally associated with the viral load. notably, patients receiving steroid therapy and not presenting gastrointestinal symptoms displayed a higher viral load ( table ) . no correlation was evident between maximum viral load and inflammatory markers, including interleukin (il)- (ρ= . , p= . ) and c-reactive protein (crp, ρ= . , p= . ). among patients with an initial positive rt-pcr test result (n= ), no patient showed a viral clearance before d . fifteen ( . %) patients displayed a all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint positive viral load greater than log copies/reaction after d , and ten patients ( . %) showed persistent viral shedding after d ( figure ). sars-cov- loads were measured in plasma samples obtained from patients ( in the non-severe group and in the severe group). plasma viral loads ranged from to . log copies/reaction. ten patients had at least one positive rnaaemia. severe patients showed a higher frequency of rnaaemia compared with non-severe patients ( % versus . %, respectively, p= . , figure a ). moreover, rnaaemia was found to be associated with mortality ( figure b ). accordingly, rnaaemia was positive in all three non-survivors tested; in contrast, only of ( %) tested survivors had positive rnaaemia (p= . ). furthermore, two non-survivors harbored high viral loads ( . log copies/reaction and . log copies /reaction), whereas other patients were characterized by low rnaaemia (< . log copies/reaction). with regard to immunosuppressive therapy, patients receiving cni tended to have more positive rnaaemia ( / versus / , respectively, p= . ). a total of samples from patients were analyzed, with a median of three sera tested per patient (iqr: − sera). all survivors were seropositive at follow-up. four non-survivors had negative serology at their time of death, which occurred on d , d , d , and d . the two patients with a negative sars-cov- rt-pcr result had positive serology, with one patient showing positive serology at the time of diagnosis. among the samples tested before d , six ( %) were seropositive, whereas all samples tested after d were seropositive ( figure ). the kinetics of the antibodies showed that a stable titer of igg antibodies was maintained until d , suggesting persistence of immunity for at least until two months after infection all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint ( figure ). notably, igm and igg antibody level and delays in seroconversion were not correlated with covid- severity (figure a and b). in this retrospective study conducted in a sample of immunocompromised ktr hospitalized for covid- , we precisely determined the temporal evolution of nasopharyngeal and plasma sars-cov- loads, as well as the serological response to the virus. all parameters were correlated with patient characteristics, disease severity, and clinical outcomes. in our study, the viral load in the respiratory specimens of most patients was at the peak at the time of diagnosis. this finding is in line with those previously reported for the general population. - based on the data we analyzed, the viral load at the time of diagnosis did not predict the severity of the disease. moreover, viral loads were not related to inflammatory markers that have been previously associated with covid- severity. reports on the relationship between viral load and disease severity are contradictory; three studies showed no correlation between the severity of the disease and the viral load in respiratory specimens, , , whereas liu et al. described a higher viral load in patients with more severe disease. in our immunocompromised population with a median follow-up of days, the duration of viral detection in the respiratory tract was longer compared to that in the general population. more than a third of our patients displayed a high viral load after d . furthermore, almost a quarter of them still had viral shedding at d ; in contrast, in immunocompetent populations, the median duration of viral shedding was days. thus, even if a positive rt-pcr result does not indicate an infectious virus, we should be cautious about viral spread in vulnerable populations and extend isolation after a sars-cov- infection. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint fourth week after symptom onset, which is concordant with our findings. these results suggest that the anti-sars-cov- humoral response is not significantly impaired in our immunocompromised population. , , notably, the delay after transplantation was long in our cohort, and only one patient had undergone depleting induction therapy during the year preceding covid- . the level of igg remained steady until two months after onset of symptoms. although the neutralizing effect of the antibodies was not studied here, it is encouraging that igg levels are correlated with a neutralizing effect in the general population. , despite some limitations of this study, including the small sample size and lack of data from some patients, this is the first report that provides a precise assessment of sars-cov- virological and antibody response kinetics in an immunocompromised population, with a follow-up for two months after symptom onset. taken together, our data indicate that ) (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. nasopharyngeal maximal viral load are presented as median (interquartile range) abbreviations: bmi, body mass index; raas, renin-angiotensin-aldosterone system; mmf, mycophenolate mofetil; mpa, mycophenolic acid, mtor: mammalian target of rapamycin. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. non-severe disease all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint a novel coronavirus from patients with pneumonia in china rna respiratory viral infections in solid organ transplant recipients: guidelines from the american society of transplantation infectious diseases community of practice aspergillosis in critically ill patients world health organization. molecular assays to diagnose covid- : summary table of available protocols virological assessment of hospitalized patients with covid- temporal profiles of viral load in posterior detectable serum sars-cov- viral load (rnaaemia) is closely correlated with drastically elevated interleukin (il- ) level in critically ill covid- patients impaired type i interferon activity and exacerbated inflammatory responses in severe covid- patients. medrxiv viral dynamics in mild and severe cases of covid- clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study. the lancet clinical features of patients infected with novel coronavirus in wuhan, china. the lancet viral load dynamics and disease severity in patients infected with sars-cov- in zhejiang province, china key: cord- - hxau vt authors: richard, a; tulasne, d title: caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis date: - - journal: cell death dis doi: . /cddis. . sha: doc_id: cord_uid: hxau vt viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. on one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. on the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. the existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. the present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host–apoptosis–virus triangle. when a viral infection threatens cells, one of the first measures they take is to induce apoptosis to restrict viral replication and spread. [ ] [ ] [ ] dying this way, host cells are likely to generate specific signals aiming at triggering the immune system with innate and/or adaptative responses allowing the eradication of the invader. , on the other hand, viruses have evolved a huge arsenal of strategies meant to either counteract or deal with this destructive process to ensure their survival. [ ] [ ] [ ] apoptotic cell death is accompanied by characteristic morphological changes (cellular rounding-up and volume reduction, chromatin condensation, nuclear fragmentation, plasma membrane blebbingy) and at a molecular level by the activation of first initiator and then effector cysteinyl aspartate proteinases or caspases. , activated caspases act through a catalytic cys that hydrolyzes peptide bonds within the substrate, with a stringent specificity for asp residue at p position (the nature of residues at positions p , p and p depending on the caspase). caspase substrates include a large number and variety of cellular proteins that participate through their cleavage to the strong apoptosisrelated morphological changes, as well as other physiological processes. interestingly, viral proteins are also likely to be cleaved by caspases but until , only four of them were reported as such and suggested as advantaging the associated viruses through their cleavage. since, their number has greatly increased and at least viruses are now known to express proteins that undergo caspase cleavage. here, we aim at updating these cleavages and discussing their biological relevance. from apoptosis inhibition to the improvement or attenuation of viral amplification, caspase cleavage of vps highlights a potential new fascinating viral strategy to handle apoptosis induction. apoptosis induction by infected cells is meant to jeopardize viral replication. therefore, many viruses have developed strategies to inhibit this process through various mechanisms. among them, inhibiting caspase activity through the cleavage of viral proteins constitutes an effective way to achieve apoptosis suppression (table ) . baculoviruses are invertebrate viruses that encode several antiapoptotic products including iaps, p and p . when lacking p , the baculovirus prototype autographa californica multicapsid nucleopolyhedrovirus (acmnpv) induces apoptosis and fails to replicate, whereas p rescue inhibits cell death and restores viral replication. interestingly, p is proteolytically cleaved on infection , and the dqmd k cleavage site is required for p -mediated apoptosis suppression. p directly inhibits many caspases, including insect sf-caspase , human caspases , , , , and and mouse caspase . , indeed, p cleavage products remain irreversibly associated to the caspase through a covalent thioester bond between p d residue and the caspase catalytic cysteine. , although being %-related to p , p yet exhibits a different caspase cleavage site ( tvtd k) that confers to p its antiapoptotic properties. caspase suppression by p also involves its cleavage by and stable association to the targeted caspases although acting as a dimer. besides effector caspases, p also affects initiator caspases that p fails to suppress, like insect sf-caspase x and human initiator caspase . rather similarly, the product of the orf gene expressed by the crustacean-infecting white spot syndrome virus (wssv) was also shown to exert antiapoptotic properties. insect cells sf stably expressing orf gene strongly resist both viral-and actinomycin d-induced apoptosis. like p and p , orf (also referred as wssv or aap- ) is suggested to act as an inhibitor substrate and is able to block several caspases, including human caspases and as well as insect sf-caspase in vitro. orf ability to suppress caspase activity is associated with consensual caspase cleavage sites: devd k that targets caspase , and vetd k and lehd k that are both required for caspase inhibition. although each site specifically targets one protease, the three of them are needed to reach maximal caspase inactivation. for baculoviruses and wssv, inhibiting apoptosis is crucial to achieve their life cycle. these different invertebrateinfecting viruses encode proteins exhibiting active caspase cleavage sites, just as cellular substrates do, but with the special ability to irreversibly freeze the protease activity. this leads to a very potent and broad caspase inhibition, which keeps the host cell machinery available and fit for viral amplification. caspase cleavage as a way for viruses to replicate and spread: live and let die unlike the expression of antiapoptotic products, the requirement of apoptosis for viruses to survive is a way more counterintuitive strategy that yet has been evolved by several viruses and involves caspase cleavage (table ) . parvovirus aleutian mink disease virus (amdv) can lead, at a single cell level, to either permissive infection, namely high levels of both viral dna replication and production of viruses, or persistent infection with low viral dna replication and almost no production of progeny virions. on permissive infection, amdv induces caspase activation that is necessary for viral amplification. this requirement was associated with ns protein being cleaved through two caspase sites leading to five ns -related products. when one site is mutated within amdv molecular clones, the viral production is strongly reduced and even aborted when both sites are disrupted. interestingly, wild-type (wt) ns protein, which exerts replicative and transcriptional functions, is mostly nuclear, but when one of its cleavage sites is disrupted, the protein remains cytosolic resulting in a dramatic decrease in ns -dependent viral protein (vp) expression. ns -related c-terminal products, also nuclear, are suggested to be actively human papillomavirus (hpv) is mostly known to infect epithelial cells of the genital tract and cause cervical cancers. on epithelial differentiation, hpv induces a dna damage response that leads to caspase -dependent apoptosis. interestingly, hpv e , a protein involved in viral dna replication, is a target for caspases and at a site that is conserved in all genital hpvs and preventing e cleavage reduces viral amplification. when compared with chemically induced apoptosis, hpv-induced apoptotic markers are limited. interestingly, hpv increases the levels of both antiapoptotic bcl and survivin proteins. hpv pro-and antiapoptotic properties might achieve a caspase activity threshold that is sufficient for e cleavage and viral amplification, but not lethal for the host cell. besides e , hpv e protein is cleaved by caspases as well, but the functional consequences remain unknown. human astroviruses (hastv) are responsible for gastroenteritis. in yuc strain, orf encodes the precursor of viral capsid proteins, vp , whose processing yields vp . besides, hastv activates caspases , , , , and . , in vitro, caspases , and are able to target vp but only caspase or silencing reduces vp generation on infection. interestingly, both intracellular vp processing into vp and progeny virion release are lost in the presence of a pan caspase inhibitor. however, in opposition to what was first suggested, viral release needs caspase activation but does not need vp processing into vp to be achieved. but yielding fully infectious viruses does require shorter polypeptides to be created through vp further processing by trypsin. thus, first cleaving vp to generate vp likely represents a crucial intermediate event during hastv infection. sars-coronavirus (sars-cov) is a fairly new rna virus responsible for a severe acute respiratory disease. sars-cov leads to either permissive or persistent infection, with strong cytopathic effects and high viral titers or the opposite, respectively. interestingly, the nucleocapsid protein (n), that has both structural and non-structural functions, is proteolytically processed in cells undergoing a lytic cycle or ectopically expressing the protein. using a pharmalogical approach, n protein was demonstrated to be targeted by caspases and . on permissive infection, n protein localizes in both cytosol and nucleus while remaining cytosolic in cells undergoing persistent infection. moreover, preventing n from translocating to the nucleus by mutating its nuclear localization signal abolishes its cleavage by caspases. thus, permissive infection is associated with n being translocated to the nucleus and possibly its ensuing caspase cleavage. nevertheless, the mechanisms underlying viral replication, n subcellular localization and n caspase processing are unknown so far. in sharp contrast with baculoviruses and wssv, several very different viruses evolved to hijack apoptosis with caspase cleavage being required to reach optimal viral amplification. based on the well-described case of amdv, we can in some cases, apoptosis induction by the host cell leads to exactly what is expected, namely viral attenuation, but surprisingly with the help of viral protein cleavages. the possible viral advantage resulting from such an event is discussed (table ) . kaposi sarcoma-associated herpesvirus (kshv) establishes long-term infections leading to kaposi sarcomas and can go through either latent or lytic cycle. when kshv is reactivated, orf (or mta or ks-sm) is detected by western blot as a doublet, with the smaller product being abolished by a pan caspase inhibitor. in vitro, caspase , which is activated on kshv reactivation, and to a lesser extent caspases and , are able to process orf at detd k. interestingly, coexpression assays show that orf cleavage product (i.e. orf lacking residues - ) is no longer able to promote viral lytic gene expression. accordingly, complementing a stable cell line containing an orf -null kshv genome with ectopic wt or uncleavable orf promotes lytic gene expression, while orf lacking residues - does not. consistently, the number of cell-free virus particles dramatically increases when caspase is inhibited. thus, orf cleavage would prevent proper expression of its downstream targets and subsequent full reactivation of kshv, suggesting that it allows kshv to maintain a persistent infection. as described above, amdv ns protein is cleaved by caspases, which allows full viral replication. interestingly, amdv capsid proteins vp and are also processed by caspases on viral infection or ectopic expression. the cleavage generates a stable -kda product, vpx, and can be partly prevented by the pharmacological inhibition of caspases , , and , and completely by a pan caspase inhibitor. besides, vp protein expression in either transfected or amdv-infected cells mainly activates caspase , followed by caspases , , and , with caspase as the more efficient in vp cleavage in vitro. altogether, these data argue for the involvement of several caspases in vp cleavage. amdv-g strain grows more efficiently at . c than c. the alteration of the caspase site within amdv-g increases both vp and genomic copies production at the non-optimal temperature of c and shows no significant effects at . c, suggesting that vp caspase cleavage attenuates amdv-g life cycle. in vivo amdv-g infection might thus be regulated by active caspases because it only occurs at c. interestingly, ns cleavage favors viral dna replication while vp processing would limit the packaging of progeny virions. as it is possible that both ns and vp cleavages do not inevitably occur simultaneously depending on the pattern of activated caspases, it would result in either effective or attenuated viral replication and allow the virus to spread or persist. h- parvovirus (h- pv) displays oncotropic and oncolytic features meaning it preferentially replicates in and kills transformed cells, mainly in a non-apoptotic manner. however h- pv is able to activate caspases in non-transformed cells, leading to the cleavage of ns , a non-structural protein (ns) notably involved in viral dna replication and gene expression by transactivating p promoter, which controls the synthesis of capsid proteins. cleaved ns protein (named ns -nterm) lacks its c-terminal transactivation domain. when ectopically expressed, ns -nterm acts as a dominant negative on ns -driven p promoter transactivation, and dramatically decreases viral production. moreover, a molecular clone expressing an uncleavable version of ns tends to generate more virions. as cancer cells are often refractory to proper apoptotic induction, we propose that h- pv oncotropism is due, at least in part, to the inability of cancer cells to cleave ns protein, thus allowing strong virus production. on the other hand, ns cleavage would act as a sensor of antiviral defenses and accordingly attenuate viral amplification, likely to favor persistent infection (our unpublished results). crimean-congo hemorrhagic fever virus (cchfv) causes severe coagulation dysfunction in humans. cchfv nucleocapsid protein (np) is an important structural protein also involved in viral replication and, when ectopically expressed with an apoptotic factor such as bax protein, it generates two fragments through cleavage at a consensual devd k motif. a caspase inhibitor and cells lacking caspase both allows a one log-increase in the production of virions, which suggests that caspase activation is involved in cchfv attenuation. thus, np caspase cleavage and/or np-related products may participate to this attenuation although the mechanisms remain to be investigated. interestingly, the caspase cleavage site is conserved within all the strains that were checked, suggesting that it is an important feature for cchfv regulation. caspase cleavages of viral proteins can result in the attenuation of the virus. rather than a weakness of the virus, this might be considered as a way for the virus to adapt to the cellular context: as a result of such viral attenuation, the host response is largely reduced while the virus keeps its ability to replicate to some extent. although the consequences of several caspase protein cleavages have been elucidated, they remain elusive so far for others (table ) . influenza viruses are structured into ribonucleoprotein segments consisting of viral rna and viral proteins, the major one being the np. unlike avian np, np proteins from human influenza a and b viruses are long known to be caspase targets. , human influenza with uncleavable ('avian-like') np is dramatically less lethal for mice, along with lower viral titers and faster clearance than its wt counterpart. this argues for the involvement of np caspase cleavage in the virulence of influenza virus. but making avian np cleavable (i.e. making the protein 'human-like' by introducing the same caspase cleavage site) does not enhance the viral virulence as could have been expected. thus, modulating influenza pathogenicity through np caspase cleavage would be a specificity of human strains that cannot be extrapolated to avian ones. however, avian influenza pathogenicity might still be regulated by caspase cleavage. the viral ionic channel m protein is also cleaved by caspases, likely caspase and/ or , in both human and avian influenza viruses and was shown to be associated with avian influenza pathogenicity. altogether, these studies highlight that modulating influenza caspase sites attenuates the virus although through currently unknown mechanisms. , hepatitis c virus (hcv) often leads to chronic infection evolving to cirrhosis and possibly hepatocellular carcinoma. hcv core protein induces activation of caspases and , and interacts with viral ns a protein that is cleaved by the activated caspases. [ ] [ ] [ ] [ ] several studies point to a role of ns a cleavage in its subcellular localization. the protein is mainly found in the cytosol while exerting nuclear functions. ns a caspase cleavage was first suggested to allow the removal of a c-terminal cytoplasmic retention signal along with the translocation of the c-terminal deleted ns a to the nucleus, reminding what was already described for amdv ns protein. however, a study more recently reported a cytosolic localization of these c-terminal truncated forms of ns a and no obvious role of ns a caspase cleavage in its trafficking. although its characterization is well admitted, the functional relevance of ns a cleavage is still debated. transmissible gastroenteritis coronavirus (tgev) causes acute and fatal diarrhea in newborn piglets. tgev infection is able to induce caspases , , , and activation , and the cleavage of the structural np n, leading to a stable -kda fragment (n ). n protein is targeted in vitro by caspases and , and less efficiently by caspase . in hrt jap cells, the infection causes apoptosis but is not productive, suggesting that caspase activation (and possibly n caspase cleavage) prevents progeny virion generation. however, caspase inhibition in this context does not restore any viral production. owing to the lack of satisfactory tools, human hrt jap cells were used as a study model but might not be appropriate to reveal n caspase cleavage relevance, notably for virus production. further digging will thus be needed to understand the biological significance of n processing by caspases on tgev infection. feline calicivirus (fcv) causes upper respiratory tract disease in cats. fcv capsid protein is synthesized as a precursor further cleaved by the viral protease. however, a -kda product (p ) is also usually observed at late stages of fcv infection and can be abolished with a pan caspase or a caspase / inhibitor. accordingly, fcv-infected cells undergo apoptosis as well as caspases , and activation. only caspase and far less efficiently caspase process fcv capsid protein in vitro at cleavage sites that have not been described yet. as suggested for instance for amdv, cleavage of the capsid protein might impair particle assembly and participate to viral persistence but this has not been investigated either. adenoviruses gather more than a species with about being able to infect humans. adenovirus early region a (ade a) encodes two major proteins, s and s, that are involved in the control of early viral gene expression through their interactions with the host cell machinery. in vitro, caspases and are able to cleave both s and s proteins, although the cleavage efficiency depends on the caspase cleavages of viral proteins were recapitulated in tables according to the functional consequences on viral life cycle they were shown or suggested to exert. the name of the virus, nature of its genome and caspase(s) induced on infection are indicated. the characteristics of the cleavages were also summarized, with identity of the cleavage site(s), caspase(s) able to cleave in vitro and/or in cellulo (sometimes different from caspases induced in cells undergoing infection) and suggested functions of the caspase-related products as often as possible. vertical black arrows indicate where cleavage occurs viral serotype. furthermore, when apoptosis is chemically induced in ade a-transformed cells, ade a levels decrease but can be restored using a pan caspase inhibitor. however, ade a levels remain stable on adenovirus infection despite tnfa-induced caspase activation. thus, although ade a caspase cleavage is likely to occur, its existence on infection as well as its biological relevance for adenoviruses remains elusive so far. interestingly, ade a-binding abilities to transcription factors such as cbp and tbp are affected by the cleavage, suggesting that it might modulate the expression of early viral genes. molluscum contagiosum virus (mcv) is a poxvirus that, like variola, exclusively infects humans. mc p, an mcv protein, exhibits antiapoptotic properties by protecting cells from fas-induced apoptosis through caspase inhibition but is not targeted by caspases. on the other hand, mc p is not associated with any antiapoptotic abilities but in vitro, is cleaved by caspases and . when expressed through vaccinia virus, mc p is also specifically cleaved by caspases on fas-induced apoptosis while mc p remains stable, as expected. mc p and mc p being thought to be produced simultaneously, a balance might take place between mc p antiapoptotic response and mc p caspase cleavage. however, the pathophysiological significance of this balance is difficult to address because mcv does not grow in tissue culture or experimental animals. herpex simplex virus (hsv- ) causes lifelong infections affecting between and % of the global population. hsv- exerts antiapoptotic properties against numerous stimuli. but some variants including d mutant induce apoptosis and under these conditions, infected cell protein no. (icp or m r , protein) leads to an additional product (m r , ) that can result in vitro from icp cleavage by caspase . neither the identity of the caspase site nor the biological relevance of icp cleavage has been further investigated yet. viruses face up to apoptosis: die harder. there are clear evidences concerning the relevance of viral protein caspase cleavages in the physiology of viruses ( figure ) . coherently, such a modification is used to directly fight apoptosis and acmnpv as well as wssv express proteins acting as strong and broad inhibitor caspase substrates. for other viruses, caspase cleavage probably allows the removal of specific regions that reveals or eliminates functional domains or signals. in some cases, this leads to full viral amplification (amdv ns , hpv e ), with the striking example of amdv ns protein that needs to be cleaved by caspases to translocate to the nucleus and eventually exert its functions. on the contrary, caspase cleavage can result in the attenuation of the virus by generating new viral products acting as dominant negatives of ns proteins (h- pv ns , kshv orf ) or unfit for virus packaging when structural proteins are concerned (amdv vp and possibly fcv capsid protein). interestingly, such attenuation at a cellular level might hold importance regarding the possibility for viruses to establish permissive and/or persistent infection at a higher scale (i.e. multicellular organisms). the functional consequences of some viral protein caspase cleavages still remain debated or elusive because of the difficulty of investigating hazardous entities (influenza, hcv), the multitude of strains and genotypes (influenza, hcv, adenovirus) or the lack of appropriate tools such as cellular or animal models allowing to recapitulate the viral life cycle (tgev, mcv). when caspase cleavages of viral proteins work in too mysterious ways, it is sometimes concluded that they constitute a mechanism of degradation resulting from the host fighting back the infection. however, the caspase-related products detected are mostly stable, suggesting they could be somehow involved in the viral life cycle as mentioned right above. besides, knowing how high the mutation rates of viruses can be, or that some viral oncoproteins (like v-rel) adapted to resist caspase cleavage through evolution unlike their cellular counterparts, keeping caspase cleavage sites seems very unlikely if viruses do not somehow profit from them. as caspase cleavage of viral proteins occurs in all sorts of proteins (structural or non-structural) and in all sorts of viruses (dna as well as rna), this suggests that it constitutes a strong strategy to handle apoptosis. by managing to use the molecular effectors of apoptosis to protect themselves from eradication without any additional genetic information required, viruses prove once again how fascinating their adaptability can be. host defense, viruses and apoptosis enter the kill zone: initiation of death signaling during virus entry cell death in the host response to infection immunogenic and tolerogenic cell death viral subversion of immunogenic cell death viral control of mitochondrial apoptosis a time to kill: viral manipulation of the cell death program to kill or be killed: how viruses interact with the cell death machinery caspases in apoptosis and beyond human caspases: activation, specificity, and regulation caspase activation during virus infection: more than just the kiss of death? prevention of apoptosis by a baculovirus gene during infection of insect cells apoptotic suppression by baculovirus p involves cleavage by and inhibition of a virus-induced ced- /ice-like protease inhibition of ice family proteases by baculovirus antiapoptotic protein p interaction of the baculovirus anti-apoptotic protein p with caspases. specificity, kinetics, and characterization of the caspase/p complex crystal structure of baculovirus p : role of a novel reactive site loop in apoptotic caspase inhibition covalent inhibition revealed by the crystal structure of the caspase- /p complex baculovirus apoptotic suppressor p is a substrate inhibitor of initiator caspases resistant to p in vivo reactive-site cleavage residues confer target specificity to baculovirus p , a dimeric member of the p family of caspase inhibitors functional analysis of the orf gene of the white spot syndrome virus molecular mechanism of the interactions between white spot syndrome virus anti-apoptosis protein aap- (wssv ) and shrimp effector caspase caspase activation is required for permissive replication of aleutian mink disease parvovirus in vitro caspase cleavage of the nonstructural protein ns mediates replication of aleutian mink disease parvovirus human papillomaviruses activate the atm dna damage pathway for viral genome amplification upon differentiation human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification degradation of hpv e by p : delta np alpha and mutant p r w protect the wild type p mediated caspase-degradation role of individual caspases induced by astrovirus on the processing of its structural protein and its release from the cell through a non-lytic mechanism caspases mediate processing of the capsid precursor and cell release of human astroviruses cell typespecific cleavage of nucleocapsid protein by effector caspases during sars coronavirus infection caspase- cleavage of kaposi sarcoma-associated herpesvirus orf confers a cellular function against viral lytic gene expression the capsid proteins of aleutian mink disease virus activate caspases and are specifically cleaved during infection induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during crimean-congo hemorrhagic fever virus infection nucleoproteins of animal influenza viruses, in contrast to those of human strains, are not cleaved in infected cells caspase-dependent n-terminal cleavage of influenza virus nucleocapsid protein in infected cells the role of the n-terminal caspase cleavage site in the nucleoprotein of influenza a virus in vitro and in vivo ns protein of influenza a virus downregulates apoptosis influenza virus pathogenicity is determined by caspase cleavage motifs located in the viral proteins alterations in caspase cleavage motifs of np and m proteins attenuate virulence of a highly pathogenic avian influenza virus the hepatitis c virus core protein interacts with ns a and activates its caspase-mediated proteolytic cleavage the ns a protein of the hepatitis c virus genotype a is cleaved by caspases to produce c-terminal-truncated forms of the protein that reside mainly in the cytosol calcium-dependent calpain proteases are implicated in processing of the hepatitis c virus ns a protein cleavage of hepatitis c virus nonstructural protein a by a caspase-like protease(s) in mammalian cells transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and - during tgev-induced apoptosis caspase-mediated cleavage of the feline calicivirus capsid protein apoptosis in cultured cells infected with feline calicivirus caspasemediated cleavage of adenovirus early region a proteins molluscum contagiosum virus inhibitors of apoptosis: the mc v-flip protein blocks fas-induced activation of procaspases and degradation of the related mc protein infected cell protein no. is subject to proteolytic cleavage by caspases activated by a mutant that induces apoptosis three mutations in v-rel render it resistant to cleavage by cell-death protease caspase- viral mutation rates requirement for shrimp caspase in apoptosis against virus infection the authors declare no conflict of interest.acknowledgements. this work was supported by the french institutions cnrs, institut pasteur de lille and inserm, and by grants from 'conseil régional nord-pas de calais' and 'ligue contre le cancer, comité nord'. ar was supported by a fellowship from 'association pour la recherche sur le cancer'. we are grateful to dr. gauthier goormachtigh for his careful reading and the helpful comments he made about this paper. key: cord- - yc ribg authors: morehouse, zachary p.; proctor, caleb m.; ryan, gabriella l.; nash, rodney j. title: a novel two-step, direct-to-pcr method for virus detection off swabs using human coronavirus e date: - - journal: virol j doi: . /s - - -y sha: doc_id: cord_uid: yc ribg background: currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (pcr) based assays. this process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a pcr assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. methods: using human coronavirus e (hcov- e) as a model system, we spiked swabs in vitro for proof-of-concept testing. swabs were spiked in serial dilutions from . × ( ) to . × ( ) copies/ml and then placed in ml tubes with viral transport media (vtm) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. after homogenization, μl of lysate was processed using rt-qpcr for amplification of the nucleocapsid (n) gene, qualifying viral detection. results: hcov- e in vitro spiked swabs were processed in a novel two-step, direct-to-pcr methodology for viral detection. after running swabs, we confidently determined our limit of detection to be . × ( ) viral copies/ml with . % sensitivity. conclusion: we have proven that the shaker-mill homogenization-based two-step, direct-to-pcr procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in pcr for the detection of hcov- e. this finding allows for reductions in the time and resources required for pcr based virus detection in comparison to the traditional extraction-to-pcr methodology. as the number of viral diseases are on the rise, it is critical to continue to innovate and advance diagnostic, treatment, and surveillance methods surrounding viral infections. herein, we are proposing a novel method for viral pathogen detection off swabs as an improvement or alternative to the current pcr based assays commonly used for viral detection [ , ] . the traditional protocol for preparing a sample for pcr based detection often involves procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify its nucleotides, and then amplify the purified genetic material via pcr for detection of a gene product needed to confirm the patient's suspected diagnosis [ ] . in the face of the covid- pandemic, attempts have been made to perform pcr based diagnostics for sars-cov- with reduced time and cost, especially as the plastics and reagents needed for traditional viral nucleotide extractions have become scarce in the face of an exponentially increased global demand [ ] [ ] [ ] [ ] . though some success has been seen with thermal and enzymatic digestion attempts on viral samples to expose the genetic material for extraction-less pcr detection, no method has shown high viral lysis in under min when dealing with clinical concentrations of virus on swabs [ , , ] . shaker-mill homogenization as a form of mechanical lysis has been proven time and again as a successful method for disrupting tissues, microorganisms, and biologic samples for downstream molecular analysis; however, these downstream applications often require the use of additional purification, isolation, or extraction procedures before those analyses can be completed [ ] [ ] [ ] [ ] . while, shaker-mill homogenization procedures have not yet been applied directly to diagnostic technologies, its ability to lyse organisms and tissues far tougher than a virus have been well categorized [ ] [ ] [ ] [ ] . building off the increased need for novel viral diagnostic methodologies, which reduce the resources and time required for accurate viral pathogen detection, we felt it was time to examine the capabilities of shaker-mill homogenization for viral lysis to be used for downstream detection. using human coronavirus e (hcov- e) as our model organism, we developed a novel two-step methodology of optimized shaker-mill homogenization parameters that allowed for direct-to-pcr viral detection. hcov- e is an enveloped, positive-sense, singlestranded rna virus [ ] . as a known human pathogen, it hcov- e is one of the four most common circulating coronaviruses associated with mild to moderate respiratory illness globally [ ] . it is commonly included in commercial respiratory viral panel screening as a source of the common cold [ ] . hcov- e was the chosen model for this project as a biosafety level pathogen with similar genetic and protein composition to the current sars-cov- virus for developing novel diagnostic approaches, until the process is proven in vitro [ , ] . the linear genome and enveloped structure of hcov- e allows for this virus to be a sufficient in vitro substitute for sars-cov- during the early stages of methodology development described within this manuscript [ ] . no. - ), incubated at °c with % co [ ] . the cell culture supernatant was harvested at h post infection when % cytopathic effect (cpe) was observed. viral transport media (vtm) was produced following the us cdc (atlanta, ga, usa) guidelines, found freely available on their website for formulation of vtm in a laboratory as an alternative to commercial vtm purchases. ml of hanks balanced salt solution (hbss) x with calcium and magnesium ions (no phenol red) (fisher science, cat. no. sh ) was supplemented with % heat inactivated fetal bovine serum (gemini bioproducts, cat. no. - ). mg of gentamicin (gemini bioproducts, cat. no. - p) and μg of amphotericin b (gemini bioproducts, cat. no. - p) was added to the mixture and mixed thoroughly to create a final product of viral transpot media, % fbs, μg/ml gentamicin, . μg/ml amphotericin b. this viral transport media was used for storage and processing of all swab samples in this manuscript. sterile cotton swabs (fisher science, cat. no. - - ) were submerged for s in viral solutions ranging from . × to . × viral copies/ml [ ] . the swabs were exposed in a serial dilution pattern, with three swabs being exposed at each concentration log to evaluate the detection capabilities of this method. the saturated swabs were then placed in a ml screw capped tube (omni international, cat. no. - ) prefilled with ml of viral transfer buffer [ ] . the stem of the swab was then broken off at a level even with the top of the tube to allow for the cap to be screwed on for transporting and processing. the samples were prepared at °c and then incubated for h at °c prior to processing. to maintain optimal levels of biosafety, the following shaker-mill processing was completed in a biosafety cabinet to protect the user from any potential aerosol production during processing. twenty-four ml screw cap tubes containing the virally spiked swabs were processed on the omni bead ruptor elite (omni international, cat. no. - e) for s at . m/s. this processing generated froth within the tube which was allowed to settle prior to removal of μl of lysate for rt-qpcr (fig. ) . hcov- e nucleocapsid gene (n gene) was selected as a target for rt-pcr from vabret et al. [ , ] . the n gene was targeted with forward primer ′-aggcgcaa gaattcagaaccagag- ′ and reverse primer ′-agcaggactctgattacgagaaag- ′ [ ] . μl of sample lysate was added to create a final reaction volume of μl using the proportions of primers, sample, sybr, rt, and depc-treated h o as laid out in the new england biologics luna rt-qpcr kit (neb, cat. no. e s). amplification of lysate was performed for cycles and the resulting amplicons were loaded into a % agarose (bio-rad, cat. no. ) gel for product visualization. out of abundance of caution, the loading of the pcr plate with viral lysate should be completed in a biosafety cabinet to protect the user from any potentially viable virus particles remaining following shaker-mill homogenization. hcov- e was quantified with standard plaque assay protocols [ ] . once the cells achieved % confluence, μl of hcov- e stock was added to the media of the first well. the media was gently mixed and μl of the infected media was transferred into the adjacent well. this was repeated to create serial dilutions throughout the plate. after h of incubation, the virally infected media was removed from each well and replaced with ml of dmem infused with % heat inactivated fbs and % agarose (bio-rad, cat. no. ). the plate was incubated for an additional days at °c with % co and plaques were counted to determine viral concentration in plaque forming units/ml (pfu/ml). we have successfully demonstrated that shaker-mill homogenization using the omni bead ruptor elite fig. graphical depiction of general two-step, direct-to-pcr in vitro testing method that was employed in all experiments discussed in this manuscript fig. two-step, direct-to-pcr results from serial dilution of viral concentration spiked onto swabs for limit of detection testing. red lines represent . × viral copies/ml spiked swabs. brown lines represent . × viral copies/ml spiked swabs. pink lines represent . × viral copies/ml spiked swabs. navy lines represent . × viral copies/ml spiked swabs. teal lines represent . × viral copies/ml spiked swabs. olive lines represent . × viral copies/ml spiked swabs. black lines represent . × viral copies/ml spiked swabs. green lines represent virus free, negative control swabs provides sufficient viral lysis from spiked swabs to allow for viral detection via direct rt-qpcr of the lysate. using this novel method (fig. ) at our optimized run parameters of . m/s for s, we found the lower limit of reliable detection to be . × viral copies/ml (figs. , ). this limit of detection was evaluated for reproducibility by testing in vitro spiked swabs from two different viral stock solutions, using rt-qpcr (fig. ) . the results were confirmed via amplicon visualization (fig. ) and demonstrated a sensitivity of . % when processing with this two-step, direct-to-pcr method. in addition to the strong method sensitivity shown with in vitro testing, we also consistently showed greater than % viral lysis after shaker-mill homogenization for s at . m/s. plaque assays were used to determine percent lysis following homogenization as shown with decreased pfu counts between stock and lysate ( table ). the cost, time, and availability of current pcr assays have been considered prohibitive measures in the timely diagnosis and subsequent treatment of viral infections [ ] . there is a critical need for the development of diagnostic technologies that help to improve access and reduce cost-this is especially important for increasing the treatment of infectious diseases in socioeconomically disenfranchised populations. while still in its infancy, it is our hope that this novel methodology can be applied as one potential solution for this need. the authors acknowledge that there are crucial next steps for clinical validation of this methodology but feel that the presented data is compelling and provides a strong starting point for this to occur. while these studies was completed using hcov- e as an in vitro model for methodology development geared towards sars-cov- applications, it is also our hope that this methodology can be further evaluated at both the benchtop and the bedside to be applied to other respiratory viruses also commonly tested for via nasopharyngeal or oropharyngeal swabs and pcr based detection. hcov- e n transcript detection using rt-qpcr following shaker-mill homogenization off swabs spiked in vitro with . × viral copies/ ml. orange lines represent viral stock spiked swabs at . × viral copies/ml. grey lines represent viral stock spiked swabs at . × viral copies/ml. the green line represents a negative control, virus-free swab in addition to the improvements to access of care, this two-step, direct-to-pcr methodology also has the potential to improve laboratory safety. as shown in table , shaker-mill homogenization results in greater than % lysis of the virus in a sample. this high percentage of viral lysis demonstrates that there would be few, if any, viable viral particles left in the sample tube when the laboratory technician opens it for transfer to the rt-qpcr reaction. by using our two-step, direct-to-pcr methodology, the lab technician is also able to significantly reduce exposure to viral particles that would normally be increased when opening and closing sample tubes and adding buffers during traditional nucleic acid extraction processes. the low levels of viable and infectious viral particles when the sample is opened result in decreased exposure potential through laboratory accidents or aerosolization during sample opening that could result in laboratory acquired infections. in contrast to the many benefits provided by this novel methodology, the authors do acknowledge that introducing a new approach to existing methods is difficult. we expect to see resistance from laboratories with currently validated testing methods, researchers with years of experience using the traditional extraction-to-pcr process, and other commercial entities involved in the currently accepted processes for pcr based viral diagnostics. however, it is our opinion that when advancements in technology and methodology can be made that will improve access to care, reduce cost, and improve safety, it is our responsibility to the public to explore these options. in the face of increasing virally caused infections globally, it is our duty to do everything we can to shake every tree and turn over every stone to prepare society to combat these diseases. we have successfully proven that shaker-mill homogenization provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in pcr based assays for the detection of virus. this novel two-step, directto-pcr method for viral detection off swabs has shown a lower limit of reliable detection at . × viral copies/ml with . % sensitivity in vitro when screening for hcov- e. herein, we have demonstrated the success of this methodology in vitro and propose it as a novel approach to viral detection that allows for decrease run time in comparison to traditional pcr based viral detection assay protocols, as well as a reduction in the materials needed for successful viral detection. bypassing standard extraction methods, while maintaining a lower limit of detection one log below the reported viral load on clinically obtained swabs positive for coronavirus [ ] , we demonstrated that this novel method has warranted further clinical evaluation for its potential to reduce the cost and time needed for each test. direct diagnosis of human respiratory coronaviruses e and oc by the polymerase chain reaction detection of the human coronavirus e, hku , nl , and oc between and in yamagata rt-qpcr testing of sars-cov- : a primer perforrmance of abbott id now covid- rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a new york city academic institution a novel reverse transcription loopmediated isothermal amplification method for rapid detection of sars-cov- an alternative workflow for molecular detection fo sars-cov- -escape from the na extraction kit-shortage rapid implementation and validation of a cold-chain free sars-cov- diagnostic testing workflow to support surge capacity fast sars-cov- detection byrt-qpcr in preheated nasopharyngeal swab samples assessing species biomass contributions in microbial communities via metaproteomics a conserved rna seed-pairing domain directs small rna-mediated stress resistance in enterobacteria mapping interactions of microbial metabolites with human g-protein-coupled receptors treatment of human immunodeficiency virus infection with tenofovir disoproxil fumaratecontaining antiretrovirals maintains low bone formation rate, but increases osteoid volume on bone histomorphometry host and sources of endemic human coronaviruses properties of coronavirus and sars-cov- characterization of a human coronavirus (strain e) c-like proteinase activity viral load of sars-cov- in clinical samples nasopharyngeal swab collection in the suspicion of covid- clinical validation of a sars-cov- real-time reverse transcription pcr assay targeting the nucleocapsid gene two detailed plaque assay protocols for the quantification of infectious sars-cov- overcoming barriers in hpv vaccination and screening programs publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to acknowledge pete tortorelli, karl jahn, and erik masefield for their financial support and organizational support of the experiments needed for this publication. additionally, we would like to acknowledge rachel true, rachel nash, and leah proctor for their continued support of our work.authors' contributions zp morehouse: conceptualization, methodology, validation, formal analysis, investigation, data curation, writingoriginal draft, review, and editing. cm proctor: methodology, validation, formal analysis, investigation, data curation, writingreview and editing. gl ryan: data curation, writingreview and editing. rj nash: conceptualization, methodology, investigation, data curation, writingreview and editing, supervision, funding acquisition. the author(s) read and approved the final manuscript. this work was supported solely through private funding by omni international inc.availability of data and materials all data supporting this manuscript can be accessed by reaching out to the corresponding authors rj nash and zp morehouse directly at rnash @gsu. edu and moreho @msu.edu.ethics approval and consent to participate because no animals or patients were involved in this study, ethics approval is not applicable in this section. not applicable.competing interests zp morehouse, cm proctor, gl ryan, and rj nash all are currently employed by omni international inc. in some capacity but have no personal financial interests in the success of the company. key: cord- - g klpyg authors: guajardo-leiva, sergio; chnaiderman, jonás; gaggero, aldo; díez, beatriz title: metagenomic insights into the sewage rna virosphere of a large city date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g klpyg sewage-associated viruses can cause several human and animal diseases, such as gastroenteritis, hepatitis, and respiratory infections. therefore, their detection in wastewater can reflect current infections within the source population. to date, no viral study has been performed using the sewage of any large south american city. in this study, we used viral metagenomics to obtain a single sample snapshot of the rna virosphere in the wastewater from santiago de chile, the seventh largest city in the americas. despite the overrepresentation of dsrna viruses, our results show that santiago’s sewage rna virosphere was composed mostly of unknown sequences ( %), while known viral sequences were dominated by viruses that infect bacteria ( %), invertebrates ( %) and humans ( . %). interestingly, we discovered three novel genogroups within the picobirnaviridae family that can fill major gaps in this taxa’s evolutionary history. we also demonstrated the dominance of emerging rotavirus genotypes, such as g and g , that have displaced other classical genotypes, which is consistent with recent clinical reports. this study supports the usefulness of sewage viral metagenomics for public health surveillance. moreover, it demonstrates the need to monitor the viral component during the wastewater treatment and recycling process, where this virome can constitute a reservoir of human pathogens. viruses are the most abundant biological entities on earth, with an estimated particles worldwide [ ] . urban environments impacted by human activity, such as wastewater treatment plants (wwtps) and sewage are not an exception. sewage and wwtps form an ecosystem that supports thriving microbial communities (prokaryotic and eukaryotic), plants and animals, such as rodents, birds and bats [ ] . in these environments, viruses associated with the biological waste of a city are mixed with viruses from all the organisms living in the wwtp, thus forming an untapped source of viral diversity [ , ] . in general, the sewage virosphere can be considered a mixture of human viruses excreted in the feces, urine and skin peeling, and viruses from animals, invertebrates, plants, fungi and bacteria [ , ] . sewage has been historically used to monitor known human viral pathogens, such as noroviruses, hepatitis viruses, enteroviruses, rotaviruses and adenoviruses [ , ] . the presence of viral pathogens in wastewater reflects ongoing infections being transmitted in the human population served by the given wwtp [ ] [ ] [ ] . likewise, sewage can reveal new and unknown viral genomes that could, in the future, be associated with idiopathic human diseases [ ] . nowadays, decreased water availability due to global warming and increased human water consumption have turned water scarcity into a cyclical problem. to solve this, recycled water derived from wwtp effluent has been intensively used for industrial operations, agricultural irrigation and even recreational activities [ ] . in this way, recycling of treated sewage generates a potential public health risk, as well as a critical risk for agricultural and animal production industries, due to insufficient removal of pathogenic viruses [ , , ] . current regulations have promoted improved treatment guidelines, which now combine mechanical, biological and chemical processes, such as flocculation, sedimentation, filtration, chlorination and uv-radiation [ , , ] . these treatments have significantly reduced microbiological contamination by inactivating and removing bacteria and protozoa, but they have little effect on human viruses, such as adenoviruses and enteroviruses, which are later dispersed in effluent waters [ ] [ ] [ ] . in latin america, the sanitary systems, which are still under development, have a low sewage treatment coverage ( %), generating a potential health risk for human and animal populations [ ] . chile is a middle-income country with a population of million inhabitants, of which approximately million inhabitants are concentrated in the capital, santiago de chile, making it the seventh largest city in the americas. most of santiago's sewage is decontaminated by three wwtps: el trebal, la florida and la farfana. el trebal serves . million equivalent inhabitants, and water decontaminated by this wwtp provides irrigation for , agricultural hectares of land that is mainly used for the production of fruits and vegetables consumed in santiago and exported according to international environmental standards. the sewage virosphere has been monitored world-wide using molecular techniques, such as pcr and quantitative pcr (qpcr) [ , ] . these methods can only provide information about the presence and abundance of known and characterized viruses because there is no universal marker for viruses, such as the s rrna gene for bacteria [ , ] . despite the increasing application of high-throughput sequencing techniques (hts) for viral metagenomics, their use in identifying viruses in sewage has not been well explored [ , , ] . however, the few existing studies have demonstrated that the study of the viral metagenomics of sewage is an excellent tool to monitor, identify and explore the diversity of the viral communities circulating among human and livestock populations [ , , , , , ] . this is especially important for rna viruses that, in the last decade and along with viral metagenomics, have undergone a revolution in terms of their discovery, thereby contributing to our understanding of viral diversity [ ] . to our knowledge, few studies [ , ] have used viral metagenomics to investigate rna viral communities in sewage around the world, none of which have been conducted in latin america. with this in mind, we conducted a pilot study to investigate the rna virosphere using a single sample equivalent to the sewage treated during one day in santiago (el trebal wwtp) using viral metagenomics. our main results, despite bias related to the overrepresentation of dsrna viruses (in comparison to other studies), show that the rna virosphere of el trebal is mainly composed of unknown sequences (microbial and viral dark-matter). the known viral sequence fraction was dominated by viruses that infect bacteria and invertebrate hosts. a high diversity and novelty were discovered within the picobirnaviridae family, which can fill major gaps in the evolutionary history of this group. likewise, we unveiled abundant and emergent rotavirus a genotypes never before recorded in chile, thus representing changes in the prevalence of the classical genotypes. the latter discovery provides evidence for the benefits of using viral metagenomics to aid public health surveillance based on excreted viruses in sewage. additionally, this study reveals the importance of analyzing viral dark matter through self-clustering of the sequences independent of their direct comparison to databases, which can result in the discovery and classification of new viral sequences. el trebal (hereafter referred to as trebal) is a wwtp ( • . " s • . " w) located in santiago, chile (figure ), that serves a population equivalent to . million inhabitants. a composite sample ( l), representing h of raw sewage, was obtained on jun . the sample was sequentially filtered through -and -µm pore size polycarbonate filters (isopore, mm diameter, millipore, milford, ma, usa) using a swinex filter holder (millipore) and then a . -µm pore size filter (sterivex pes, millipore). particles in the . -µm filtrate were concentrated by ultracentrifugation to a final volume of approximately ml, as described in [ ] . briefly, the . -µm filtered sample was centrifuged at , × g for h. the pellet containing viral particles was resuspended in glycine buffer and then incubated on ice for min. finally, after an additional ultracentrifugation at , × g for h, viruses were recovered by resuspending the viral pellet in ml of pbs. viruses , , x for peer review of el trebal (hereafter referred to as trebal) is a wwtp ( ° ′ . ″ s ° ′ . ″ w) located in santiago, chile (figure ), that serves a population equivalent to . million inhabitants. a composite sample ( l), representing h of raw sewage, was obtained on jun . the sample was sequentially filtered through -and -μm pore size polycarbonate filters (isopore, mm diameter, millipore, milford, ma, usa) using a swinex filter holder (millipore) and then a . -μm pore size filter (sterivex pes, millipore). particles in the . -μm filtrate were concentrated by ultracentrifugation to a final volume of approximately ml, as described in [ ] . briefly, the . -μm filtered sample was centrifuged at , × g for h. the pellet containing viral particles was resuspended in glycine buffer and then incubated on ice for min. finally, after an additional ultracentrifugation at , × g for h, viruses were recovered by resuspending the viral pellet in ml of pbs. the resuspended viral particles ( ml) were treated with dnase i ( u) to remove the remaining free dna from the cellular fraction. the mixture was incubated for h at °c, followed by inactivation at °c for min. viral rna was extracted using the high pure viral rna kit (roche, basel, switzerland) according to the manufacturer's instructions, but without the use of the poly(a) carrier. bacterial dna contamination was checked by s rrna gene pcr amplification using a universal bacterial primer set ( f: '-gtgycagcmgccgcggtaa- ' and r: '-ggactacnvgggtwtctaat- ') https://earthmicrobiome.org/protocols-and-standards/ s/. bacterial (e. coli jm ) dna was used as a pcr spike control to check for pcr inhibition of viral rna. the purified rna sample was then sequenced using illumina hiseq technology (roy j. carver biotechnology center, urbana, il, usa). briefly, the rnaseq library was prepared with the illumina truseq stranded mrna sample prep kit (illumina, san diego, ca, usa). the library was quantitated by qpcr and sequenced from one end of the fragment in a single lane for cycles on a hiseq . fastq files were generated and demultiplexed with the bcl fastq v . . . conversion software (illumina). raw metagenomic reads were quality filtered using cutadapt v . [ ] , leaving only sequences longer than bp (-m ), and conducting ′ end trimming for bases with a quality below (-q ) and hard clipping of the first five leftmost bases (-u ). finally, sequences representing simple the resuspended viral particles ( ml) were treated with dnase i ( u) to remove the remaining free dna from the cellular fraction. the mixture was incubated for h at • c, followed by inactivation at • c for min. viral rna was extracted using the high pure viral rna kit (roche, basel, switzerland) according to the manufacturer's instructions, but without the use of the poly(a) carrier. bacterial dna contamination was checked by s rrna gene pcr amplification using a universal bacterial primer set ( f: -gtgycagcmgccgcggtaa- and r: -ggactacnvgggtwtctaat- ) https://earthmicrobiome.org/protocols-and-standards/ s/. bacterial (e. coli jm ) dna was used as a pcr spike control to check for pcr inhibition of viral rna. the purified rna sample was then sequenced using illumina hiseq technology (roy j. carver biotechnology center, urbana, il, usa). briefly, the rnaseq library was prepared with the illumina truseq stranded mrna sample prep kit (illumina, san diego, ca, usa). the library was quantitated by qpcr and sequenced from one end of the fragment in a single lane for cycles on a hiseq . fastq files were generated and demultiplexed with the bcl fastq v . . . conversion software (illumina). raw metagenomic reads were quality filtered using cutadapt v . [ ] , leaving only sequences longer than bp (-m ), and conducting end trimming for bases with a quality below (-q ) and hard clipping of the first five leftmost bases (-u ). finally, sequences representing simple repetitions, which are usually due to sequencing errors, were removed using prinseq v . . [ ] at a dust threshold of (-lc_method dust, -lc_threshold ). details of the obtained sequences are shown in supplementary table s . viral rna metagenomes were assembled using de bruijn graphs, as implemented in the megahit v . . [ ] and idba-ud v . assemblers [ ] in metagenomic mode. only contig sequences > pb were further analyzed. both assemblies were merged by clustering contigs at % identity and % coverage of the shortest sequence, leaving the largest contig using the nucmer algorithm implemented in mummer v [ ] . after assembly, prodigal software [ ] was used to predict protein-coding regions, with options (-p meta -n). the predicted proteins were aligned against the ncbi nr database using diamond v . . [ ] (-e-value . ) and parsed using the lowest common ancestor algorithm in megan [ ] (lca score = ) using ncbi taxonomy tree to obtain the taxonomic annotation of each viral protein. species classification of viral proteins was used to infer putative hosts based on the virus-host db [ ] , as described in [ ] . the abundance of mapped proteins was quantified through read recruitment via bowtie [ ] , with parameters (-end-to-end-very sensitive-n ). the resulting sam file was parsed by the bbmap pileup script (bushnell b.-sourceforge.net/projects/bbmap/) and the relative protein abundances were normalized by gene length. the predicted proteins were functionally annotated using the pfam v database [ ] through hmmscan options (-cut_ga) implemented on hmmer [ ] . proteins annotated as rna-dependent rna polymerase (rdrp; predicted proteins) were taxonomically identified as described before, and the species classification was used to infer putative hosts. pairwise genetic distances were calculated for trebal rdrp nucleotide sequences and refseq (release ) using word-based alignment-free distances implemented on alfree software [ ] , with options (-s -d braycurtis-v counts). the pairwise distances between samples were analyzed by hierarchical clustering (hclust function in r) using a minimal increase in the sum of squares method (ward's method). the resulting dendrogram was cut (cutree function in r) into groups that represent the main clusters based on the "unrooted" dendrogram representation. the trebal proteins annotated as rdrp (> aa) using the pfam database [ ] and classified as picobirnaviridae were aligned by mafft v [ ] using default parameters, with the option -globalpair. a phylogenetic rdrp gene tree was constructed using the maximum likelihood method implemented with iqtree (-bb , -nm , -alrt -abayes) and , ultrafast bootstraps to evaluate branch robustness [ ] . the amino acid substitution model used in the phylogenetic analysis (vt + f + i + g ) was determined by modelfinder [ ] . reference sequences were obtained from ncbi refseq based on the phylogenetic analysis of the picobirnaviridae family from the international committee on taxonomy of viruses (ictv) [ ] , and a reference genome of the genus alphapartitivirus was used as an outgroup. a ribosomal binding site (rbs) prediction of the rdrp-predicted proteins used in the phylogenetic analysis was performed using prodigal software [ ] , as described before. nucleotide sequences of trebal proteins annotated as rotavirus vp , vp and vp using the pfam database [ ] were aligned against the ncbi nt database using blastn [ ] . the best hit (e-value < . ) classification of each nucleotide sequence was used to assign rotavirus species and type (g and p). raw sequences are available under ncbi sra bioproject prjna . assembled contigs and their annotation are available in the mg-rast server under the accession number mgm . and can be accessed at the following link https://www.mg-rast.org/linkin.cgi? project=mgp . the trebal rna viral fraction, sequenced on the illumina hiseq platform, yielded~ m high-quality reads (supplementary table s ). assembly of the rna metagenomic reads resulted in , contigs, which included . m reads and , predicted proteins. as with most viral metagenomes, only a small fraction (~ %) of the viral proteins matched to large databases [ , ] such as ncbi nr, representing~ % of the assembled reads. the high number of unmapped contigs ( % of the assembled reads) are most likely derived from novel and uncharacterized microbial and viral sequences, as has been observed in other wastewater studies [ , ] . a taxonomic survey of the predicted proteins shows that the known trebal reads ( figure a ) were assigned mostly to the virus domain ( %), followed by bacteria ( %) and eukarya ( %). cellular contamination of viral enriched fractions is a common feature of viral metagenomes [ ] [ ] [ ] . in this study, this could correspond to cellular transcripts, probably due to the lack of rnase treatment during nucleic acid preparation, or the presence of some residual dna after the dnase treatment, although the s rrna gene was not amplified by pcr. viral sequences can also be misannotated to homologous cellular genes [ , ] , which relies on the low number and diversity of viral sequences in the databases. additionally, horizontal gene transfer between viral and host genomes can lead to incorrect annotation based on the closest homologous sequences [ , ] . misleading annotation is a frequent phenomenon in underexplored communities, such as environmental rna viruses where new schemes of classification are needed [ ] . viruses , , x for peer review of "unclassified rna viruses shim- " category in the ncbi taxonomy (~ % abundance; figure b ) and totiviriade family were also highly abundant in treated and untreated sewage samples from the eu [ , ] . the cystoviridae family (~ % abundance; figure b ) is the only ictv-recognized bacteriophage family detected at more than % abundance. these bacteriophages are known to be abundant in the gastrointestinal tract of vertebrates and as part of raw sewage samples [ ] . only recently was this family reported in metagenomic assessments of sewage samples using previously published viral metagenomes from pittsburgh, barcelona and addis ababa [ , ] . finally, the reoviridae family, represented by the genus rotavirus, accounted for ~ . % abundance. these human pathogenic viruses are routinely found in raw sewage samples using amplification techniques such as rt-pcr and rt-qpcr [ , ] ; however, they have been detected by hts in a recent investigation in wales, uk [ ] . since some human pathogenic viruses are seasonal (e.g., rotavirus and norovirus), their presence in wastewater can also vary, which could explain the absence of rotavirus in previous metagenomic surveys of sewage [ ] . the assignment of viral species through the last common ancestor allowed us to classify the known viral sequences by their putative host using the virus-host database [ ] , as described in [ ] . most of the sequences belong to viruses putatively infecting bacteria ( %) and invertebrates (~ %) in further sections, we only analyze proteins classified as having viral origin. most of the viral proteins in the trebal rna viral metagenome ( figure b ) were classified as belonging to the picobirnaviridae family ( %), followed by partitiviridae (~ %) and other families, such as totiviridae (~ %), cystoviridae (~ %) and reoviridae (~ %). in summary, most of classified viral sequences ( %) belong to families comprising group iii (dsrna viruses) of the baltimore classification. nevertheless, ssrna (e.g., virgaviridae and leviviridae), dsdna (e.g., myoviridae and siphoviridae), and ssdna (microviridae) viral families were detected, but their relative abundances were below % (supplementary table s ). it is important to emphasize that since we did not perform any amplification step (e.g., mda) before library construction, it is not expected that relative viral abundances were affected by amplification bias in any steps before library preparation. thus, a possible explanation for the low abundance of ssrna viruses is that the inclusion of an inactivation step using dnase at • c could potentially enhance the effect of natural rnases present in the sample, as has been described before [ ] . however, this must be confirmed experimentally using an ssrna virus as spike control subjected to the indicated conditions in the same type of matrix. despite possibly missing some viral types during the extraction procedure, the characterized viral rna metagenome still harbors a vast diversity of viruses within each family. in general, rna viral families identified here have been identified in other previous studies that describe the rna virus diversity of wastewater [ , , , [ ] [ ] [ ] . however, the viral community's specific taxonomic composition in the trebal has not been reported in other sewage studies. picobirnaviruses (pbvs) were dominant in sewage influent samples from wales, uk [ ] . likewise, pbvs were prevalent in sewage samples across the usa [ ] and have been proposed as a potential marker of fecal pollution [ ] . viral sequences identified as partitiviridae-like viruses included in the "unclassified rna viruses shim- " category in the ncbi taxonomy (~ % abundance; figure b ) and totiviriade family were also highly abundant in treated and untreated sewage samples from the eu [ , ] . the cystoviridae family (~ % abundance; figure b ) is the only ictv-recognized bacteriophage family detected at more than % abundance. these bacteriophages are known to be abundant in the gastrointestinal tract of vertebrates and as part of raw sewage samples [ ] . only recently was this family reported in metagenomic assessments of sewage samples using previously published viral metagenomes from pittsburgh, barcelona and addis ababa [ , ] . finally, the reoviridae family, represented by the genus rotavirus, accounted for~ . % abundance. these human pathogenic viruses are routinely found in raw sewage samples using amplification techniques such as rt-pcr and rt-qpcr [ , ] ; however, they have been detected by hts in a recent investigation in wales, uk [ ] . since some human pathogenic viruses are seasonal (e.g., rotavirus and norovirus), their presence in wastewater can also vary, which could explain the absence of rotavirus in previous metagenomic surveys of sewage [ ] . the assignment of viral species through the last common ancestor allowed us to classify the known viral sequences by their putative host using the virus-host database [ ] , as described in [ ] . most of the sequences belong to viruses putatively infecting bacteria ( %) and invertebrates (~ %) ( figure c ). other relevant groups belong to known viruses that infect humans (~ . %). finally, there was a small contribution of viruses infecting plants, fungi, unicellular eukaryotes, non-mammal vertebrates and non-human mammals ( figure c ). most bacterial viruses belong to the picobirnaviridae ( %), cystoviridae (~ . %) and levoviridae (~ . %) families, while invertebrate viruses are associated with the partitiviridae-like sequences in the "unclassified rna viruses shim- " group and totiviridae family. human viruses were composed exclusively of sequences from the reoviridae family. viruses , , of as discussed above, most of these viral families and their hosts have been reported in previous metagenomic wastewater studies [ , , , , ] . interestingly, the most abundant viruses in these studies belong to the virgaviridae family and the caudovirales order that infect plants and prokaryotes, respectively; however, these viruses appeared in low abundance in our sample [ , ] . the only rna-based metagenomic study that was methodologically similar to our study also reported a high abundance of pbvs related sequences and rotaviruses [ ] . the presence of pbvs sequences in the trebal wastewater, which were genetically close to those found in animal feces, could be related to farm runoff, since the location of this wwtp (figure ) is outside the urban zone of santiago and surrounded by many irrigation channels. even though farm waste should not end up in the trebal, whose exclusive purpose is the treatment of sewage from santiago city, negligent handling of this waste could cause this result. the presence and high abundance of bacterial viruses (bacteriophages) is frequent in wwtps. their numbers are in part due to the release of phages that infect intestinal bacteria by human or animal defecation, but also from new infections of bacteria whose natural niche is the sewage ponds and sludge [ ] . bacterial rna viruses are poorly understood in comparison with their dna counterparts that are commonly found in sewage viral metagenomic studies [ , ] . the international committee on taxonomy of viruses (ictv) has only recognized two rna bacteriophage families, the ssrna family leviviridae and the dsrna family cystoviridae [ ] . both families are represented by only genomes in the ncbi refseq database (january ), which is low when compared to the viral dna genomes in the same database. however, it has been proposed that the picobirnaviridae is a new family of rna bacteriophages based on the presence of bacterial ribosome binding sites (rbss) upstream of the coding sequences, and also due to the lack of any consistent epidemiologic association with animal and human diseases [ , , ] . in contrast to previous studies [ , ] , most of the sequences associated with rna bacteriophages that we found correspond to the ictv-unrecognized picobirnaviridae family and the cystoviridae family, with only a small fraction associated with the leviviridae family. all the known members of the cystoviridae family infect pseudomonas species, which are commonly present in eutrophic environments, such as sewage and the human body [ ] . therefore, the abundance of these viruses in the trebal metagenome can expand the known sequence space associated with this family (only genomes are currently available in the ncbi database) and contribute to a better understanding of the bacteriophage biology related to rna genomes. invertebrate virus categories mostly include sequences of those that putatively infect annelids and arthropods, which was expected since these phyla have high densities in sewage stabilization ponds and aquatic environments of wwtps [ , ] . in previous studies, a high prevalence of these invertebrate rna viruses was not reported since only recently was their vast diversity discovered and their sequences made available in the databases [ ] . human viruses (which in trebal correspond to rotavirus a and c) are common in sewage and come from feces, urine, and respiratory secretions of infected hosts [ ] . the most commonly identified viral pathogens in wastewater are adenovirus, enterovirus, hepatitis a and e viruses, norovirus, sapovirus, and rotavirus a [ ] . these viruses are considered a potential public health risk because they are usually found at high concentrations in raw sewage, and their removal efficiency in wwtps is not commonly assessed [ , ] . moreover, since wastewater is composed of the excreta of thousands to millions of inhabitants, it is a representative sample that can be used for epidemiological surveillance purposes [ ] . therefore, metagenomic analysis of wastewater can highlight the presence of viral strains that circulate within a population, while enabling the discovery of new viruses that spread between humans and that are outside surveillance programs. to uncover the untapped viral diversity present in the trebal metagenome, which was not possible to recover through direct mapping of viral sequences against databases, we searched for rdrp homologous sequences using hidden markov models (hmms) for the protein. subsequently, we estimated the genetic diversity of the rna viruses using an alignment-free comparison of the retrieved rdrp sequences with those available in the ncbi refseq database (figure ). the rdrp is the most essential and conserved protein in rna viruses [ ] . it catalyzes rna synthesis from rna templates and is responsible for viral genome replication and transcription processes [ ] . viruses , , x for peer review of figure . hierarchical clustering analysis of rna-dependent rna polymerase (rdrp) predicted protein sequences from trebal and ncbi refseq database based on bray-curtis amino acid distance (k = ). dendrogram was divided in main cluster based on the "unrooted" dendrogram. pie charts represent the frequency of sequences in each cluster classified by the putative host trough lca algorithm. bar charts represent the source (ncbi or trebal) from which sequences were retrieved inside each cluster. to assess the novelty of the most abundant viral sequences observed in trebal, namely those of the picobirnaviridae family, we performed a phylogenetic analysis. first, we identified the rdrp protein sequences inside the picobirnaviridae family and then filtered them by size to include only proteins ≥ aa, which is the size of the smaller full-length pbv rdrp in the ncbi nr database. next, we reconstructed a phylogenetic gene tree (figure ) using rdrps that met the filtering criteria and reference pbv sequences. our results show that seven trebal sequences were associated with the known pbv genogroups [ ] i and ii associated with vertebrate stools. interestingly, the rest of the environmental sequences ( ) formed three separate monophyletic clades. two of these exclusive trebal monophyletic groups, tg and tg ( sequences), could be considered sister clades of the known pbv genogroup three (giii) associated with invertebrate samples [ ] . in contrast, the third monophyletic group, tg , which contains ten trebal sequences, is between the pbv genogroups one (gi) and two (gii) [ ] , but closer to gii. therefore, tg can represent a highly divergent version of viruses that infect bacteria of the gastrointestinal tract from other vertebrates, such as domestic, farm or wild animals. this is highly probable since pbvs are ubiquitous in the feces of a vast range of animal species worldwide [ , , ] , including cattle, monkeys, dogs, cats, bats, horses, poultry and chickens [ ] . for instance, pbv rdrps sequences recovered from a metagenomic survey of bat stools in cameroon showed a large group of highly divergent sequences that were closely related to the pbv figure . hierarchical clustering analysis of rna-dependent rna polymerase (rdrp) predicted protein sequences from trebal and ncbi refseq database based on bray-curtis amino acid distance (k = ). dendrogram was divided in main cluster based on the "unrooted" dendrogram. pie charts represent the frequency of sequences in each cluster classified by the putative host trough lca algorithm. bar charts represent the source (ncbi or trebal) from which sequences were retrieved inside each cluster. we identified predicted proteins as rdrps using protein hmms in the pfam database [ ] . most of the rdrp sequences ( %) were classified as unknown since they do not align with any known protein in the ncbi nr database under standard cutoff parameters (e-value ≤ × − and score ≥ ). the latter is expected since the software that was used is designed to detect remote homologs in the most sensitive way, based on the strength of the underlying probability models (hmms) [ ] . the remaining % of the rdrps corresponded to known viruses that putatively infect invertebrates ( %), bacteria ( %), unicellular eukaryotes ( %), fungi ( %), humans ( %), and plants ( %). hierarchical clustering analysis based on the genetic distances of rdrps showed well-defined genetic clusters, five of which were formed exclusively by ncbi sequences and five of which were formed mostly by the trebal sequences ( figure ) . moreover, the sequences were clustered by a single host only in three of the clusters (e.g., human viruses of cluster c , and plant viruses of clusters c -c ). additionally, most of the rdrps formed a continuous sequence space represented by highly heterogeneous clusters (e.g., c -c and c -c ). the latter feature was expected due to the orthologous nature of viral rdrps and their degree of structural conservation inside the riboviria viruses , , of realm [ , ] . despite this, a closer inspection of the clusters with stricter cutoffs could reveal more specific associations. the animal virus cluster c was formed exclusively by ncbi reference sequences inside the astroviridae and coronaviridae families, but no further precision regarding the host was feasible. astroviruses are a commonly known cause of viral gastroenteritis in animals and humans [ ] . specifically, sequences of cluster c corresponded to avian astrovirus associated with poultry and wild aquatic birds from a study in asia [ ] . coronaviruses have a global distribution and infect a wide range of mammals and birds. they can cause respiratory and enteric infections that are usually mild, but severe infections of the respiratory system can develop, such as severe acute respiratory syndrome (sars) and the infection currently responsible for a global pandemic, sars-cov- [ ] . coronaviridae sequences from cluster c were described in two studies from that investigated the viral population in bats in china and vietnam [ , ] . plant virus clusters c -c exclusively represent ncbi reference sequences of the luteoviridae (c ) and bromoviridae (c ) families. both viral families have a global distribution and are transmitted by specific aphid vectors [ , ] . these two viral families have a broad host range of genera within many plant families, causing necrosis in most of their hosts [ , ] . finally, the human virus cluster c was formed exclusively by ncbi sequences of the norovirus genogroup ii (nov gii). noroviruses are a genetically diverse genus within the caliciviridae family that can cause acute gastroenteritis in mammalian hosts [ ] . most of the human noroviruses belong to genogroups gi and gii, where nov gii is usually the causal agent of acute gastroenteritis outbreaks [ , ] . these pandemic characteristics are probably related to the nov gii epidemiology, which resembles that of influenza a viruses, with the emergence of new variants every - years that replace the previously established variant [ ] . interestingly, bacterial viruses form two clusters, c and c , which group picobirnaviridae reference sequences from the ncbi, recovered from animal stools and trebal new pbvs, together with unknown sequences that escaped our analyses using direct mapping against ncbi databases. this reflects the great diversity within the picobirnaviridae family, which is not represented in current databases. taken together, our results show that metagenomic surveys of rna viruses in sewage samples and the use of hmms could uncover extraordinary viral diversity through the detection of remote homologs in these human-impacted environments. additionally, the use of alignment-free genetic distances, such as the bray-curtis distance used here [ ] , can provide a reliable method for clusterization and classification based on related sequences for a large number of sequences, such as those generated by metagenomics methods. to assess the novelty of the most abundant viral sequences observed in trebal, namely those of the picobirnaviridae family, we performed a phylogenetic analysis. first, we identified the rdrp protein sequences inside the picobirnaviridae family and then filtered them by size to include only proteins ≥ aa, which is the size of the smaller full-length pbv rdrp in the ncbi nr database. next, we reconstructed a phylogenetic gene tree ( figure ) using rdrps that met the filtering criteria and reference pbv sequences. our results show that seven trebal sequences were associated with the known pbv genogroups [ ] i and ii associated with vertebrate stools. interestingly, the rest of the environmental sequences ( ) formed three separate monophyletic clades. two of these exclusive trebal monophyletic groups, tg and tg ( sequences), could be considered sister clades of the known pbv genogroup three (giii) associated with invertebrate samples [ ] . in contrast, the third monophyletic group, tg , which contains ten trebal sequences, is between the pbv genogroups one (gi) and two (gii) [ ] , but closer to gii. s ). we found that all except one of the full sequences (those not predicted in contig edges) contain an rbs, being aggagg and aggag, which are the most frequent motifs. this matches with other sewage pbvs that have the aggagg motif in % of the full rdrp [ ] . this result is relevant because only prokaryotic viral families contain species whose genomes are highly enriched in rbs sequences [ , ] . finally, pbvs have been assumed to be animal pathogens based on inferences from a few studies reporting the virus in diarrhea stool samples. however, they have not been cultured in animal cell lines, nor do they have any consistent epidemiologic association with diarrhea [ ] . rotavirus species are classically defined by their major capsid protein vp , whereas rotavirus genotypes are defined based on their outer capsid proteins vp and vp [ ] . in chile, over the last therefore, tg can represent a highly divergent version of viruses that infect bacteria of the gastrointestinal tract from other vertebrates, such as domestic, farm or wild animals. this is highly probable since pbvs are ubiquitous in the feces of a vast range of animal species worldwide [ , , ] , including cattle, monkeys, dogs, cats, bats, horses, poultry and chickens [ ] . for instance, pbv rdrps sequences recovered from a metagenomic survey of bat stools in cameroon showed a large group of highly divergent sequences that were closely related to the pbv giii [ ] , as is the case of tg and tg . this last evidence provides a probable origin for tg and tg since sewage ponds are known to be a feeding area for insectivorous bats [ ] . new evidence of pbv sequences from sewage samples shows that sewage-recovered rdrps were spuriously distributed between many pbv genogroups [ ] . the latter reinforce the idea that pbvs do not infect mammals but are a new family of rna bacteriophages, due to the consistent presence of bacterial rbss upstream of the coding sequences [ , , ] . to test this hypothesis, we searched for prokaryotic rbs motifs in the rdrp sequences from the pbvs (supplementary table s ). we found that all except one of the full sequences (those not predicted in contig edges) contain an rbs, being aggagg and aggag, which are the most frequent motifs. this matches with other sewage pbvs that have the aggagg motif in % of the full rdrp [ ] . this result is relevant because only prokaryotic viral families contain species whose genomes are highly enriched in rbs sequences [ , ] . finally, pbvs have been assumed to be animal pathogens based on inferences from a few studies reporting the virus in diarrhea stool samples. however, they have not been cultured in animal cell lines, nor do they have any consistent epidemiologic association with diarrhea [ ] . rotavirus species are classically defined by their major capsid protein vp , whereas rotavirus genotypes are defined based on their outer capsid proteins vp and vp [ ] . in chile, over the last ten years, globally common rotavirus genotypes, such as g p ( ), g p( ), g p( ) and g p( ) have alternated in their dominance, while emerging genotypes, such as g p ( ), have only recently been reported [ ] . here, using pfam annotation of the trebal predicted proteins, we recovered vp , vp and vp human rotavirus proteins. local alignment-based classification ( figure ) shows that the most abundant rotavirus species belongs to human rotavirus a, which is the most common cause of hospitalization due to viral gastroenteritis worldwide [ ] . however, it is interesting to note that the presence (in low abundance; . %) of human rotavirus c sequences that are closely associated to asian strains, have, to date, only been reported in chile through personal communication. likewise, the most abundant rotavirus genotypes were g ( %), g ( %) and g ( %) and p ( %) and p ( %). the segmented nature of rotavirus genomes does not allow us to infer the g-p genotype classification because we do not know which combinations of vp and vp were inside the same viral particle. however, it is highly probable that the most abundant g genotypes were combined with the most abundant p genotypes-for example, g p( ), g p( ) or g p ( ) . of these genotypes, g p( ) was recently reported as an emergent rotavirus strain in a medium-sized city near santiago between and [ ] . this strain has not been previously reported in south or north america, but has a highly similar identity to sequences detected between and in asia [ ] . in the same line of evidence, the g sequences found in trebal also shared a % nucleotide identity with sequences detected in in thailand [ ] and between and in japan [ ] . the rotavirus g genotype is also an emergent strain, with most of the reports concentrated in europe between - , but spurious circulation has been reported since the s [ , ] . in our analysis, the g recovered from the trebal sequences are closely related ( % nucleotide identity) to a g p( ) strain detected in germany in and to a g p( ) strain detected in belgium in ; yet, to our knowledge, this is the first report of rotavirus g in chile. these results emphasize the relevance of sewage viromes as epidemiological surveillance tools. likewise, our study demonstrates the advantage of using viral metagenomics for this task, which, despite using short sequences, can deliver reliable results that can later be confirmed by other methodologies. the latter point is especially relevant for rotavirus since surveillance based on pcr has shown serious bias related to primer specificity [ ] . these results are especially significant for chile, as it is one of the few south american countries that has not implemented a national rotavirus vaccination program [ ] . therefore, identification of genotypes that have not been previously reported in chile represents a first step in rotavirus prevention. furthermore, this can be used to generate valuable information to improve or implement new vaccination programs against this disease worldwide. this study explored the use of viral metagenomics to discover rna viruses in sewage and it is the first insight into the wastewater virosphere from a large city in south america. we have demonstrated the utility of metaviromics for the discovery of new groups and viral genotypes associated with known families. this is especially important due to the underrepresentation of pbvs in databases and the presence of uncommon rotavirus genotypes that are usually beyond the range of pcr-based surveillance. 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diarrhea in bulgaria this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to thank to christina ridley for her help in the language editing of this manuscript and for her valuable opinion on it. we are grateful to aguas andinas staff, marcela etcheberrigaray, jacqueline pizarro, and christian sepulveda, for their invaluable support in obtaining the sewage samples. the authors declare no conflict of interest. key: cord- -l bnxi authors: huang, jianping; mao, tingting; li, shufei; wu, lianpeng; xu, xueqin; li, huanzheng; xu, chenyang; su, feifei; dai, jianyi; shi, jichan; cai, jing; huang, chongquan; lin, xuan; chen, dong; lin, xiaoling; sun, baochang; tang, shaohua title: long period dynamics of viral load and antibodies for sars-cov- infection: an observational cohort study date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: l bnxi abstract objective to investigate the dynamics of viral rna, igm, and igg and their relationships in patients with sars-cov- pneumonia over an -week period. design retrospective, observational case series. setting wenzhou sixth peoples hospital participants thirty-three patients with laboratory confirmed sars-cov- pneumonia admitted to hospital. data were collected from january to april , . main outcome measures throat swabs, sputum, stool, and blood samples were collected, and viral load was measured by reverse transcription pcr (rt-pcr). specific igm and igg against spike protein (s), spike protein receptor binding domain (rbd), and nucleocapsid (n) were analyzed. results at the early stages of symptom onset, sars-cov- viral load is higher in throat swabs and sputum, but lower in stool. the median (iqr) time of undetectable viral rna in throat swab, sputum, and stool was . ( . - ) days, ( . - . ) days, and ( . - ) days, respectively. in sputum, patients ( . %) had undetectable viral rna within days (short persistence), and ( . %) had persistent viral rna more than days (long persistence). three patients ( . %) had a detectable relapse of viral rna in sputum within two weeks of their discharge from the hospital. one patient had persistent viral rna for days or longer. the median (iqr) seroconversion time of anti-s igm, anti-rbd igm, and anti-n igm was . ( . - . ) days, ( - ) days, and ( - ) days, respectively. the median (iqr) seroconversion time of anti-s igg, anti-rbd igg, and anti-n igg was ( . - . ) days, ( - ) days, and ( - ) days, respectively. by week after symptom onset, igm were negative in many of the previously positive patients, and igg levels remained less than % of the peak levels in more than % of the patients. in about % of the patients, anti-rbd igg levels were -times higher in convalescence than in acute phase. sars-cov- rna coexisted with antibodies for more than days. anti-rbd igm and igg levels, including anti-rbd igm levels at presentation and peak time, were significantly higher in viral rna short persistence patients than in long persistence patients. conclusion this study adds important new information about the features of viral load and antibody dynamics of sars-cov- . it is clear from these results that the viral rna persists in sputum and stool specimens for a relatively long time in many patients. anti-rbd may also serve as a potential protective antibody against sars-cov- infection, as viral persistence appears to be related to anti-rbd levels. earlier treatment intervention also appears to be a factor in viral persistence. there are several reports about the serum antibodies against sars-cov- . however, most of them evaluate diagnostic accuracy. only two articles report dynamics of sars-cov- viral rna and antibodies with serial samples, but the observation periods are within days. none of the studies investigate the profiles of sars-cov- viral load and antibodies in a long period. three reports investigate profiles in respiratory samples, but there are no reports on the dynamics of the viral load in stool samples. in both sputum and stool, sars-cov- rna persists for a long time. the anti-rbd antibodies may involve in the clearance of sars-cov- infection. after eight weeks from symptom onset, igm were negative in many of the previously positive patients, and igg levels remained less than % of the peak levels in more than % of the patients. in about % of the patients, anti-rbd igg levels increased -time higher in convalescence than in acute phase. long persistence of sars-cov- viral rna in sputum and stool presents challenges for management of the infection. the igm/igg comb test is better than single igm test as a supplement diagnostic tool. anti-rbd may be a protective antibody, and is valuable for development of vaccines. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint introduction a novel coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ), has spread worldwide. [ ] [ ] [ ] as of april , , more than , , confirmed cases and more than , deaths have been reported. the clinical characteristics of sars-cov- pneumonia have been well defined. according to a large-scale epidemiological study in china, among the confirmed cases, . % were aged between - years, . % were men, . % were considered as mild pneumonia, and . % have died. common symptoms include fever, cough, fatigue, and lymphopenia etc. [ ] [ ] [ ] on admission, half of the patients present typical radiological ground-glass opacity on chest computed tomography (ct). clearance of viral rna and appearance of specific antibodies are essential for recovery from viral infection. sars-cov- viral load in respiratory samples rapidly increases soon after symptom onset and peaks at around - days. [ ] [ ] [ ] during the convalescence period, the clearance of viral rna in patients' stool is delayed compared to oropharyngeal swabs. as recommended by the national health commission of the people's republic of china, the criteria for discharge from the hospital are relieved symptoms and two successive negative viral nucleic acid results from respiratory samples separated by at least a hour sampling interval. it has been reported that some patients have had a relapse of the viral rna after discharge from the hospital. , however, this report included only four patients, and thus cannot show a complete picture of the patients discharged from hospitals. at present, reports about the profile of sars-cov- viral load have been mainly focused on relatively short periods (less than one month). the long-term dynamics of the viral load remain unclear. serological responses in patients are essential for understanding the immunological mechanisms and the recovery process of viral infection. it is also important for the development of serological diagnostic tools. several assays have been developed for the detection of specific igm and igg against sars-cov- . [ ] [ ] [ ] these studies have focused either on the evaluation of detection methods, or the diagnostic value of igm and igg for sars-cov- infection. , only two studies investigated the dynamics of igm and igg. those studies reported that the median seroconversion time of igm and igg was and days, respectively, and more patients had earlier seroconversion for igg than igm. due to the lack of serial results from the same cohort of patients over a longer period, the profile of specific igm and igg against sars-cov- remains unclear. until now, antibody persistence after patients have been discharged from the hospital has been rarely reported, though this information is all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint crucial for evaluating the immune profile of the recovered patients. thus, the relationship between viral clearance and serological response has not been well investigated. in this study, we investigated the profiles of viral rna, igm, and igg in a group of patients with confirmed sars-cov- pneumonia over an -week period after symptom onset. baseline characteristics and serial results of viral rna in throat swabs, sputum, and stool specimens were monitored. specific igm and igg against spike protein (s), spike protein receptor binding domain (rbd), and nucleocapsid (n) were also measured. in this way, the long-term relationship between viral rna persistence and the dynamics of sars-cov- antibodies have been more clearly established. this retrospective study was completed in wenzhou sixth people's hospital, wenzhou central hospital group, one of the designated hospitals to treat patients with sars-cov- pneumonia. a total of patients admitted to wenzhou sixth people's hospital, wenzhou central hospital group, from january to february , were included in this study. among the patients, ( . %) were moderate, and two were severe sars-cov- pneumonia according to interim guidance issued by the who and national health commission of the people's republic of china. the patients were followed up for - days (median, days) after discharge from the hospital. this study was approved by the ethics commission of wenzhou central hospital (l - - ). patient consent form was signed by patients themselves. a team of trained physicians and medical students reviewed all available electronic medical records, radiological findings, and laboratory examinations for all hospitalized patients with confirmed sars-cov- infection. data were recorded on a standardized data collection form. demographic data, symptom onset time, clinical features, radiological findings, routine laboratory results, and the results of sars-cov- viral rna in throat swabs, sputum, and stool samples were recorded during hospitalization and follow-up. results of specific igm and igg antibodies against sars-cov- s, rbd, and n were measured during hospitalization and follow-up. any missing or uncertain records were clarified through communication with the physicians responsible for the patients in question, or with the patients themselves. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. continuous variables were presented as medians with interquartile ranges (iqrs) and means with standard deviations (sd). for categorical variables, percentages of patients in each category were analyzed. differences between patients with sputum viral rna short-persistence (viral rna undetectable within days) and those with long-persistence (viral rna persists more than days) were assessed by two sample t test or the wilcoxon ranks sum test, depending on parametric or nonparametric data for continuous variables. spearman's correlation analysis was used to assess relationships between parameters. all analyses were done with spss version . (ibm, armonk, ny, usa). tests were two-sided with significance assessed at the . level. the funders had no role in this study. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. a total of patients admitted to wenzhou sixth people's hospital, wenzhou central hospital group, from january to february , , were included in this study. the median age of the patients was years old (range, - ) and ( . %) of them were less than years old. among the patients, ( . %) were men, and ( . %) had comorbid illness (supplemental table ) the most common symptoms were fever ( , . %), cough ( , . %), expectoration ( , . %), fatigue ( , . %), and diarrhea ( , . routine laboratory parameters at admission and the first return visit time were available for analysis. most of the laboratory parameters at admission were normal in all of the patients. abnormal parameters observed in the patients at admission were abnormal white blood cell count ( , . %), neutrophil all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint count ( , . %), lymphocyte count ( , . %), platelet count ( , . %), c-reactive protein (crp) ( , . %), creatinine kinase (ck) ( , . %), lactate dehydrogenase (ldh) ( , . %), blood urea nitrogen (bun) ( , . %), and blood creatinine (cr) ( , . %). almost all of the patients had normal laboratory parameters upon their return visit one week after discharge (supplemental table ). all of the patients received antiviral treatment during hospitalization. atomized interferon, lopinavir and ritonavir, arbidol, ribavirin, and lianhuaqingwen (chinese traditional medicine) were used in combination. atomized interferon was used in all patients. lopinavir and ritonavir was used in ( . %) of the patients. lianhuaqingwen, which was believed to relieve fever and cough, was used in for more than days. three patients ( . %) showed a detectable relapse of viral rna in sputum when they were tested two weeks after discharge from the hospital. one patient had persistent viral rna for days or longer. sars-cov- viral loads at the first detection after onset were not corelated with age all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (figures , , and ) . when comparing the igg levels between the acute and all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint convalescent phases, a -fold increase was observed in patients ( . %) for anti-s, patients ( . %) for anti-rbd, and patients ( . %) for anti-n, respectively. since detection of sars-cov- rna in sputum is a routine test during treatment, and is commonly used in clinical settings, the relationship between the persistence of sputum viral rna and antibodies was analyzed (figures , , and ). compared to the viral rna long persistence group, patients in the short persistence group had higher anti-s ig g (p= . ), anti-rbd igm (p= . ), and anti-rbd igg levels (p= . ). anti-rbd igm levels at seroconversion (p= . ) and the peak level of anti-rbd igm (p= . ) were also significantly higher in the short persistence group. it was also observed that the time from symptom onset to hospital admission was significantly different between the short persistence and long persistence groups (p= . ). we investigated the serial viral load and dynamics of antibodies from patients infected with sars-cov- over an eight-week period following the onset of symptoms. in throat swab specimens, the sars-cov- viral load was high at the beginning of infection but declined quickly. the viral load in stool samples was low in the initial period but declined slowly. in both sputum and stool, viral rna persisted for a long time. simultaneous seroconversion of igm and igg was observed in most of the patients. igm peaked in the fourth week (except for anti-n igm, which peaked in the second week), and igg peaked between the fourth and fifth week after the onset of symptoms. high anti-rbd antibody levels were associated with short persistence of sars-cov- rna in sputum. sars-cov- viral load varies across different types of specimens. in this study, we observed that in throat swab samples, the viral load was high in the early stages of infection, but it became undetectable by three weeks following symptom onset. pan et al previously reported the viral load in several types of specimens from two patients , and to and colleagues have reported the viral load in posterior oropharyngeal saliva samples. combining those studies with our own results here, we can conclude that in upper respiratory samples, sars-cov- viral load is high shortly after symptom onset but declines quickly. the detection of sars-cov- rna in respiratory specimens is included in the guidelines for monitoring treatment. according to these guidelines, patients will be discharged from hospitals if they have experienced relief of symptoms and had two successive negative viral rna tests from respiratory all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint specimens across at least a h sampling interval. we observed that in sputum, viral load declined with time, and most patients were undetectable by week after the onset of symptoms. however, positive viral rna was detected in about % of our patients two weeks after their discharge from the hospital. in addition, one patient in our cohort had positive viral rna for days after symptom onset and would go on to persist even longer. this means that sars-cov- may persist for a long time in some patients. whether "relapsed patients" are truly relapsed, or the result of false negative viral rna tests, needs to be explored further. our study also investigated sars-cov- viral load in serial stool specimens. the viral load in stool was lower than what was observed in throat swabs and sputum at the beginning of infection but declined slowly. many patients had persistent viral rna in stool for more than five weeks. since fluctuations of viral rna in respiratory specimens were observed in most of the patients, false negative results will be inevitable in such specimens. detection of viral rna in stool specimens may thus act as a useful complement for monitoring treatments. currently, serological tests are widely used for diagnosis, and sars-cov- igm or igm/igg tests have been approved under emergency use authorization (eua) in many countries. in most of the known viral infections, seroconversion of igm occurs earlier than seroconversion of igg. [ ] [ ] [ ] [ ] hence, specific igm is often used as a diagnostic marker for acute viral infection. our results indicate that in sars-cov- infected patients, simultaneous seroconversion of igm and igg was observed in % of the patients. almost no patient had earlier seroconversion of igm than igg. nearly % of the patients had negative igm across the entire study period. to and colleagues also reported that in most of sars-cov- infected patients, seroconversion of igg was earlier than igm, and they thought that this might be due to low sensitivity of their igm test. combining the results together, it appears that a single igm test may not be a reliable index for an auxiliary diagnosis of sars-cov- infection. convalescent igm/igg testing will likely be better than single igm for patients for whom nucleic acid tests are not available, or those who have typical sars-cov- symptoms but are negative for nucleic acid detection. the coexistence of sars-cov- viral rna and specific antibodies has been reported by several studies, , , and has been observed in patients with severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome (mers). , our results shown that in some patients, sars-cov- rna coexisted with antibodies for more than days. these results raise concerns about all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint the existence of protective antibodies against sars-cov- , since one of the key roles of protective antibodies is to clear viral infection. to and colleagues reported that anti-sars-cov- -n or anti-sars-cov- -rbd igg levels correlated with virus neutralization titre. in this study, we found that anti-sars-cov- igg peaked within about days after symptom onset. in about % of the patients, anti-rbd igg increased more than four-fold within one to two weeks after seroconversion and persisted for more than four weeks. we also found that anti-rbd igm and igg levels, including anti-rbd igm levels at presentation and peak time, were significantly higher in patients showing viral rna short persistence than in patients showing long persistence. these data imply that the anti-rbd antibodies may be involved in the clearance of sars-cov- infection and would be potential candidates for a protective antibody. further studies are needed to identify whether rbd is a useful epitope for developing vaccines against sars-cov- . we also observed that the length of time from symptom onset to hospital admission was associated with sars-cov- viral rna clearance. in addition, the seropositive rate for anti-s and anti-rbd igm was significantly higher in viral rna long persistence patients. based on current evidence, the relationship between late treatment and seropositivity for anti-s or anti-rbd igm is undetermined. it is possible that the higher seropositive rate is due to a longer infection time and delayed treatment. however, our data imply that shorter delay is linked to better recovery. "early identification, early hospitalization" is thus likely to be an efficient strategy for the prevention and management of sars-cov- infection. our study has some limitations. first, the patient number in our study is relatively small, and only patients were included in this study. this is, of course, a common weakness of studies of emerging infectious diseases. second, samples for viral rna and antibody detection were collected weekly and not on a daily basis. it was also difficult to collect samples at exactly one-week intervals. in some patients, initial samples were even missed due to the emerging situation for sars-cov- infection. in conclusion, this study adds important new information about the features of viral load and antibody dynamics of sars-cov- . it is clear from these results that the viral rna persists in sputum and stool specimens for a relatively long time in many patients. anti-rbd may also serve as a potential protective antibody against sars-cov- infection, as viral persistence appears to be related to anti-rbd levels. earlier treatment intervention also appears to be a factor in viral persistence. further all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint studies are needed to better understand the virological and immunological features of sars-cov- infection, which are important for the prevention, treatment and control of the spread of this disease. contributors: sht designed the study, and had full access to all data in the study. jph and ttm wrote the report and contributed equally to this paper. sht patient consent: written informed consent was waived due to the emergency state of the disease. no additional data available. the lead author and the manuscript's guarantor affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. a comparison of viral loads across different types of specimens is also shown (d). each column represents the mean levels of the antibody in one week. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint a novel coronavirus from patients with pneumonia in china emergence of novel coronavirus and covid- : whether to stay or die out? covid- in europe: the italian lesson clinical features of patients infected with novel coronavirus in wuhan novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of coronavirus disease in china sars-cov- viral load in upper respiratory specimens of infected patients viral load of sars-cov- in clinical samples temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients national health commission of the people's republic of china. guidelines for the diagnosis and treatment of novel coronavirus infected pneumonia (version ) recurrence of positive sars-cov- rna in covid- : a case report positive rt-pcr test results in patients recovered from covid- development and clinical application of a rapid igm-igg combined antibody test for sars-cov- clinical significance of igm and igg test for diagnosis of highly suspected covid- infection antibody responses to sars-cov- in patients of novel coronavirus disease clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance epidemiologic features and clinical course of patients infected with sars-cov- in singapore profile of specific antibodies to the sars-associated coronavirus longitudinal profile of immunoglobin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus sars-cov and emergent coronaviruses: viral determinants of interspecies transmission identification of a novel coronavirus in patients with severe acute respiratory syndrome evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted april , . all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- - muareue authors: kidszun, andré; klein, lena; winter, julia; schmeh, isabella; gröndahl, britta; gehring, stephan; knuf, markus; weise, kerstin; mildenberger, eva title: viral infections in neonates with suspected late-onset bacterial sepsis—a prospective cohort study date: - - journal: am j perinatol doi: . /s- - sha: doc_id: cord_uid: muareue objective the aim of our study was to evaluate the occurrence of viral infections in infants with suspected late-onset bacterial sepsis in a neonatal intensive care unit. methods in a prospective study, infants with suspected late-onset bacterial sepsis underwent viral testing alongside routine blood culture sampling. using a multiplex reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay, nasopharyngeal aspirates were analyzed for adenovirus, respiratory syncytial virus (rsv), influenza virus a and b, h n virus, parainfluenza virus to , metapneumovirus, coronavirus, and picornavirus. stools were examined for adenovirus, rotavirus, norovirus, and enterovirus. results between august and march , data of infants with episodes of suspected late-onset bacterial sepsis were analyzed. six infants were diagnosed with a respiratory viral infection ( × rsv, × picornavirus). blood culture–proven bacterial sepsis was detected in infants. neither viral–bacterial coinfections nor polymerase chain reaction positive stool samples were found. conclusion respiratory viruses can be detected in a considerable number of neonates with suspected late-onset bacterial sepsis. in contrast, gastrointestinal viral or enterovirus infections appear uncommon in such cases. substantiated these findings and focused on respiratory viruses as frequent pathogens. [ ] [ ] [ ] [ ] besides, viral outbreaks in nicus have been reported, indicating significant impact on health outcomes and costs. , the emergence of new detection methods such as the multiplex reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay (multiplex rt-pcr elisa) has tremendously facilitated the detection of viruses. , multiplex rt-pcr elisa allows screening for a large number of different viruses from a single specimen. there is first evidence that viral infections in term and preterm neonates during birth hospitalization have a significant impact on short-and long-term morbidity. [ ] [ ] [ ] in a preceding feasibility study, we demonstrated that this technique can be used to detect respiratory viruses in cases of clinical deterioration in the nicu. the aim of the present study was to further elucidate the occurrence of viral infections in neonates with suspected late-onset sepsis. we performed a single-center study in the nicu of the university medical center of the johannes gutenberg university mainz. this level iii unit has intensive care cots, which are allocated to three rooms. two rooms have five and four cots each; the other is a single room. the unit usually admits patients from the delivery room and the postnatal ward. both are located nearby in the same building. previously discharged patients or patients from the special care unit are only readmitted in exceptional cases. the unit is / open to visitors. due to limited space, the number of simultaneous visitors is restricted to two per patient. parents and other visitors are instructed in proper hand hygiene and are repeatedly asked to wear a facemask in case of signs of a minor respiratory tract infection. siblings of all ages may visit the nicu if clinical examination verifies no signs of infection. this prospective cohort study was conducted from august to march . the local ethics committee approved the study. parents were approached for informed consent on admission. infants were eligible for the study from hours onward after admission, whether following birth or later in life. infants in whom intravenous antibiotic treatment was initiated due to suspected late-onset bacterial sepsis were enrolled consecutively. enrolled infants could be tested repeatedly in the event of independent episodes of suspected sepsis. the decision to start intravenous antibiotics was at the clinician's discretion. blood culture sampling was mandatory prior to starting antibiotics. one positive blood culture was considered sufficient to diagnose a bacterial bloodstream infection, also in cases of coagulase-negative staphylococci. enrolled infants were tested for respiratory and gastrointestinal viruses within the next hours. nasopharyngeal aspirates (npas) were analyzed using an in-house multiplex rt-pcr elisa as described previously. , this assay included adenovirus, respiratory syncytial virus (rsv), influ-enza virus a and b, h n virus, parainfluenza virus - , metapneumovirus, coronavirus, and picornavirus. stool samples were tested for rotavirus, adenovirus, and norovirus using commercially available antigen tests (ridascreen, r-biopharm, darmstadt, germany) and for enterovirus via inhouse pcr at the local institute for virology. in case of a positive antigen test, a confirmation test using pcr was performed from the same sample. only virus detections via pcr were considered as positive results. the infants' clinical and demographic characteristics, laboratory markers, and clinical presentation were immediately documented in a written case report form. following pseudonymization, study personnel transferred the data into a prespecified microsoft excel database (microsoft office - , versions . - . ). before saving, data of all infants with pathogen detection were additionally checked for correctness and completeness by the corresponding author using the original patient files. the results of the viral tests were not blinded to the clinicians in the unit. in case of a positive test, infants were isolated or hospitalized in cohorts whenever possible. to avoid feelings of guilt especially in affected parents, we resigned the idea of screening close contacts intentionally. during the study period, , infants were admitted to our nicu. parents had given their informed consent in all enrolled cases. in total, infants were eligible for enrollment in the study but infants had to be excluded due to incomplete or nonanalyzable samples. finally, patients and sepsis episodes were analyzed. this analysis included the sepsis episodes reported in our feasibility study. clinical and demographic data of enrolled cases are presented in ►table . several infants experienced multiple episodes of suspected late-onset sepsis. a total of infants underwent one, infants two, and infants three or more sepsis evaluations. in % of all episodes with suspected lateonset bacterial sepsis, neither a bacterial nor a viral pathogen was detected. a positive respiratory virus test was observed in six infants ( . %). rsv was detected in two and picornavirus in four infants. one infant had three and another infant two consecutive episodes with a positive pcr result for picornavirus. these episodes were each identified as new clinical deteriorations. the infants had recovered and antibiotics had been stopped between all episodes. viral testing was not performed during asymptomatic intervals. the two infants with rsv infection had not yet received rsv immunization. the characteristics of the virus-positive infants are summarized in ►table . a virus and a positive blood culture were never detected simultaneously. however, one infant was diagnosed with both, a viral infection (picornavirus, days postnatal) and in the later course with a proven bacterial bloodstream infection (klebsiella oxytoca, days postnatal). respiratory viral infections did not occur in clusters in the nicu and none of the virus-positive infants had been discharged home previously. stool examinations showed two positive antigen tests for adenovirus and one positive test for norovirus. when retested via pcr, none of the three positive antigen tests could be confirmed. enterovirus and rotavirus were not detected in any of the studied infants. during the study period, no vaccinations for rotavirus had been performed. characteristics of the infants with a blood cultureproven sepsis are presented in ►table . during the study period, no fungal bloodstream infection was diagnosed. virus detection in the nicu is an important issue that has been considered to contribute to successful antibiotic stewardship, provided that specific, validated algorithms can be implemented in clinical routine. although evidence is still lacking that the use of multiplex rt-pcr elisa to detect respiratory viruses provides a medical or economic benefit to neonatal or pediatric patients, potential benefits from virus detection may arise from early implementation of effective isolation and infection control measures to prevent viral spread. this is of particular importance since modern, family-centered neonatal care that includes kangaroo care and units that are always open to parents and siblings bears an increased risk of virus transmission. in our study, viral infections always presented with respiratory symptoms, albeit their clinical characteristics were highly heterogeneous. when compared with bacterial infections, nosocomial viral infections occurred rather late during birth hospitalization. it is unclear whether this is due to physiological factors, such as decreasing maternal passive immunity or simply due to long-term hospitalization. affected infants were either extremely premature infants or term infants with a pulmonary or thoracic disease that presented with respiratory distress. these might be the patients at high risk in whom viral testing should definitely be considered in cases of suspected late-onset sepsis. multiplex rt-pcr elisa from nasopharyngeal specimens has been previously used to detect respiratory viruses in nicus. [ ] [ ] [ ] our results are very similar to those recently published by ronchi et al. their study had a comparable design and included a similar number of infants. a comparable incidence of % of viral respiratory infections in cases of sepsis evaluation in the nicu was observed. likewise, rhinovirus, which is a member of the picornavirus family, was the dominating pathogen. bennett et al performed a surveillance study of viral respiratory infections in two nicus. all infants of a gestational age < weeks were tested twice a week using multiplex rt-pcr elisa. the authors found that % of all tested infants had a positive virus screen at least once during their birth hospitalization. in contrast to our study, they identified infants with a positive virus screen who had no or only minimal clinical symptoms. another study by gonzalez-carrasco et al used a mixed design of weekly surveillance and additional tests in the event of respiratory symptoms. in that study, . % of all samples were positive. a total of infants were affected, and of them were symptomatic. again, rhinovirus was most frequently found ( % of the cases). it can be doubted whether the detection of a virus in npas really proves an active respiratory viral infection. this is in contrast to previous studies suggesting that picornaviruses/rhinoviruses are major causative pathogens in infants with lower respiratory tract infection. , rhinovirus may be detectable for up to to weeks following infection, but shorter clearance of around weeks has also been described. since active shedding and clearance occurs, repeated detection of rhinovirus may rather reflect resolving or subclinical upper respiratory tract infection than colonization. it remains unclear how these results should be applied to preterm infants during birth hospitalization. in large-scale future studies, a screening cohort of asymptomatic infants and serial quantitative pcr analyses might help to evaluate whether clinical deteriorations may be caused by a reactivation of a persisting subclinical viral infection. to allow for discrimination of harmless colonization and true infection, a future study would ideally consist of three cohorts: ( ) asymptomatic virus negative, ( ) asymptomatic virus positive, and ( ) symptomatic virus positive. the additional implementation of host-based rt-pcr gene expression assays into clinical practice might offer a significantly improved tool to diagnose viral infections. furthermore, the range of viruses, which can be screened for, should be expanded. quite recently, a study with a comparable design investigated human parechovirus in blood samples of all cases of suspected late-onset sepsis in the nicu and found a high infection rate of %. further research should also focus on modes of virus transmission, patients at risk, and the impact of preventive measures and long-term outcomes of viral infections during birth hospitalization. the main results of the present study have been orally presented at the th international conference on clinical neonatology (torino, italy, september - 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nau l authors: wu, chang-jer; chan, yi-lin title: antiviral applications of rnai for coronavirus date: - - journal: expert opin investig drugs doi: . / . . . sha: doc_id: cord_uid: nau l until the appearance of severe acute respiratory syndrome (sars), caused by the sars coronavirus (sars-cov) in early , coronavirus infection was not considered to be serious enough to be controlled by either vaccination or specific antiviral therapy. it is now believed that the availability of antiviral drugs effective against sars-cov will be crucial for the control of future sars outbreaks. recently, rna interference has been successfully used as a more specific and efficient method for gene silencing. rna interference induced by small interfering rna can inhibit the expression of viral antigens and so provides a new approach to the therapy of pathogenic viruses. this review provides an overview of current information on coronavirus and the application of small interfering rna in viral therapeutics, with particular reference to sars-cov. coronaviruses are large, enveloped, positive-stranded rna viruses. they have a broad host range and are capable of infecting many species, particularly the tissues of the upper respiratory and gastrointestinal tracts, the liver and cns [ , ] . coronaviruses, such as human coronavirus (hcov)- e and hcov-oc , are the major causative agent for - % of common colds in humans each year. colds are usually mild and self limiting, with a clear increase in neutralising antibody titre soon after infection; however, a certain percentage of the population can be reinfected by the same virus. colds due to coronavirus infection are particularly prevalent in the winter. in fact, outbreaks of two known human coronaviruses, occurring in alternating years, has become a common seasonal event [ ] . coronavirus infections were not previously considered to be serious enough to require treatment by either vaccination or specific antiviral therapy [ ] . the situation may have altered since the quasi-pandemic outbreaks during late and early of severe acute respiratory syndrome (sars), caused by a newly identified coronavirus, sars-cov [ ] [ ] [ ] [ ] [ ] [ ] . the disease has typical influenza-like symptoms such as high fever, myalgia, dyspnea, lymphopenia and lung infiltrates (pneumonia). then, as it develops further, acute breathing problems that raise the overall mortality to % can result. a total of , probable sars cases were reported to the who between november and july , of which who died were shown to be sars-cov-positive [ , ] . owing to this abrupt pandemic outbreak, certain empirical measures, such as antibiotics, antiviral agents (ribavirin, oseltamivir and hiv- protease inhibitors), corticosteroids, ifns and normal human immunoglobulin preparations, were applied to treat those suffering from sars [ ] [ ] [ ] . researchers continue to test all kinds of regimens to treat sars, including neutralising antibodies, fusion inhibitors, natural products, vaccines and rna interference (rnai) [ ] [ ] [ ] . a large-scale re-emergence cannot be ruled out even though only sporadic sars infections have been reported since june . as a result, antiviral drugs with high activity against sars-cov are in high demand. rnai is an innate, adaptive defence mechanism triggered by double-stranded rna (dsrna). recently, small interfering rna (sirna) has shown promise in the protection from viral invasion, as it can inhibit the expression of viral antigens and accessory genes as well as control the transcription and replication of the viral genome. this review focuses on the current information available on viral gene expression and coronavirus genome replication. based on this knowledge, rnai strategies that have been designed to interfere with viral replication in the treatment of sars-cov infection are reviewed. coronaviruses, a genus of the coronaviridae family, are large, enveloped, positive-stranded rna viruses, and are the causative agent of many infectious diseases in humans and animals [ , ] . the viral envelope is studded with long, petal-shaped spikes, which give the virus its characteristic crown appearance from which it was named [ ] . before the sars outbreak, coronaviruses were antigenically divided into three groups and viruses within each group were further classified on the basis of host, nucleotide sequence and serological relationships [ ] . members of group i include hcov- e, porcine transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), feline infectious peritonitis virus (fipv), feline enteritis virus (fecov) and canine coronavirus (ccov), whereas hcov-oc , mouse hepatitis virus (mhv) and bovine coronavirus (bcov) belong to group ii. group iii includes avian infectious bronchitis virus (ibv) and turkey coronavirus (tcov). gene analysis of the entire sars-cov rna genome ( , nucleotides) [ , ] suggests that sars-cov should be included in the coronaviridae family. although sars-cov reacts with antibodies against group i viruses [ ], sars-cov clearly represents a new group of coronavirus [ , , , ] . the virion of coronavirus is a spherical particle - nm in diameter and contains a capped, polyadenylated, single-stranded, positive-sense genomic rna, which is - kb in length, and is the largest known rna virus genome [ , ] . coronavirus-infected cells contain a characteristic ′ coterminal and nested mrna. the mrna has a capped leader sequence consisting of ∼ nucleotides that is derived from the ′ end of the genome [ , ]. an untranslated region (utr) of - nucleotides follows this leader sequence. another utr at the ′ end of the rna genome is followed by a polya tail of variable length. both the ′-and ′-utrs are important in rna replication and transcription, as is the transcription-regulatory sequence (trs), which is a typical feature of coronaviruses [ ] . this short motif is usually near the beginning of each open reading frame (orf) and the ′ end of the leader sequence (figure ). in addition, the consensus sequence, ′-cuaaac- ′, is found immediately in front of the spike (s) protein and membrane (m) protein genes and orf [ ] . gene organisation in most coronaviruses follows the same pattern of genes coding for polyproteins a and -b, s, m, envelope protein (e) and nucleocapsid protein (n; figure ) [ , ] . some group ii coronaviruses have a fringe of short spikes consisting of haemagglutinin esterase (he). coronavirus genomes also contain a variety of additional orfs that encode - nonstructural proteins with no known function; these genes are not conserved among coronaviruses. polyproteins a and -b are required for viral rna synthesis and believed to be the only viral proteins that are synthesised directly from the original viral (other viral proteins are translated from subgenomic mrnas). once viral rna synthesis takes place, more product of gene are translated from the newly synthesised genomic rna. the primary gene products (orf a and -b), predicted to be ∼ - kda, undergo co-or post-translational processing into various proteins as a result of their own protease activity. the virus-encoded proteases, papain-like cysteine protease (plpro) and c-like cysteine protease ( clpro), cleave the polypeptide into small polypeptides that are required for replication and transcription [ , ] . rna-dependent rna polymerase (rdrp) and helicase are essential components of the replicase complex, which, presumably, contains other viral and cellular proteins. the replicase complex is responsible for transcribing: the full-length negative and positive rnas; the ′ coterminal set of nested subgenomic mrnas; and subgenomic negative rna strands [ , , ] . the entry of enveloped coronaviruses usually involves the three steps of attachment, receptor binding and virus-cell fusion, mediated by viral envelope proteins [ ] . the s glycoproteins, which make up large, petal-shaped spikes on the surface of the virion, bind to a cellular receptor, promoting fusion of the viral and cellular membranes, and this event probably explains the primary viral tropism [ ] . this highly glycosylated protein, with a molecular mass of ∼ - kda, can be divided into three structural domains: a large external n-terminal domain (divided into subdomains s and - ); transmembrane domain; and short c-terminal cytoplasmic domain [ ] . in many cases, the cell receptors for coronaviruses have been identified. the mhv receptor is a murine biliary glycoprotein belonging to the carcinoembryonic antigen family of the ig superfamily [ , ] . the cell membrane-bound metalloproteinase, aminopeptidase n (cd ), is probably the receptor for tgev, hcov- e and ccov [ ] [ ] [ ] . cd is widely distributed in cells in many tissues, including respiratory, enteric epithelial, neuronal and glial cells. the receptor for sars-cov is considered to be angiotensin-converting enzyme (ace ), required for binding to permissive cells and the s subunit [ ] . no receptors for type iii viruses have yet been identified. the integral membrane proteins, m and e, are the minimum protein units required for virus assembly [ ] . m protein is found not only on the viral envelope but also in the viral internal core [ ] , and associates with the golgi complex in the cell, thereby dictating the site of virus assembly [ ] . the expression of m and e proteins may be sufficient enough to trigger the formation of virus-like particles (vlps). when s protein is coexpressed with the m and e proteins, it is incorporated into vlps with (presumably) an authentic conformation that is able to infect cells [ - ]. as they do not contain viral nucleic acid, vlps are an ideal candidate for vaccine preparation. the final structural protein, n protein, with a molecular mass of - kda, probably associates with viral rna to form a long and flexible helical nucleocapsid [ , ] . n protein may also play a role in viral rna synthesis, as coronavirus rna polymerase activity is inhibited in vitro by the anti-n protein antibody [ ] . . introduction dsrnas usually play pivotal roles as intrinsic intermediates during the genomic replication of many viruses, but they are rare in eukaryotic cells. higher eukaryotes respond to dsrna regardless of whether it is produced by viruses, introduced artificially or formed in immune defence responses. in mammalian cells exposed to dsrna, the major immune response is an increase in serum ifn levels, which can suppress viral spread by inhibiting viral gene expression and initiating the apoptosis of infected cells [ , ] . in contrast to mammalian cells, many other higher eukaryotes, including plants, nematodes and insects, are not able to mount an ifn response. nonetheless, rnai was first described in plants, in which it acts as a natural immune mechanism against viral infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] . triggering post-transcriptional gene silencing (ptgs) by sirna molecules has been observed in a wide variety of species [ ] [ ] [ ] . rnai, which is a powerful technique, has been widely used to silence specific genes in mammalian and human cells [ , ] . however, dsrnas > bp can be destroyed as they can induce an immune response in mammalian cells and activate dsrna-dependent kinase and ′- ′-oligoadenylate synthetase, leading to the induction of ifn expression [ ] . because of their relatively small size, synthetic sirnas can escape the immune response [ ] . as a result, researchers are developing many rnai-based drugs to prevent and treat diseases such as viral infection, tumours and metabolic disorders [ ] [ ] [ ] [ ] [ ] . recent studies indicate that rnai exerts it effects via a two-step mechanism: an initiation and effector step [ , ] . in the initiation step, long dsrnas are introduced either directly or via a transgene or virus. subsequently, the host protein, dicer, an atp-dependent ribonuclease and member of the rnase iii family, binds with high affinity to the introduced dsrna and cleaves it into - nucleotide fragments referred to as sirna duplexes [ , ] . in the effector step, the sirna duplexes complex with multinucleases to form the so-called rna-induced silencing complex (risc) [ ] , which then undergoes an atp-dependent activation step that results in the unwinding of the double-stranded sirna component to form a single-stranded guide rna that leads the risc to other complementary mrnas. after the mrna is bound, an unidentified ribonuclease component of risc cleaves the bound mrna at the centre of the region complementary to the guide rna. after this reaction has taken place, risc is released to seek out other mrna homologues, at the same time as the cleaved mrna is degraded by cellular exonucleases [ ] . the effectiveness of rnai has been improved using rna-directed rna polymerase, which produces a large number of copies of the triggering rna [ ] . specifically targeting sirnas is very important because slight positional changes in the sirna relative to the mrna can have drastic effects on silencing, indicating that the secondary structure of the target mrna plays a role in sirna accessibility [ ] . however, an exact sequence match in the sense strand of sirna duplexes may not be necessary, as a single-stranded antisense sirna has been shown to initiate the cleavage of target rna [ ] and as many as five mismatches in the sense-strand rna can be tolerated [ ] . in contrast, a single basepair mismatch on the antisense strand corresponding to the target rna can significantly reduce sirna-mediated message degradation [ ] . genetic mutation in the regions coding for major antigenic proteins may limit the inhibitory effect of sirna. to circumvent the sequence variation among viral strains, highly conserved sirna target sequences must be selected. if possible, the target sequences should be chosen from beyond the coding regions, where they may have structural roles [ ] . general guidelines for the choice of sirna sequences have been reported [ ] [ ] [ ] [ ] : and a g:c bp at the ′ end of the sense strand, a:t bp and a-t-rich sequence at the ′ end of the antisense strand, and no g-c stretch > bp are highly recommended. sirnas can be prepared enzymatically, which has the advantages of improved efficiency and lower costs [ ] . in addition, it can provide a large panel of sirnas, cover a long segment of mrna and avoid the re-emergence of escaped mutants. another method is to use rna polymerase to transcribe sirnas from short dna templates in which the encoded sirnas are downstream of the rna promoter [ ] . a major limitation when using either chemically or enzymatically synthesised sirnas is that they are unstable and may only have a transient effect; however, the use of chemically protected sirnas and viral vectors may avoid this problem [ ] [ ] [ ] . rnai technology is an ideal tool for inhibiting viral replication in host cells as the sirna can interact with certain viral genes and silence their expression. it can also be used to probe the steps during the viral cycle, not only for plus-strand rna viruses (figure ) but also for many other types of virus [ , ] . genes encoding vital proteins in reproducing sars-cov virions can be chosen for chemotherapeutic intervention (e.g., those coding for s, c-like protease [ clpro], rna-dependent rna polymerase and possibly other gene products involved in viral-protein-mediated processes) [ ] first demonstrated that sirna was able to silence the replicase of sars-cov ( a region of the genome) and that this approach was effective in vitro against sars-cov. wang et al. [ ] subsequently observed that vector-based sirnas could inhibit the replication of sars-cov, and showed that expression in the plasmid, psuper, of sirnas specifically targeting viral rna polymerases could block the cytopathic effects of sars-cov on vero cells. these plasmids were also able to block viral replication, as shown by both the titre and levels of viral rna and protein. zhang et al. [ ] reported that sirna can reduce the cytopathic effects when used to transfect veroe cells immediately before sars-cov infection. moreover, zhang et al. [ ] showed that dna vector-driven sirna can selectively silence s gene expression in sars-infected t cells, and reported that sirna targeting the leader sequence decreased both mrna levels and protein levels of reporter genes in t cells. in addition, the steady expression of sirna in veroe cells decreased the transcription of sars-cov gene mrna. furthermore, these researchers pointed out that sirna specific for the leader sequence has a stronger inhibitory effect on sars-cov replication than that targeting either the s gene or antisense oligodeoxynucleotides [ ] . zheng et al. [ ] showed that three chemically synthesised sirna duplexes targeting viral rna polymerases, and one targeting the s gene potently inhibited sars-cov infection and replication in fetal rhesus kidney cells (frhk- ) . they observed a prolonged prophylactic effect of sirna duplexes, with ≤ % inhibition of transcription, lasting for ≥ h. combinations of active sirna duplexes targeting different regions of the viral genome resulted in ≤ % inhibition. the authors [ ] focused on four regions in the sars-cov genome: the leader sequence; trs, ′-utr; and the s protein-coding sequence. purpose-designed sirnas were prepared to test their specificity for the sars-cov viral rnas and examine their inhibitory effects on viral protein expression and replication. the results showed that s gene-targeted sirnas profoundly reduced levels of both rna transcripts and viral antigens, although ′-utr-oriented sirnas were not as effective, the two other sirnas had no effect. the sirnas results mentioned above were obtained in cell culture studies. recent studies using mouse models have demonstrated that airway infections caused by influenza virus and respiratory syncytial virus can be treated prophylactically by intranasal delivery of sirnas [ ] [ ] [ ] [ ] . li et al. [ ] administered chemically synthesised sirna duplexes intranasally in the rhesus macaque sars model and found a reduction in sars-cov infection-induced fever, sars-cov viral levels and acute diffuse alveoli damage [ ] ; accumulated dosages of sirna - mg/kg did not result in any sirna-induced toxicity. these results provide strong evidence that these sirna agents are potent both in the prophylactic and therapeutic treatment of sars infection as well as lacking toxicity in this nonhuman primate model. these encouraging findings suggest that sirnas may be applicable against sars-cov in man, and that suppression of the viral cycle or expression of viral antigen using rnai treatment shows promise for the therapy of viral infection. conventional drugs and vaccines used for the treatment of viral diseases may have many adverse effects, such as toxicity, cost and resistance, and complicated administration protocols. considerable information regarding the mechanism and use silencing the virus receptor of the host cell with sirna may prevent entry of the virus into the cell. s protein is also a good target for rnai. the rna genome of the virion is released into the cytoplasm after the virion attaches to the host receptor, and the virus takes advantage of the host translational machinery to translate orf a and - b into polyproteins. cleavage by virally encoded proteases yields the components that are required to assemble the viral replicase complex, which synthesises full-length, negative-strand rna. the protease and polymerase involved in viral replication are potential sirna targets. a discontinuous transcription strategy is taken by the virus during negative-strand synthesis through which a set of nested subgenomic and minus-sense rnas are formed. the resulting mrna has a base leader sequence at the ′ end and a polya tail at the ´ end (shown as circles and squares, respectively). viral mrnas are then translated to protein (indicated on the right). these negative strands act as templates of the synthesis for their positive counterpart. these subgenomic rnas are good targets for sirna silencing. n protein and the newly synthesised genomic rna associate into a helical nucleocapsid. m, e and s proteins are incorporated into the lipid bilayer of the endoplasmic reticulum and transported to a budding compartment. n protein then binds to m protein, initiating virion assembly. the virus is finally released from the host cell by the fusion of virion-containing vesicles with the plasma membrane. the steps of the viral replication cycle that can be inhibited by rnai are highlighted with arrows. budding e c re of rnai as a tool for manipulating gene expression and inhibiting viral replication has been obtained in recent years. the marked advantages of sirna can be attributed to the relative ease of its design, construction and testing, but, in particular, its low cost. more importantly, sirna is a short length of nucleic acid and believed not to generate an unfavourable immune response when administered to patients, thus making it an ideal candidate for the treatment of viral infection. sirna normally carries a negative charge under physiological conditions, which makes it difficult for the molecule to traverse the cell membrane, but carriers such as liposomes and many viral vectors and electroporation can efficiently deliver sirnas into mammalian cells. the current challenge for the success of sirna-based treatment is how to precisely deliver the sirnas in an efficient and safe way to the target cells and organs. considerable effort will be required for a better understanding of virology and the further development of this technique. sirna-based drugs may soon be deployed to relieve or even eradicate the intractable viral infections that currently afflict mankind. current concepts in sars treatment advancements in the battle against severe acute respiratory syndrome antivirals and antiviral strategies medicine. caution urged on sars 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therapy • this review shows that ptgs already appears to be a promising new therapeutic tool to fight viral multiplication and dissemination through the host, and to prevent inflammation and virus-induced pathogenesis inhibition of sars-associated coronavirus infection and replication by rna interference inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering rna silencing sars-cov spike protein expression in cultured cells by rna interference sirna targeting the leader sequence of sars-cov inhibits virus replication synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of sars-associated coronavirus inhibition of sars-cov replication by sirna inhibition of influenza virus production in virus-infected mice by rna interference protection against lethal influenza virus challenge by rna interference in vivo inhibition of respiratory viruses by nasally administered sirna inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns gene • this paper provides strong evidence that these sirna agents are potent for the prophylactic and therapeutic treatment of sars infection an animal model of sars produced by infection of macaca mulatta with sars coronavirus tml an animated tour introduces the process of rnai this work was funded by the national science council, taiwan. the authors are greatly indebted to y-s chang of chang-gung university, t-l li of national taiwan ocean university for many invaluable suggestions, and t barkas for critical evaluation of the english. papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers. key: cord- -vqorb j authors: kumar, krishna; lupoli, tania j. title: exploiting existing molecular scaffolds for long-term covid treatment date: - - journal: acs med chem lett doi: . /acsmedchemlett. c sha: doc_id: cord_uid: vqorb j [image: see text] discovery and development of covid- prophylactics and treatments remains a global imperative. this perspective provides an overview of important molecular pathways involved in the viral life cycle of sars-cov- , the infectious agent of covid- . we highlight past and recent findings in essential coronavirus proteins, including rna polymerase machinery, proteases, and fusion proteins, that offer opportunities for the design of novel inhibitors of sars-cov- infection. by discussing the current inventory of viral inhibitors, we identify molecular scaffolds that may be improved by medicinal chemistry efforts for effective therapeutics to treat current and future coronavirus-caused diseases. disease (covid- ) have focused on the repurposing of approved drugs. this approach is warranted, as we seek quick solutions to a pandemic, caused by severe acute respiratory syndrome coronavirus (sars-cov- ). the recent announcement of the antiviral remdesivir as the first u.s. food and drug administration (fda) approved drug for the treatment of patients suffering from severe symptoms of covid- affirms this approach. anthony fauci, the director of the national institute of allergy and infectious disease (niaid) said that the completed clinical trial with remdesivir proved "that a drug can block this virus", although this drug alone is not a "magic bullet" for covid - . without the ability to accurately predict a timeline for a vaccine, we need additional clinical candidates to build an arsenal against sars-cov infections. related coronaviruses, sars-cov- and mers-cov (middle east respiratory syndrome coronavirus), caused serious, albeit less widespread, health epidemics starting in and . the occurrence of multiple outbreaks further motivates us to annotate therapeutic targets for other coronavirus-caused diseases that might develop in the future. similar to the treatment of influenza, we would benefit from multiprong defense tactics that include both vaccines and therapeutic agents. many recent scientific reviews and essays have outlined vaccine efforts, as well as viral and host targets that are the focus of current campaigns aimed at redirecting clinically used compounds for covid- . here, we outline viral pathways that contain druggable targets currently overlooked and propose general mechanisms to disrupt these pathways. we begin with a short primer on the sars-cov- viral life cycle and an overview of existing drugs that are the subject of current clinical trials around the world. we then focus on alternative druggable targets largely within the viral replicase machinery, as well as viral entry and proteolytic processing pathways. our proposed strategies for blocking these pathways are informed by past and current work on inhibitors of sars-cov and mers-cov, as well as other successful antivirals. we hope to engage the medicinal chemistry community with ideas for novel therapeutic targets in order to develop new drugs to specifically disarm coronaviruses in the host. the genome organization of sars-cov- is similar to that of other members of the β-coronavirus family that also include sars-cov- and mers-cov. the viral rna-encoded proteins can be divided into two main classes: structural and nonstructural proteins (nsps). the structural proteins (spike, envelope, membrane, nucleocapsid, and hemagglutinin esterase) form the viral particle and play additional functional roles during infection. viral infection begins with an interaction between the viral spike glycoprotein (s protein) and a receptor, angiotensin-converting enzyme (ace ), on the host cell surface (figure ). ace is expressed in various cell types, including those in the lungs. host proteases in the membrane are important in this process; namely, a cellular serine protease, called transmembrane serine protease (tmprss ) is known to prime the trimer of s protein on the viral particle surface prior to cell entry. cleavage of s protein into two subunits is required for the process of viral and host membrane fusion prior to viral uptake by an endocytic mechanism. following engulfment and subsequent release from the endosome, viral genetic material is released into the host cytoplasm prior to translation of the single stranded viral rna into long polypeptides that contain the nsps. viral polypeptides are predicted to be cleaved into individual nsps through an autoprocessing mechanism. there are two cysteine-like proteases expressed as part of these polypeptides: one is a papain-like protease (plp), known as the accessory protease; the other is a chymotrypsin-like protease ( cl pro ), known as the main protease. cl pro cleaves itself and processes the remaining polypeptide into nsps − , which make up the rna replication-transcription complex (rtc). several components of the rtc aid the rna-dependent rna polymerase (rdrp), which is responsible for replicating additional copies of the rna genome and transcribing multiple mrna fragments that encode either structural or accessory proteins. following multiple cycles of replication and translation, the viral particle assembles and exits the cell though a budding mechanism known as scission. it is thought that β-coronaviruses rely on the host cell's endosomal sorting complex required for transport (escrt), but the exact method of egress is still not known. full scission from the host cell releases the virus to infect more cells and continue to replicate. the world health organization (who) and federal agencies are largely focused on clinical trials for preapproved drugs that are proposed to target some aspect of the viral life cycle described above (figure ). the nih now lists over a thousand ongoing clinical trials for treatments relating to covid- . the who is currently conducting a worldwide trial ("solidarity") by focusing on four promising covid- treatments: remdesivir; lopinavir/ritonavir with and without interferon β- a (to help stimulate the immune system); and hydroxychloroquine (this last treatment has currently been paused). the fda-approved covid- drug, remdesivir, is a nucleotide analog originally developed to treat ebola infections (caused by another single-stranded rna virus) and recently shown to inhibit the sars-cov- rdrp. the fda has issued an emergency use authorization of hydroxychloroquine and chloroquine, both of which are approved to treat malaria and various autoimmune disorders, and might function by disrupting endosome-mediated entry or egress of the virus. lopinavir-ritonavir are hiv protease inhibitors that are hypothesized to inhibit sars-cov cl pro . in addition to small molecule inhibitor candidates, various clinical trials are exploring the effect of known antibody therapies on covid- progression or plan to test antibodies raised against viral proteins. the success of remdesivir in clinical trials, although limited, suggests that inhibition of viral replication is a viable strategy for the treatment of covid- . importantly, it takes more than a single polymerase to replicate the viral genome and ensure pathogenicity. sars-cov- is thought to involve up to nine nsps in the rtc. key players include the rdrp (nsp ), helicase (nsp ), and proposed polymerase cofactors/ primase(s) (nsp , ). to our knowledge, these proteins are not targets in current covid- clinical trials. the viral rtc proteins are expressed in the cytoplasm and so are more accessible that their host counterparts in the nucleus. coronavirus helicases are motor proteins necessary for unwinding double-stranded rna, which form secondary structures, in order for replication and translation to occur. since viral helicases typically share low homology with human dna helicases, specific helicase inhibitors are unlikely to be toxic to the host. the amino acid sequence of sars-cov- helicase is identical to that found in sars-cov- ; hence, a solved crystal structure of the sars-cov- helicase provides opportunities for rational drug design. the lupoli and zhang laboratories (nyu, chemistry) are currently using this structure to design small molecule ligands for computationally predicted binding pockets. in addition to a nucleotide binding site, the sars-cov helicase contains a zinc binding domain with three zinc fingers, and mutation of residues linking this domain to the helicase core domain were shown to cause a deficiency in nucleic acid unwinding. as a result, there are multiple sites for disruption of helicase activity. unique among other rna viruses, coronaviruses are believed to have two nsps with rna polymerase activity. the canonical rdrp is primer-dependent for optimal activity, while the second polymerase (nsp ) is hypothesized to be a pubs.acs.org/acsmedchemlett viewpoint primase. the primary sequences of nsp , and rdrp are ≥ % identical in sars-cov- and cov- . a solved cryoelectron microscopy structure of the sars-cov- rtc complex has shown that interactions between rdrp, nsp and are essential for activity. interestingly, the main rdrp also has an n-terminal kinase-like domain that may carry out nucleotidyltransfer reactions. disruption of this site or any member of this complex represents a novel opportunity for chemical interference. inhibition of helicase or primase activities has been pursued as an antiviral approach in the past and has been reviewed in detail elsewhere. importantly, herpes simplex virus (hsv) helicase ul is a member of the same helicase super family (sf ) as sars-cov- nsp . in , boehringer-ingelheim and bayer independently reported on inhibitors of the hsv helicase−primase complex that were comparative to approved antivirals for hsv. boehringer-ingelheim's aminothiazoylphenyl-based inhibitors of hsv helicase−primase, namely, bils ( figure , compound ) , exhibit nanomolar antiviral activity and are selective for hsv infection in mouse models. the clinically used amenamevir ( ), another herpes antiviral, also targets in the helicase−primase at nanomolar concentrations. the most promising inhibitor of sars-cov- helicase, ssya - ( ), which shows micromolar activity against sars-cov- and mers-cov infections in vitro, was awarded a us patent in . , given the conservation of helicase sequences in sars-cov- and cov- , this inhibitor will likely serve as a useful starting point for future design of coronavirus helicase modulators. ■ novel strategies for targeting viral proteases although many host proteases are deemed essential for latestage processing of proteins translated from viral rna, viral cl pro (also called m pro ) and plp have emerged as popular targets for covid- . hiv protease inhibitors lopinavir and ritonavir, included in the solidarity trial despite mixed reviews in the clinic, have been predicted to bind sars-cov- and cov- cl pro ( % sequence identity) based on computational studies. while hiv proteases are aspartic proteases, coronavirus proteases are members of the cysteine protease family. given that approximately a quarter of human proteases are predicted to belong to this class, design of specific viral inhibitors remains a challenge. fortunately, to aid in the rational design of inhibitors, a crystal structure of sars-cov- cl pro was reported recently. as part of this work, peptidomimetic compounds (α-keto amides), previously designed against βand α-coronaviruses, were modified for stability. the most promising mimetic ( ) inhibited sars-cov- replication in human lung cells at micromolar concentrations and therefore requires further improvement. carboxamide and acetamide scaffold structures have been shown to inhibit cl pro of sars-cov- , along with other inhibitors that have been reviewed elsewhere. another viable viral target, plp, shows lower sequence conservation between sars-cov- and cov- ( % homology). notably, plp possesses additional activities beyond polypeptide processing, such as deubiquitination and cleavage of the ubiquitin-like modifier, isg . these plp-dependent activities contribute to viral immune evasion in the host. inhibitors of plp-mediated isg cleavage activity, such as compound , have ec values of ∼ um against sars-cov- infection in cell culture with no associated cytotoxicity. structural information and the noted unique features of sars-cov cysteine proteases can be exploited for selective inhibition, but existing scaffolds still require optimization to disable sars-cov- infections in the host. the s proteins of sars-cov- and cov- share % overall sequence identity, yet the receptor binding domain (rbd) of the latter has − -fold higher affinity to the human ace receptor protein. after the rbd binds ace , two heptad repeat domains, hr and hr , interact to form a six-helical bundle, and in so doing bring the viral and host membrane proximal to one another resulting in fusion. this fusion process is essential for viral entry. design of "coiled coil" heptad repeatbased peptides provides an avenue for drug design that can easily be tailored to respond to mutations that will arise in future s proteins. using a recently solved crystal structure of the hr and hr domains of the sars-cov- s protein, lipidated peptide fusion inhibitors have been designed that inhibit pseudovirus infection of cells with ic values in the single-digit nanomolar range. targeting membrane fusion with peptide-based inhibitors offers opportunities and flexibility for entry inhibition in future coronavirus strains, but the cost and possible side effects may be a deterrent for widespread global use. we envision that in the coming months and years, there will be three phases of therapeutic intervention for covid- : ( ) repurposing of existing drugs as treatments and prophylactics; ( ) the development of vaccines; and, ( ) as the threat of a mutated virus looms large, the development of an arsenal of compounds that are available against other targets to sustain a defense against widespread infection. the reservoir of targets and available compounds against this and related viruses research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan. emerging microbes infect coronavirus envelope protein: current knowledge virus-encoded proteinases and proteolytic processing in the nidovirales structural basis for inhibition of the rnadependent rna polymerase from sars-cov- by remdesivir mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology why are lopinavir and ritonavir effective against the newly emerged coronavirus ? atomistic insights into the inhibitory mechanisms discovering new medicines targeting helicases: challenges and recent progress delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp upon atp hydrolysis structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors herpes simplex virus helicase-primase inhibitors are active in animal models of human disease rubsamen-waigmann, h. new helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease evaluation of ssya − as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses suppression of sars replication by sars helicase inhibitors. us patent wo crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors an overview of severe acute respiratory syndromecoronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication cryo-em structure of the -ncov spike in the prefusion conformation inhibition of sars-cov- (previously -ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion key: cord- -my ow ee authors: nelson, philipp p.; papadopoulos, nikolaos g.; skevaki, chrysanthi title: respiratory viral pathogens date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: my ow ee respiratory viruses are responsible for a variety of clinical syndromes including the common cold, acute otitis media, laryngitis, sinusitis, pneumonia, bronchiolitis, influenza-like illness, and exacerbations of asthma and chronic obstructive pulmonary disease. diagnosis of respiratory viral infections is primarily clinical and is further supported by laboratory techniques such as antigen detection, serology, and nucleic acid detection. preventive strategies are based on avoidance of risk factors and, in case of influenza, vaccination. treatment modalities include over-the-counter and non-specific remedies along with a small number of specific antiviral medications such as the influenza neuraminidase inhibitors or palivizumab against respiratory syncytial virus. orthomyxoviridae's major representatives are the influenza viruses, which are grouped into four genera with one member per genus: influenza a virus (iav, alphainfluenzavirus), influenza b virus (ibv, betainflunenzavirus), influenza c virus (icv, gammainfluenzavirus) , and the recently discovered influenza d virus (idv, deltainflunezavirus) . while iav has a wide host range with water fowl as main reservoir, ibv mainly infects humans. icv and idv have also zoonotic potential but cause mostly mild respiratory diseases (asha and . orthomyxoviridae form enveloped, spherical or pleomorphic virions with - nm in diameter. their linear, negative-sense rna genome has a total length of - kb and is divided into eight (iav, ibv) and seven (icv, idv) segments, respectively. it encodes for up to proteins, among others, in iav and ibv, hemagglutinin (ha) and neuraminidase (na) for attachment, cell entry, and release of new particles. the na and ha proteins are regularly subjected to small changes, which are capable of producing viral strains causing annual epidemics. this phenomenon is called "antigenic drift," while "antigenic shift" is the process by which a sudden major change in the ha or na proteins of iav occurs due to genetic reassortment (king, ) . instead of ha and na, which bind and cleave sialic acid (n-acetylneuraminic acid), icv and idv express one combined protein, the hemagglutinin-esterase-fution glycoprotein (hef). this protein requires an acetylated derivative of n-acetylneuraminic acid for attachment to the host cells (su et al., ) (fig. ) . rhinovirus (rv). the rhinovirus capsid is arranged in an icosahedron composed of copies of each of the three subunits vp - (shown in red, blue, and yellow). reproduced with permission from papadopoulos ng and skevaki cl ( ) viruses of the lung. in: encyclopedia of respiratory medicine, - , https:// doi.org/ . /b - - - / - . (iav) . hemagglutinin spikes (green) radiate all over the surface and are interspersed by neuraminidase (yellow) and matrix protein m (light blue). the latter are embedded in the envelope's lipid bilayer(light yellow), which in turn surrounds a layer of matrix protein m (dark blue). the segmented rna (orange) of the virus is located in the interior. human parainfluenza viruses (hpivs) are respiratory viruses in the family of paramyxoviridae. they have enveloped, pleomorphic but mostly spherical virions with a diameter of - nm. the single, linear, negative-sense rna genome consists of . - kb in a helical nucleocapsid. for efficient replication, the genome length is confined to multiples of nucleotides. among others, it encodes for the glycosylated fusion (f) protein and the glycosylated receptor-binding protein (rbp), also called haemagglutininneuraminidase (hn) protein, which binds to neuraminic acid on the cell surface. four types of hpivs are known: hpiv- and belong to the genus respirovirus in the subfamily of orthoparamyxovirinae, hpiv- and , like mumps virus, to the genus orthorubulavirus in the subfamily of rubulavirinae (rima et al., ) . viruses of the family of pneumoviridae form enveloped, spherical or filamentous virions with - nm in diameter, which contain a single, linear, negative-sense rna genome. this genome is bound in a complex with the nucleocapsid (n) protein, the polymerase (l), and a necessary co-factor (p). the glycosylated fusion (f) and attachment (g) proteins in the envelope mediate cell entry. in contrast to paramyxoviruses, almost all pneumoviruses lack a hemagglutinin and neuraminidase (rima et al., ) . human respiratory syncytial virus (hrsv or rsv) belongs to the genus orthopneumovirus. the genome of $ kb contains genes for proteins, two of them being non-strucutural proteins (g protein, two groups, rsv-a and rsv-b, are distinguished (pangesti et al., ) . another pneumovirus is human metapneumovirus (hmpv) in the genus metapneumovirus. it forms paramyxovirus-like pleomorphic to spherical particles of - nm diameter. the genome of hmpv comprises roughly kb and encodes for proteins (shafagati and williams, ) . until the beginning of the st century, only two human coronaviruses (hcov- e and oc ) were known. since then, many other members of the family of coronaviridae have been indentified, most of them infecting animals and only four others infecting humans: hcov-nl and hku cause respiratory diseases worldwide, severe acute respiratory syndrome (sars) coronavirus was discovered in an outbreak in - , and middle east respiratory sondrome (mers) coronavirus, so far constricted to the arabian peninsula. coronaviruses form enveloped, spherical virions with a diameter of - nm. the size of the single, linear positive-sense rna genome ranges between and kb, which represents the largest genome of known rna viruses. the trimeric glycosylated spike (s) protein forms characteristic - nm long protrusions, which mediate receptor binding and membrane fusion. common to all coronaviruses are also the membrane (m) and envelope (e) glycoproteins and the nucleocapsid (n) protein. depending on the species, other proteins are included, e.g., a hemagglutinin-esterase (he) for reversible attachment to o-acetylated sialic acid in hcov-oc and hku . during replication, two polyproteins are produced from the replicase gene and then cleaved into several small products; among them nsp , which is used for genus demarcation (king, ) . high estimated mutation rates and possible genetic recombinations during replication increase the probability of the emergence of new (zoonotic) coronaviruses (su et al., ) (fig. ) . hcov- e (subgenus duvinacovirus) and hcov-nl (subgenus setracovirus) belong to the genus alphacoronavirus. the four other coronaviruses belong to the genus betacoronavirus. hcov-oc and hcov-hku are subspecies of the species betacoronavirus in the subgenus embecovirus. mers-cov is a member of the subgenus merbecovirus and sars-cov of the subgenus sarbecovirus (walker et al., ) . the cell entry receptor differs between those viruses as well. hcov- e binds human aminopeptidase n (apn, cd ) while hcov-nl and sars-cov use angiotensin-converting enzyme (ace) as their receptor. according to their he protein, hcov-oc and hku use -o-acetylated sialic acids attached to cell surface proteins. mers-cov requires dipeptidyl peptidase (dpp , cd ) for cell entry (hulswit et al., ; raj et al., ) . human adenoviruses (hadv) belong to the genus mastadenovirus and are further classified into species (a-g) with more than types identified so far (lion, ). the symptoms of an infection vary between different types and range from respiratory disease to diarrhea or conjunctivitis. adenoviruses are non-enveloped, double-stranded dna viruses with a genome of - kb in icosahedral virions ranging from to nm in diameter (king, ). fiber-like protrusions extend from each of the penton base capsomers at the corners of the icosahedron. the knobs at the ends of these fibers confer attachment to the host cells, among others to coxsackie and adenovirus receptor (car) or membrane cofactor protein (mcp, cd ). while most hadvs only use one of the two receptors, group d viruses use both receptors simultaneously (khanal et al., ) . during replication $ polypeptides are produced through alternative splicing (fig. ) . although most polyomaviruses (pyvs) have been associated with different types of tumors, two recently discovered polyomaviruses of the genus betapolyomavirus, wupyv and kipyv, have been found in respiratory samples worldwide, most frequently in samples from immunocompromised patients. while both alpha-and betapolyomaviruses may infect humans, gamma-and deltapolyomaviruses do not (babakir-mina et al., ) . pyvs are non-enveloped, icosahedral viruses of - nm diameter and contain a single, circular double-stranded dna genome of $ kb. inside the virion, the genome is packed with histone proteins h a, h b, h , and h . the capsid consists of pentameric capsomers of the major capsid protein vp and the two minor capsid proteins vp and vp . typically, the two regulatory proteins large and small tumor antigen (ltag and stag, respectively), which are expressed early during infection, are used for phylogenetic analyses (moens et al., ) . even though detected frequently, a clear association between wupyv and kipyv on one hand and respiratory disease on the other hand is still missing (babakir-mina et al., ) . parvoviridae are non-enveloped, icosahedral viruses of around nm diameter. the capsid encloses a single, linear, mostly negativesense dna-genome with approximately . kb. the genome encodes for three capsid proteins (vp - ) and five non-structural proteins (ns - , np ). human bocavirus (hbov ), a member of the species primate bocaparvovirus , in the genus bocaparvovirus and the subfamily of parvovirinae, is strongly associated with upper and lower respiratory tract infections in young children. the related viruses, hbov - , are only found in stool samples. by exploiting the cellular dna repair machinery, hbov is independent from the cell cycle for genome replication (qiu et al., ) . generally, respiratory viruses are transmitted between humans by the following routes: direct or indirect contact to infected persons, via droplets, or by aerosols. the main route for each virus differs and is still in discussion (kutter et al., ) . in case of zoonotic mers-cov infection, the consumption of uncooked meat or milk from or direct contact to infected animals are other suspected routes (song et al., ) . control of the transmission of respiratory infections may be partly achieved through adequate hygiene practices and avoidance of congregation and stress. upon entering a susceptible host through eyes, nose, or mouth, respiratory viruses replicate in nasopharyngeal epithelial cells or in the epithelium of the lower respiratory tract. in more severe cases, a subsequent systemic spreading of the virus is possible. the site of the deposition of inhaled particles also depends on their size. while inhalation of aerosols with influenza virus may lead to infection of the lower respiratory tract, larger droplets are retained in the upper respiratory tract (paules and subbarao, ) . between respiratory viruses, patterns of infection differ, too. for example, epithelial infection by rvs has a "patchy" distribution and is not overtly cytotoxic, in contrast to many other respiratory viruses, including influenza viruses, paramyxoviruses, and adenoviruses, which cause extensive inflammation and epithelial shedding (royston and tapparel, ; pawełczyk and kowalski, ; shim et al., ) . several pathogen recognition receptors (prrs) contribute to the detection of viral structures within the infected cell, among them specific toll-like receptors (tlrs), which bind viral nucleic acids and stimulate the production of type i interferons (pichlmair and sousa, ) . one way of iav to suppress interferon expression and finally cell death is the production of non-structural protein (ns ) within a few hours after infection. ns binds viral rna and prevents recognition by prrs and inhibits expression of interferon-induced genes (shim et al., ) . respiratory virus infection causes an increase in both vascular permeability mediated by vasoactive amines and glandular secretion under the influence of cholinergic reflexes and neuropeptides. several cytokines and chemokines have also been implicated in virus-induced inflammation. inflammatory cells further aggravate events by release of additional mediators. humoral immunity is also activated in response to viral infections with production of serotype-specific antibodies. the nature of the response is associated with age, previous infection, and vaccination status of the host. recovery from respiratory viral illness is usually achieved prior to the detection of specific antibodies indicating that cellular and/or non-specific immune responses are primarily responsible for viral eradication. nevertheless, antibodies are able to provide protection from secondary infections. however, different aspects limit the protective effect of neutralizing antibodies against specific viruses: (a) the effect may not be long-lasting (e.g., for hmpv), (b) high mutation rates lead to the occurrence of escape-mutants (e.g., in influenza viruses), (c) many different serotypes of one virus are circulating and antibodies are lacking cross-reactivity (e.g., against other rvs). respiratory viruses not only induce a local inflammatory reaction at the level of infected epithelial cells but may also act at a distance through neuronal pathways. all parasympathetic, sympathetic, and nonadrenergic noncholinergic nervous supply of the respiratory tree may be affected by viral infections, suggesting an important neuroimmune interaction, which may contribute to the virus-mediated reactive airway disease (fig. ) . pathogenesis of respiratory viral infections. respiratory viruses enter the human body mainly through the nose. thereafter, they replicate in nasopharyngeal epithelial cells, but also in the lower respiratory tract. immune mediators such as cytokines, chemokines, and bradykinins will both elicit an "immune" response leading to effective virus eradication and contribute to the development of associated symptomatology, through vascular leakage and increased glandular secretions. neurogenic pathways are also involved in the development of clinical manifestations. reproduced with permission from papadopoulos ng and skevaki cl ( ) viruses of the lung. in: encyclopedia of respiratory medicine, - , https://doi.org/ . /b - - - / - . respiratory viruses are related to various distinct as well as non-specific clinical presentations. while acute infections are usually selflimited, severe forms may occur. there is considerable overlap between viruses and clinical presentations. although differences exist, all agents may cause any of several clinical syndromes (table ) . among these, the common cold is by far the commonest with an enormous socioeconomic impact. clinical presentation ranges from asymptomatic to upper respiratory tract (urt) symptoms such as nasal congestion, rhinorrhea, and nasopharyngeal irritation, lower respiratory tract symptoms such as cough, to systemic symptoms including general malaise, fever, headache, and sleep impairment depending on the viral culprit and host. the incubation period is - h and symptoms usually last for - days with a peak of viral replication before or together with the first clinical symptoms. the common cold is a rather benign clinical entity, which may however be complicated by secondary bacterial infections, otitis media, sinusitis, pneumonia, and asthma exacerbations; severe courses of disease and death may occur in young children and immunocompromised patients. viral urt infections often result in impairment of the function of the eustachian tube, which predisposes to the development of acute otitis media. the latter is one of the commonest infections among children and is very often attributed to viral etiology. respiratory viruses may also predispose to nasopharyngeal bacterial colonization and alteration of the host's immune response. finally, viruses may be responsible for antibiotic treatment failure in cases of concurrent bacterial and viral infections. likewise, respiratory viruses have been implicated in the development of sinusitis, either directly as an extension of urt infection or as a result of secondary bacterial infection. laryngitis and laryngotracheobronchitis-croup have more frequently been associated with the parainfluenza virus group. pneumonia in infants and young children is most commonly attributed to viruses. disease burden is also significant among the elderly and the immunocompromised. rvs and rsvs are the agents most frequently isolated in viral pneumonias, although age, season, year, and other variations can be considerable. the presence of adenovirus is a risk factor for severe disease in infants. influenza occurs in yearly winter epidemics and pandemics every - years resulting in enormous morbidity and mortality, especially among the very young, the elderly, and the immunocompromised. infections with ibv or icv are mostly milder than those with iav (paules and subbarao, ) . the zoonotic potential of idv is yet unknown. so far, this virus could not be found in patients with respiratory symptoms although a high seroprevalence in people working with cattle has been observed (su et al., ) . rsv is the major pathogen implicated in acute bronchiolitis in infants up to year and is responsible for a great number of hospitalizations and deaths in this age group. in the second year of life, rv and hbov become more frequent pathogens (jartti et al., ) . host immune response skewed towards type cytokine production seems to play a central role in the pathogenesis of the disease. bronchiolitis is further associated with recurrent wheezing. the great majority of acute asthma exacerbations in children and a considerable proportion in adults is preceded by a viral urt infection. such exacerbations may often result in hospitalization. the most frequently isolated offending agent is rv, a fact that reflects the preponderance of the virus among common cold cases. finally, exacerbations of chronic obstructive pulmonary disease may also have a viral etiology, most commonly due to rvs. wupyv and kipyv have so far only been associated with increased sputum production and wheezing; a clear role as causative agent has not been shown (cook, ) . the diagnosis of respiratory viral infections relies upon clinical criteria and is further supported by laboratory techniques such as direct viral, nucleic acid or antigen detection, culture, and serology. direct detection is based on the identification of whole virus or inclusion bodies with electron or light microscopy, respectively. viral antigens may also be detected by the use of immunofluorescence and enzyme immunoassays but an adequate viral load is required. viral cultures usually demonstrate the biological effects of an agent, such as the cytopathic effect or hemagglutination, in cultured cells. culture methods, however, have drawbacks due to table disease syndromes of common respiratory viruses in upper and lower respiratory tract. their low sensitivity, extensive time length (days to weeks), and sometimes complex culture systems. determination of specific viral antibodies in a host's blood sample and comparison between acute and convalescent phases of respiratory illness is another diagnostic modality, which is mainly used for epidemiological purposes. serological tests include immunoassays, complement fixation, and passive agglutination assays as well as hemagglutination inhibition. nucleic acid amplification tests (naats), like realtime polymerase chain reactions (real-time pcrs), are gaining more and more importance due to their high sensitivity and specificity, combined with low turnaround times. together with antigen-based diagnostic tests, naats are used in point-of-care tests (pocts), which are able to provide results much faster than lab-based tests and directly at the site of sample collection. in the field of genetic epidemiology and virus discovery, next-generation sequencing (ngs) became the method of choice, as no susceptible cell lines are required. usually, samples from the respiratory tract are taken for diagnostic purposes. these include swabs from nose or throat, nasopharyngeal aspirates, nasal washes, sputum, bronchial aspirates, and bronchoalveolar lavages. upon infection with hpevs or hadvs systemic infections or other complications, e.g., gastrointestinal or neurological symptoms, may occur. in this case, additional specimens, ranging from stool, blood, urine, and cerebrospinal fluid, are taken (olijve et al., ; khanal et al., ) . the development of effective vaccines is hampered by the large number of viral serotypes. currently, licensed vaccines are available only against influenza a and b viruses, as well as an oral live vaccine against hadvs for u.s. military personell (paules and subbarao, ; crenshaw et al., ) . following the regular recommendations of the world health organization (who), the composition of the seasonal influenza vaccines is updated every year to include the most important strains and to confer the best protection. however, as the actual frequency of the circulating influenza strains in each season may differ from this recommendation, the efficacy of these vaccines vary from year to year. mostly, tri-and quadrivalent influenza vaccines, inactivated, liveattenuated, or recombinant vaccines are used. to overcome the need for a yearly vaccination, the search for alternative approaches and universal influenza vaccines is ongoing. vaccine candidates against other respiratory viruses, like hrsvs, hpivs, hmpv, and mers-cov are in development or under clinical investigation. over-the-counter remedies for the alleviation and reduction of duration of common cold symptoms include vitamin c, zinc, and echinacea, although these agents are only moderately effective. nasal decongestants, first generation antihistamines, anticholinergic nasal sprays, oral and intranasal a-adrenergic agonists, inhalation of humidified hot air at - c, nonsteroidal antiinflammatory drugs, and cromones have been proposed for symptomatic relief with moderate results. a combination of specific antivirals with one or more anti-inflammatory drugs may offer the ideal therapeutic approach since it would both inhibit viral replication and block inflammatory events, although cost and compliance are considerable obstacles. unfortunately, only a limited number of antiviral agents against respiratory viruses is available, due to the frequent mutations resulting in the constant emergence of new and often resistant strains. nevertheless, many new drugs and experimental approaches are under investigation (papadopoulos et al., ) . the licensed antiviral chemotherapy against influenza viruses can be grouped into four classes, which prevent uncoating (adamantanes like amantadine and rimantadine), inhibit na (e.g., oral oseltamivir and inhaled zanamivir), prevent membrane fusion (arbidol), and interfere with the viral rna-dependent rna polymerase (the broad-spectrum antiviral favipiravir). adamantanes are only active against iva, however, today all circulating seasonal strains of iav are resistant against this antiviral, so the use is not recommended any more. na inhibitors are available for treatment and prophylactic purposes, but prophylactic applications are more effective as randomized controlled trials showed only marginal effects on the duration of symptoms (paules and subbarao, ) . new experimental antiviral agents target both the virus and the host, e.g., by inducing apoptosis only in infected cells (shim et al., ) . another experimental host-targeted strategy, against infections with hbov, is repurposing of kinase inhibitors developed for tumor treatments (qiu et al., ) . palivizumab is a monoclonal antibody against rsv f glycoprotein, which may be administered prophylactically to reduce the risk of severe bronchiolitis and hospitalization in specially vulnerable children, i.e. preterm infants and children with chronic lung or heart diseases (jartti et al., ) . the antivirals cidofovir and ribavirin have been used against rsv especially in immunocompromised patients, but little to no beneficial and many adverse effects have been observed. consequently, their widespread use is not recommended (khanal et al., ) . the clinical usefulness of the capsid binding antivirals pleconaril, vapendavir, and pocapavir against rvs still has to be shown. a high likelihood for drug resistances limits their clinical applicability (papadopoulos et al., ) . a long-known effective compound against a variety of respiratory viruses and the resulting common cold is interferon alpha- b, administered intranasally, before or shortly after exposure. however, due to its high cost, dosage frequency (six times daily), and side effects (mainly nasal irritation and bleeding) clinical application has been abandoned (mossad, ) . the human polyomaviruses ki and wu: virological background and clinical implications perspective on adenoviruses: epidemiology, pathogenicity, and gene therapy enteroviruses and parechoviruses human coronaviruses oc and hku bind to -o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a bronchiolitis needs a revisit: distinguishing between virus entities and their treatments the repertoire of adenovirus in human disease: the innocuous to the deadly virus taxonomy. ninth report of the international committee on taxonomy of viruses. international union of microbiological societies transmission routes of respiratory viruses among humans adenovirus persistence, reactivation, and clinical management viruses and bacteria in the etiology of the common cold proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types ictv virus taxonomy profile: polyomaviridae treatment of the common cold human parechovirus: an increasingly recognized cause of sepsis-like illness in young infants molecular epidemiology of respiratory syncytial virus promising approaches for the treatment and prevention of viral respiratory illnesses the role of human parainfluenza virus infections in the immunopathology of the respiratory tract innate recognition of viruses human parvoviruses dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc ictv virus taxonomy profile: pneumoviridae royston l and tapparel c ( ) rhinoviruses and respiratory enteroviruses: not as simple as abc human metapneumovirus-what we know now influenza virus infection, interferon response, viral counter-response, and apoptosis from sars to mers, thrusting coronaviruses into the spotlight epidemiology, genetic recombination, and pathogenesis of coronaviruses novel influenza d virus: epidemiology, pathology, evolution and biological characteristics changes to virus taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses ictv virus taxonomy profile: picornaviridae microbiologic diagnosis of respiratory illness: practical applications influenza burden, prevention, and treatment in asthma-a scoping review by the eaaci influenza in asthma task force european scientific working group on influenza (eswi) /research_projects/study_groups/respiratory_viruses-european society of clinical microbiology and infectious diseases (escmid) study group on respiratory viruses (esgrev) hadvwg.gmu.edu-human adenovirus working group. talk.ictvonline.org-international committee on taxonomy of viruses (ictv). www.picornaviridae.com-picornavirus study group. www.resvinet.org-respiratory syncytial virus network the authors would like to thank the european society of clinical microbiology and infectious diseases (escmid) study group on respiratory viruses (esgrev) for providing a platform for scientific discussion on the topic. key: cord- -y woqsii authors: tews, birke andrea; meyers, gregor title: self-replicating rna date: - - journal: rna vaccines doi: . / - - - - _ sha: doc_id: cord_uid: y woqsii self-replicating rna derived from the genomes of positive strand rna viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. the following chapter summarizes the principles how such rnas can be established and used for design of vaccines. due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature. the story of self-replicating rna started with the recognition of the infectious nature of some viral rna genomes in the s and s [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the evidence that naked rna upon introduction into cells is able to promote a full replication cycle including release of infectious virus particles represented the starting point for a new era of research on rna virus molecular biology and its application. due to the technical difficulties, rna is not amenable to site specific manipulation so that reverse genetics systems for rna viruses always rely on a cdna intermediate [ , ] . first successful approaches towards recovery of replicating viruses from cloned cdna were published for positive-strand rna viruses relying on transfection of plasmid dna containing a virus derived cdna insert [ ] . soon afterwards, in vitro transcription and transfection of viral genome-like rna was described leading to recovery of infectious progeny virus [ , ] . positive-strand rna viruses were the first rna viruses amenable to direct genetic manipulation due to their simple strategy of gene expression and replication [ ] . the genomic rna (vrna) represents an mrna able to govern the production of all viral proteins necessary for the initiation of virus replication. products of the first round of translation of the viral genomic rna assemble into a replicase complex that polymerizes a minus strand complementary to the genome (crna) as a template for the synthesis of additional mrna molecules. thus, for all positive-strand rna viruses the components of the replicase complex have to be translated directly from the genomic rna. viral polypeptides not required for rna replication, which mainly constitute structural proteins, can either also be translated from the genomic rna or from one or more subgenomic mrnas transcribed from the negative sense crna template, depending on the specific type of virus. genomes of members of the group using the former expression strategy contain one long open reading frame (orf). translation of this rna leads to a polyprotein that is co-translationally and posttranslationally processed by viral and host cellular proteases. the members of the families picornaviridae and flaviviridae belong to this first group (fig. ) . the second group comprises the families togaviridae, coronaviridae, arteriviridae, and caliciviridae. these viruses are characterized by the subgenomic rnas used for expression of part of their genes ( fig. ) . in contrast to the first group, the replicase genes of these viruses are located in the ′ part of the genome upstream of the structural genes. for all of these viruses the subgenomic rnas are ′ co-terminal with the genomic rna. is translated into one polyprotein that is subsequently processed into the viral proteins. togaviruses and caliciviruses transcribe one rna of subgenomic length encoding the structural proteins. coronaviruses and arteriviruses use multiple subgenomic mrnas for expression of structural and accessory proteins. rna in coding orientation (mrna sense) is represented by black bars whereas grey bars symbolize negative strand intermediates of viral genome replication. the location of structural and nonstructural genes in the viral genomes is indicated the possibility to recover self-replicating viral rna from cloned cdna sequences opened a window to sophisticated studies on the mechanisms of rna virus replication. moreover, this knowledge was crucial for establishment of rationally attenuated viruses as well as development of strategies for use of self-replicating rna expressing foreign genes for vaccine purposes and other applications. in this chapter, we present the technical principles used for establishment of self-replicating rna and selected examples for its application in the context of vaccine development. due to the greater instability of (single stranded) rna versus dna and the wealth of techniques for dna manipulation in contrast to the difficulties of direct rna manipulation recombinant virus systems are based on dna constructs, even in the case of rna viruses where these systems rely on cdna of the viral rna. due to the infectious nature of the positive strand rna virus genome reverse genetics systems for positive strand rna viruses need not be much more complicated than to be a way to deliver genome-like rna into cells for successful replication of said rna and for virus recovery. the history of reverse genetic systems for positive strand rna viruses highlights the pitfalls that may be encountered in the design of a reverse genetic system and show solutions how to circumvent these difficulties. some of these difficulties are covalently linked to the genome structures found in different positive strand rna virus families. the genome can be capped or linked to a so-called vpgprotein or contain a naked ′ end. the ′ end can form loop structures or be a poly-a tail as would be expected for mrnas. depending on the virus the correct ′ and ′ end is very important as they can be crucial for replication and/or translation, or the production of subgenomic rna (fig. ). the first infectious cdna clone of a eukaryotic virus was a cdna clone for poliovirus [ ] . this construct had the complete cdna-sequence including a residue poly (a) tail in the plasmid pbr and yielded infectious virus particles upon transfection in mammalian cells. this first construct did not contain a dedicated promoter to ensure the transcription of viral rna, but nevertheless led to enough rna expression for virus recovery. three years later, the performance of poliovirus cdna clones could be significantly ameliorated through the introduction of sv transcription and replication signals and transfection of the resulting construct into cells expressing the sv large t antigen [ ] , thus ensuring replication of the dna-plasmid in eukaryotic cells leading to a higher yield of viral rna and recovered virus (fig. , left part) . for other picornaviruses, cloning the cdna into a bacterial fig. different strategies to generate reverse genetic systems for positive strand rna viruses. upper part: viral rna can either be obtained from purified virus particles or from infected cells trough total rna extraction. cdna of the viral genome can be generated using a specific primer complementary to the ′ end of the viral genome if the sequence is known, oligo-d(t) primers for polyadenylated genomes or random priming in case of unknown sequences. rna can also be used in high throughput sequencing approaches to determine the viral genomic sequence including the genomic ends. middle part: to obtain efficient reverse genetics systems the cdna needs to be cloned downstream of promoter sequences. this can either be a rna polymerase ii promoter if the vrna shall be transcribed in the nucleus of transfected cells, or bacteriophage promoters like t for in vitro transcription. when possible, the cdna is assembled in one full length construct (left). alternatively, the cdna can be cloned in fragments into different plasmids to avoid instability or to break down large genomes to sizes that are more amenable to manipulation (right). lower part: from full length plasmids containing a eukaryotic promoter vrna will be transcribed by the cellular machinery upon transfection of the cdna construct. after export of the rna into the cytoplasm its translation will provide the viral proteins necessary for replication (left). full length plasmids with bacteriophage promoters are linearized before rna synthesis via run-off in vitro transcription (middle). when the viral cdna is cloned in several fragments, the complete cdna needs first to be assembled into a full length cdna template by in vitro ligation to obtain a template for in vitro transcription (right). the resulting rna is transfected into cells where it is translated. in all cases translation of the rna within transfected cells generates the viral replicase proteins that are necessary and sufficient to initiate virus replication and production of viral particles plasmid was not enough to establish constructs leading to the production of infectious virus progeny. indeed, the first cdna clone for rhinovirus type failed to produce infectious viral particles, but addition of an sp bacteriophage promoter upstream of the cdna combined with in vitro transcription of the cdna, produced rna that led to infectious progeny upon transfection into cells [ ] (fig. , middle ). an equivalent approach was also used in the reverse genetics system for brome mosaic virus, which consists of three plasmids containing the cdnas to the three viral genomic rnas immediately downstream of a λ-phage promoter to drive in vitro transcription. combined transfection of the three in vitro-transcribed rnas led to virus infection in plants [ ] . another problem encountered in the generation of reverse genetic systems was the fact that some plasmids containing viral cdna were unstable in e. coli and/or induced cytotoxicity. in many cases, cytotoxicity or instability of the viral cdna could be countered successfully by use of low copy plasmids with for example p a origins of replication restricting the plasmid copy number to or very few per cell. this approach was successful in all of the first infectious clones for pestiviruses (ncpbvdv, cpbvdv, and csfv) [ ] [ ] [ ] [ ] but failed in case of yellow fever virus (yfv). this problem led to the development of a strategy using two or more plasmids, each of which contained a different part of the virus-derived cdna. the first yfv infectious cdna clone ( d vaccine strain, first flavivirus infectious clone at all) consisted of two separated fragments corresponding to the ′ and ′ half of the genome, respectively. infectious rna was generated through in vitro ligation of the two fragments followed by in vitro transcription [ ] (fig. right part) . correct ′ end ′ ends of the viral genome are often very important for the success of a reverse genetics system, as many viruses rely on special structures at their termini for replication and/or translation. all systems described above used restriction enzyme sites introduced directly downstream of the viral cdna to linearize the plasmids before run-off in vitro transcription to obtain rna ′ ends identical or as similar as possible to those of the viral genome. with regard to the ′ end of the rna the use of bacteriophage promoters (mostly t or sp ) allowed to transcribe rna with a marginally modified or even the desired start since only a ′ g residue is necessary for efficient transcription by these enzymes. all infectious cdna clones for members of the flaviviridae were established with a t promoter directly upstream of the genomic cdna and a blunt cutting restriction enzyme with a recognition site that directly overlaps the ′ end of the genome to allow run-off in vitro transcription resulting in rna identical to viral genomic rna [ ] [ ] [ ] [ ] [ ] . as an alternative to in vitro ligation of cdna fragments an interesting approach based on reconstitution of full length viral genomic rna via intracellular rna recombination has been developed. rna recombination is a naturally occurring process and very widespread in rna viruses. it gives rise to new virus variants such as the cytopathic biotype of pestiviruses [ ] [ ] [ ] [ ] [ ] . recombination of rna of positive strand rna viruses that replicate in the cytoplasm of infected cells, is different from dna recombination or cellular rna splicing, in which dedicated cellular machinery joins the ends of the respective nucleic acids. recombination of cytoplasmic rna is thought to occur either through template switching by the rna-dependent polymerase during genome replication or through breakage of the rna and joining with other rna ends [ ] . several experiments with pestivirus and poliovirus mutants have shown that rna recombination can happen independently of active rna replication [ , ] . in these experiments rna fragments that each encoded only part of the rna-dependent rna polymerase were co-transfected into cells and were sufficient to lead to the recovery of infectious virus. the fact that intracellular recombination of viral rna occurs rather frequently has been used as a tool to manipulate viral genomes not (yet) accessible to reverse genetics systems by cdna clones or similar approaches via recombination of (mutated) genome fragments [ ] [ ] [ ] [ ] . the above mentioned instability of viral full length cdna clones is in part dependent on the size of the viral genome. the first cdna clones were established for members of the picornaviridae with genome sizes of about . kb [ , , ] . members of the flaviviridae have genome sizes of . - kb. coronaviruses have the largest rna genomes and therefore remained inaccessible to reverse genetic systems based on cdna clones for a long time. instead, targeted mutagenesis was achieved through recombination of transfected in vitro-transcribed rna representing only a part of the viral genome and full length viral rna in infected cells [ ] [ ] [ ] . it took years from the first infectious full length cdna clone of a picornavirus to a full length infectious cdna clone of a member of the coronaviridae [ ] . the latter construct was for the transmissible gastroenteritis virus (tgev) and used a bacterial artificial chromosome (bac) to propagate the large virus derived cdna with low copy number, as parts of the genome were toxic to the bacteria. furthermore, this cdna clone contained the tgev sequence downstream of a cytomegalovirus immediate early promoter and upon transfection of the dna into cells, viral rna was produced by the cellular rna polymerase ii, which then led to the production of infectious viral particles. the same year, a second cdna system for tgev was published using five separate plasmids which together contained the full length genome and needed to be assembled through in vitro ligation before rna synthesis [ ] . yet another approach followed a year later for the avian coronavirus infectious bronchitis virus in which the genomic cdna was inserted into the genome of vaccinia virus, a large dna virus from the family poxviridae [ ] . however, also strategies based on the use of rna recombination are still employed for establishment of recombinant coronaviruses [ ] . methods to generate the long viral cdna have changed in the last years. the first approaches were based on cdna libraries made from purified virion rna or rna of infected cells [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . later, full length pcr amplification of viral genomes became feasible through the availability of proofreading polymerases that allowed generation of an infectious clone after a single round of reverse transcription, followed by long-range pcr [ , ] . with the rapid development of nucleic acid synthesis and high throughput sequencing it is now possible to generate cdna clones through synthesis of the corresponding dna sequences simply with the knowledge of the sequence. this was first demonstrated once again with poliovirus, but recently a cdna clone system based on synthetic plasmids was published for the coronavirus porcine epidemic diarrhea virus [ , ] . development of a strategy for establishment of a reverse genetics system for a new virus, which allows generation of self-replicating rna and recovery of recombinant virus, requires knowledge on the molecular biology of this virus and a variety of considerations with regard to the final aim of the approach. the first step will usually be the determination of the sequence of the viral genome including the correct ′ and ′ ends. the latter information can be obtained by so-called race technology (rapid amplification of cdna ends), pcr based systems that nowadays are provided by different commercial suppliers. the knowledge of the sequence will provide the necessary information on the genome organization which helps to understand the gene expression strategy of the virus. an important question in this context concerns the mechanism promoting initiation of translation and replication of the viral genomic rna. as described above, translation of the genome is necessary to provide the components of the replicase that starts genome replication and thereby initiates the viral life cycle. positive strand rna viruses have developed a variety of strategies to ensure initiation of translation of their rna [ ] [ ] [ ] . in most systems, the infectious cdna construct can be designed in a way that cis-acting structures important for translation and replication of the genomelike rna derived from the construct will be equivalent to what is found in the viral genome. there are, however, special cases providing problems. caliciviruses have a protein called vpg covalently bound to ′ end of the viral rna, which functions as a substitute for the cap structure driving translation initiation in eukaryotic mrnas. this protein is most likely also crucial for the rna to be accepted as a substrate for rna replication but cannot be easily linked to in vitro-transcribed viral rna. replacing vpg by a standard cap structure was found to be sufficient for translation and initiation of replication of the in vitro-transcribed rna, but with quite low efficiency [ , , ] . similarly, the ′ end of the viral rna is important for successful recovery of self-replicating rna. many viruses contain a poly(a) tail at the ′ end and thereby mimic the structure of a standard eukaryotic mrna ensuring efficient translation. the poly(a) tail should also be important for replication of the viral rna since it is the sequence at which transcription has to start during minus strand synthesis. other viral genomes contain no poly(a) but for example specific secondary structures representing important cis-acting elements for both translation and rna replication. as a general rule, any virus with a genome containing a poly(a) tail should also have such a sequence in its infectious cdna construct, whereas viruses without a poly(a) tail can be expected to be very sensitive to changes in the sequence at their genomic ′ end, so that steps should be undertaken to ensure generation of the correct genomic end during transcription. when the necessary information on the viral genome and strategy of gene expression are available the next point to be decided is where and how transcription of the cdna construct should occur. for the majority of reverse genetics systems for positive strand rna viruses the genome-like rna is generated in vitro and subsequently introduced into cells via transfection. this strategy is characterized by some methodical advantages, especially the simple generation of correct end sequences through use of bacteriophage rna polymerases and "run-off" transcription. the transcription procedure was improved over the years so that highly efficient kits yielding large amounts of rna became commercially available. the most common promoters used in in vitro transcription systems are the phage promoters t and sp . these can be placed immediately upstream of the cdna sequence ensuring a correct ′ end of the resulting rna. to obtain capped transcripts either a cap analog (like m g( ′) ppp( ′)g) has to be included in the in vitro transcription reaction or the rna needs to be capped after the in vitro transcription (using vaccinia virus derived capping systems). if the genomic rna should contain a poly-a tail this needs to be either included in the template construct or added after transcription using a poly(a) polymerase. the second choice adds yet another step to the generation of the rna and thus might reduce yield. the above mentioned ways to introduction of a cap structure into in vitro-transcribed rna work with only rather low efficiency. thus, the alternative strategy relying on transfection of the plasmid dna followed by in vivo transcription of the genome-like rna can be advantageous when the production of capped transcripts is necessary, since rna produced in transfected cells is ′ capped and ′ polyadenylated by cellular enzymes. a problem with this approach is the relatively high chance of further post-transcriptional modification of the rna like splicing, which could abrogate any infectivity. to obtain correct genomic ends for non-polyadenylated viruses with this approach ribozyme sequences such as the hepatitis delta ribozyme can be added at the ends, which will cleave themselves off and leave the correct terminus [ ] . in fact, reverse genetics systems for positive strand rna viruses using direct transfection of dna into cells are much rarer than in vitro transcription based approaches. an interesting alternative combining features of the in vitro transcription system with the advantages of dna transfection is based on helper viruses like the vaccinia virus mva-t . cells infected with the latter virus contain bacteriophage t rna polymerase expressed by mva-t which upon introduction of plasmid constructs with t promoters will transcribe the desired rna in the cytoplasm of the cell which avoids nuclear location and the danger of unwanted splicing of the rna product. the defined start site of t based transcription allows for an easy production of the correct ′ end just as during in vitro transcription. insertion of ribozyme sequences into the plasmid can ensure the formation of the desired defined ′ end of the transcript. since vaccinia virus replicates in the cytoplasm it expresses enzymes that cap and polyadenylate its own transcripts efficiently, which is also true for the t transcripts. the final result is the efficient production of a capped and polyadenylated transcript with correct ends within the cell which can lead to superior performance compared to in vitro transcription/transfection of rna [ ] . as mentioned before instability of viral sequences in e. coli while propagating the cdna plasmids can be countered by different measures. it is preferable to use low copy plasmids or bacs to minimize the amount of plasmids with toxic sequences in the bacteria. moreover, bacs can carry large inserts and thus are suitable for every positive strand rna virus genome including those of coronaviruses. sequences that seem to be deleterious for the propagation in e. coli can be disrupted by strategically placed intron sequences, if virus recovery is achieved via plasmid transfection into cells and intracellular rna synthesis through rna polymerase ii. the intron will be spliced out of the produced rna regenerating the viral sequence within the cells. this approach was employed in the production of a tgev infectious clone [ ] . taken together, the establishment of systems for generation of self-replicating rnas and recovery of infectious recombinant positive strand rna viruses are in principle straight forward today but have to pay attention to the individual features of the respective system and the aims to be achieved. depending on the system, more rarely used strategies like rna recombination based generation of mutants might show up as the feasible solution. in fact, system specific problems had to be solved during development of almost any reverse genetics system that is routinely used now but the available repertoire of possible solutions for such problems will facilitate such approaches in the future. the development of techniques allowing the recovery of selfreplicating viral rna from cdna was not only a milestone for basic research on rna virus biology but also opened a door to new approaches towards modified live vaccines against viral diseases. in contrast to the traditional ways relying for attenuation on elaborate passaging of viruses in tissue culture cells or unusual host animals, reverse genetics systems allow for defined mutagenesis and rational attenuation. pestivirus are economically important pathogens of farm animals that are grouped in the family flaviviridae together with their closest relatives, the hepaciviruses. most important pestiviruses are the classical swine fever virus (csfv) and the bovine viral diarrhea virus (bvdv) [ ] . all members of the flaviviridae are enveloped viruses with positive strand rna genomes containing one long open reading frame. the economic impact of pestiviruses results at least in part from causing a wide range of pregnancy disorders and persistent infection due to their ability to cross the placenta in pregnant animals [ ] . persistently infected animals represent an important reservoir for virus spread. vaccination represents a feasible means to interrupt the cycle of transmission as long as the vaccines do not only prevent disease but also fetal transmission of the pathogens. to fulfill the latter demand pestivirus vaccines have to be very potent. the so-called csfv c-strain represents an example of a successful pestivirus vaccine. it is a traditional modified live vaccine that was attenuated via serial passages in rabbit cells resulting in a very safe and efficient vaccine virus with so far undefined basis of attenuation. the latter is also true for different live bvdv strains used for vaccination in various countries worldwide. as an important disadvantage of these vaccines the attenuated viruses can still cross the placenta and infect the fetus in pregnant animals which in case of the conventional live bvdv vaccines can even lead to abortion. using a reverse genetics approach we were able to establish a bvdv mutant with defined genomic deletions of nonessential sequences that knocked out two viral factors interfering with the host's type interferon response without significantly impairing viral replication [ ] . as a consequence of these changes affecting viral mechanisms blocking the innate immune response to bvdv infection not only complete attenuation of the mutant virus was observed but also the inability to infect the fetus in pregnant animals, the prerequisite for pregnancy disorders and persistent infection. another approach based on deletion of nonessential sequences was described for coronaviruses. members of the family coronaviridae represent important pathogens of man and animals among which sars and mers coronavirus (sars-cov and mers-cov) are best known [ , ] . as outlined above, coronaviruses have by far the largest known rna genomes which encode not only essential but also some nonessential accessory proteins. deletion of five of the eight group-specific orfs (orf a, of b, orf , orf a, and orf b), either alone or in various combinations, from the sars-cov genomic rna did not result in clear indications for attenuation in a mouse model. in contrast, a viable sars-cov mutant lacking the sequence coding for the e protein (orf ) was recovered that exhibited reduced virulence in two animal models probably by enhanced response of the immune response to the infection [ ] [ ] [ ] [ ] [ ] . e represents one of the membrane bound structural proteins of the virus and is involved in virion assembly and release. such deletion mutants are still being characterized and improved but might provide a basis for the development of coronavirus vaccines in the future. not only deletion of sequences but also exchange of genomic fragments between related viruses is easily done via reverse genetics and can lead to attenuation and other desired features. as an example, a chimeric pestivirus was established as a vaccine against classical swine fever (fig. ) . this concept was based on the replacement of the region coding for the major envelope protein e of a bvdv genome by the corresponding sequence of csfv. the resulting virus cp _e alf was only able to infect pigs and thus displayed the fig. generation of a chimeric viral genome from two parental rnas. on the level of a cdna construct, one protein-coding sequence is replaced by the corresponding gene of the other virus (principle used for the pestivirus vaccine cp _e alf [ ] ). ires internal ribosome entry site tropism of csfv. the chimeric virus was fully attenuated but nevertheless induced strong protective immunity [ ] [ ] [ ] . as an important further advantage, the configuration of this chimera allows for serologic differentiation between vaccinated animals and those having been infected by a csfv field virus, a feature of major importance for control and eradication programs in veterinary medicine. similar approaches as for cp _e alf were also used for members of the genus flavivirus in the family flaviviridae. the first approach employed the yellow fever virus (yfv) vaccine strain d, a virus developed in by empirical passage. yfv d is a very effective and safe vaccine that was found to highly trigger the innate immune response which helps driving the adaptive immune response to long lasting protective immunity [ ] [ ] [ ] [ ] . therefore, yfv d was chosen as backbone for a chimeric japanese encephalitis/yellow fever virus vaccine (chimerivax™-je) in which the surface protein prm/e coding region of yfv was replaced by the corresponding but modified jev sequence resulting in a safe and effective vaccine launched by the end of (trade name imojev™) [ ] . similar constructs in the d background were established with prm-e sequences from west nile virus or the four dengue virus (denv) genotypes and tested as vaccine candidates [ ] [ ] [ ] [ ] [ ] . also chimeric denv composed of sequences from two different denv genotypes and encompassing attenuating mutations were established and tested, as well as chimeras of denv with tick-borne encephalitis virus sequences [ , ] . the chimeric approach has also been followed in vaccine trials in alphaviruses, another group of positive strand rna viruses belonging to the family togaviridae. low virulent sindbis virus provided the backbone for these approaches that used exchange of the complete structural protein coding regions with sequences from highly virulent alphaviruses like eastern or western or venezuelan equine encephalitis virus (eee or wee or vee). the chimerization process itself led to significant attenuation of the resulting viruses that were found to be highly immunogenic (for review, see ref. ). nevertheless, the safety issue is a major concern in such approaches since biomarkers for the attenuation of sindbis virus are not known and small animal models for testing virulence in most cases not adequate to evaluate attenuation in humans. further research is needed to fully evaluate these vaccine candidates. it has to be stressed that all the approaches described above employ self-replicating rnas that represent either full length viral genomes or such rna with deletion of nonessential sequences. accordingly, these constructs allow the recovery of infectious virus particles. as presented above, introduction of the in vitrotranscribed recombinant rna into a cell via transfection starts its autonomous replication leading to release of infectious viruses that after amplification in tissue culture serve as vaccine. upon administration, the immune response is triggered because the vaccine virus mimics all steps of a field virus infection but without induction of significant symptoms. the use of fully replication competent recombinant viruses bears a certain risk of reversion to virulence. depending on the type of attenuating mutations this risk can be significant or only theoretically relevant as for viruses containing more than one deletion. introduction of deletions into rna virus genomes can lead to recovery of attenuated viruses in some special cases but will in most cases result in rnas that are no longer able to promote the generation of infectious progeny. as long as the deletions do not concern the sequences responsible for replication of the rna, such mutant rnas will behave as replicons that amplify autonomously when introduced into a cell and lead to translation of significant amounts of the encoded proteins. a typical replicon approach is based on deletion of sequences coding for one or more structural components of the virus (fig. ) . such replicons were important tools for research on rna replication of for example pestiviruses and hepaciviruses [ , ] . for pestiviruses, replicons have also been tested in vaccination approaches [ ] . in all cases, essential sequences were deleted from the genomes so that the vaccine candidates need complementation in trans by stably transfected cells providing the missing factors. infection of a host organism with the virus particles secreted from these complementing cells represents a dead-end since no infectious virus can be released from non-complementing cells. thus, these vaccines cross the border between live attenuated viruses and killed vaccines exhibiting safety features at least very close to killed vaccines. the big advantage over killed vaccines is the ability to express viral proteins within cells leading to mhc presentation of viral peptides and activation of a t-cell response in addition to the humoral response. fig. genome structure of a replicon. deletion of a structural protein-coding region from the viral genome (represented by a dotted line) without interrupting the reading frame results in an rna able to replicate autonomously within cells but deficient in production of infectious progeny an interesting example of a further developed replicon approach is found in flaviviruses with the so-called pseudoinfectious vaccines. integration of deletions of different length into the capsid protein coding region of the viral genomes results in autonomously replicating rnas that are no longer able to promote the generation of infectious virus particles [ , , ] . however, cells harboring these replicons will secrete large amounts of immunogenic prm/e particles. propagation of such replicons in stably transfected cells providing the missing c protein in trans leads to virus-like particles able to conduct a single round of infection with highly efficient establishment of cells producing the prm/e particles. as a further approach, a dna based vaccination has been developed that relies on two separate plasmids, one containing a cdna representing the capsid-deleted viral genome and another expressing the missing capsid protein [ ] . cells that have taken up both plasmids will not only translate and present viral sequences but will also release infectious virus particles that can infect further cells leading to an enhanced stimulus of the immune system. again, chimeric approaches with replicon backbones derived from one flavivirus species and prm/e coding sequences from another species have been tested successfully [ ] . the chimeric systems described above represent a special case of a more general approach towards vaccines based on self-replicating rna that contain foreign sequences. similar to the chimeric constructs mentioned before the replacement of viral protein-coding sequences by foreign genes can be used to express the desired proteins for immunization. in contrast to the chimeric viruses with a structural protein exchange between closely related viruses, such constructs will usually not yield autonomously propagating infectious viruses but replicons harboring non related sequences instead of the original structural proteins. alternatively, the foreign sequences can be inserted into the viral genome as additional information without loss of essential viral functions so that fully replication-competent recombinant viruses can be produced. a prerequisite for the successful establishment of such self-replicating rnas expressing foreign genes is the development of a strategy for integration and expression of the latter sequences without disturbing the autonomous replication of the rna. due to the fact that positive strand rna viruses use expression strategies based on translation of polyproteins and subsequent proteolytic processing, the integration of foreign sequences into a viral open reading frame has to be combined with a specific processing step. a common approach avoiding fusion of significant numbers of unwanted residues to the proteins of interest is to place the foreign sequence at the ′ end of the viral orf and insert the foot and mouth disease virus (fmdv) a-coding region between the foreign sequence and the viral polyprotein (fig. ) . fmdv a is a short peptide of amino acids that is able to induce an irregular stop and restart of translation [ , ] . in fact, a provokes an interruption of the polyprotein translation at its own carboxy terminus leading to release of an upstream protein fragment with a at its c-terminus and restart of translation with the proline following a in the polyprotein, so that the viral proteins following downstream are free of any added residues. an elegant approach avoiding any fusion of unwanted residues relies on the establishment of bicistronic rnas. in such constructs the foreign sequence is usually also placed at the ′ end of the orf with a stop codon at the desired end of the translated region. instead of a protein coding region ensuring processing of proteins an internal ribosome entry site (ires) is integrated between foreign sequence and the viral orf (fig. ) [ , ] . the foreign sequence is expressed using the strategy that initiates translation of the viral proteins in the wt virus. its translation terminates at a stop codon at the ′ end. the ires recruits ribosomes to the start site at the ′ end of the viral orf and thereby promotes translation of the proteins necessary for replication of the recombinant rna. an alternative arrangement has been published for bvdv, in which ires and foreign sequence are placed in the ′ ntr (fig. ) [ ] . similarly, viruses like alphaviruses that use subgenomic rnas for expression of their structural proteins can be adapted to expression of foreign sequences with an approach relying on the standard genome organization and expression strategy of the viruses. the viral rna contains promoter sequences that recruit the viral rna polymerase to internal sites of the minus strand rna replication intermediate and start transcription of a mrna of subgenomic length [ , ] . replacing the viral structural protein coding sequence downstream of this internal promoter with the desired foreign sequence will lead to a replicon that transcribes an mrna coding for the foreign protein (fig. ) . alternatively, the subgenomic rna promoter can be duplicated and inserted together fig. strategies to express foreign sequences in alphaviruses. on top, a standard alphavirus genome is shown. it contains two open reading frames, the second of which is expressed from as subgenomic mrna transcribed under control of the subgenomic promoter (sg prom). replacement of the structural protein coding second orf by a foreign sequence will lead to a replicon expressing the desired protein (middle), whereas insertion of the foreign sequence downstream of a duplicated sg promoter generates a recombinant virus expressing the desired sequence via a second sg mrna (bottom). see also fig. with the desired foreign sequence as an additional cistron into the viral genomic rna thereby preserving its ability to drive the generation of infectious replication competent virus particles. based on these principles, a variety of vaccine strategies has been developed [ ] . as a matter of fact, basically all approaches using self-replicating rna for vaccination employ packaging of the rna into virions or virus-like particles. the reason for this preference over naked or stabilized rna is based on the extraordinary performance of the viral infection machinery resulting in highly efficient delivery of the rna into cells. since self-replicating rnas derived from viral genomes exhibit the intrinsic property for specific packaging into viral particles, the use of this strategy is easy and straight forward. it has, however, to be mentioned that many virus particles display quite limited stability so that approaches relying on stabilized rna could well be advantageous in certain situations especially when a cold chain during transport and delivery cannot be provided. an interesting opportunity for the future could be a vaccine formulation containing the rna genome of a fully replication competent attenuated virus in stabilized form so that infectious virus is generated in the vaccinee upon administration. this approach would combine the superior resistance of stabilized rna with the efficacy of a modified live viral vaccine. infectivity of ribonucleic acid from ehrlich ascites tumour cells infected with mengo encephalitis infectivity of ribonucleic acid isolated from virus-infected tissues infectious ribonucleic acid derived from enteroviruses recovery from virus and assimilation by cells biological, physical, and chemical studies characterization and classification of echo -rhinovirus-coryzavirus agents biophysical studies of a rhinovirus. extraction and assay of infectious ribonucleic acid rna-based viral 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induces integrated multilineage and polyfunctional immune responses yellow fever vaccine yf- d activates multiple dendritic cell subsets via tlr , , , and to stimulate polyvalent immunity systems biology approach predicts immunogenicity of the yellow fever vaccine in humans understanding the role of innate immunity in the mechanism of action of the live attenuated yellow fever vaccine d chimeric live, attenuated vaccine against japanese encephalitis (chimerivax-je): phase clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated japanese encephalitis antigen live attenuated chimeric yellow fever dengue type (chimerivax-den ) vaccine: phase i clinical trial for safety and immunogenicity: effect of yellow fever preimmunity in induction of cross neutralizing antibody responses to all dengue serotypes recombinant chimeric yellow feverdengue type virus is immunogenic and protective in nonhuman primates west nile virus vaccine a live, attenuated recombinant west nile virus vaccine traditional and novel approaches to flavivirus vaccines chimeric tick-borne encephalitis and dengue type viruses: effects of mutations on neurovirulence in mice recombinant, chimeric, live, attenuated vaccines against flaviviruses and alphaviruses nucleic acid-based infections and pseudoinfectious flavivirus vaccines characterization of an autonomous subgenomic pestivirus rna replicon replication of subgenomic hepatitis c virus rnas in a hepatoma cell line packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response mimicking live flavivirus immunization with a noninfectious rna vaccine flavivirus immunization with capsid-deletion mutants: basics, benefits, and barriers the 'cleavage' activities of foot-and-mouth disease virus a site-directed mutants and naturally occurring ' a-like' sequences analysis of the aphthovirus a/ b polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip establishment and characterization of cytopathogenic and noncytopathogenic pestivirus replicons stable recombinants of bovine viral diarrhea virus containing a hepatitis c virus insert noncytopathic sindbis virus rna vectors for heterologous gene expression the alphaviruses: gene expression, replication, and evolution key: cord- - re l w authors: bolin, steven r.; grooms, daniel l. title: origination and consequences of bovine viral diarrhea virus diversity date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: re l w the potential consequences of bvdv genetic and antigenic diversity are far ranging. the complexity of clinical presentations associated with bvdv likely arises from factors encoded by the virus genome. more importantly,prevention and control of bvdv may be complicated by diagnostic and immunization failure resulting from virus diversity. evolutionary pressures will continue to drive further diversity, making control of bvdv challenging. current and the potential for future bvdv strain diversity should be considered when designing bvdv control programs both at the individual farm and national herd level. the purpose of this article is to provide an overview of genetic, antigenic, and biologic diversity among bovine viral diarrhea viruses (bvdv), and to discuss the impact of that diversity on disease manifestations, diagnostic testing, and disease control strategies. the genetic diversity that occurs among isolates of bvdv is characteristic of rna viruses that exist in nature as quasispecies (a swarm of viral mutants). the basis for the viral quasispecies phenomenon will be discussed briefly and related to recent evidence for the existence of bvdv as a quasispecies. the genetic diversity that occurs among bvdv is reflected in the antigenic diversity found among viral isolates worldwide. the persistently infected (pi) animal is considered important for maintaining bvdv in nature, and as being a primary source of virus for other cattle. pi cattle may also serves as a source of viral genetic variants that may be ''selected'' by non-pi cattle when infected with virus. the emergence and establishment of genetic and antigenic variants of bvdv also is affected by selective pressure applied to the virus by the innate and adaptive host immune responses. the array of disease manifestations seen during infection with bvdv, and the corresponding pathogenic processes, may be attributed to viral diversity; however, the definitive viral markers for tissue tropism or virulence have yet to be identified. quasispecies compared with dna viruses, rna viruses are highly mutable. positivestrand rna viruses, like bvdv, are subject to genomic modifications that involve point mutations or recombination of rna. the latter may be homologous (involving recombination of viral rna [self-rna]) or nonhomologous (involving recombination of rna from another bvdv [nonself] or from the infected host). point mutations are a regular occurrence in rna viruses, which have mutation frequencies that approach ÿ base substitutions per base site. this means that any given base in the viral genome is expected to undergo mutation once in every , replications of the viral rna. at that frequency of mutation, a , base rna virus (bvdv has about , bases in its genome) is essentially guaranteed at least one point mutation (single base change) per replication cycle of the viral rna [ ] [ ] [ ] [ ] . the high frequency of point mutations primarily is attributable to the error-prone viral rna polymerases responsible for replication of viral rna. as an explanation for the above, the parent rna virus (virus that infects a cell) must undergo two rounds of replication of its rna to produce viable progeny. the first round of replication produces an rna that is complementary to the rna contained in the parent virus. the complementary rna then serves as the template for a second round of replication that produces the rna that is packaged into the progeny viruses. multiple copies of complementary rna are produced from each virion that infects a cell, and multiple copies of progeny rna are produced from each strand of complementary rna. a point mutation that occurs during replication of the complementary strand of viral rna will carry over to each strand of progeny rna produced from that template, creating a clone of viral progeny expressing that particular mutation. a point mutation that occurs during replication of the progeny rna from the complementary rna template will be unique to that individual progeny (fig. ) . because there are two rounds of replication of rna for each cycle of viral replication, and each round of replication of viral rna has a mutation frequency of ÿ , the expected number of point mutations per viral genome per replication cycle of virus may exceed (mutation rate > ). thus, each progeny virus will differ from the parent virus by or more point mutations, and a swarm of viral mutants is created with each cycle of viral replication. the swarm of viral mutants forms a quasispecies. virus replicates in many cells in the infected host and several replication cycles may occur in day. the number of times that a point mutation can occur at one base site in day of viral replication can exceed million during the peak of infection, when the viral load may be to infectious particles per milliliters of plasma [ ] . the potential to create new viruses is tremendous; however, under neutral conditions that do not provide a selective advantage for one of the mutants, the mutant swarm tends to maintain a master base sequence that reflects the base sequence of the parent virus. this is because most point mutations are either deleterious for survival of the virus, and are not carried forward, or they do not give the viral mutant a competitive advantage that allows it to dominate the mutant swarm. however, the ability to constantly generate mutants allows rna viruses to adapt quickly to host responses and, in some cases, establish chronic or persistent infections using a variety of mechanisms [ , ] . the ability to evade the immune response and establish a chronic infection would prolong shedding of virus and enhance viral survival in nature. viruses that exist as a quasispecies have the potential to create a series of mutants that stay one step ahead of the adaptive immune system, thereby prolonging infection and extending the time when the virus may be transmitted to a new host. such viruses also have the potential to infect a host that has an existing immune response due to prior exposure with virus. the larger the dose of virus that is in the initial inoculum during transmission to a new host, the greater the chances are that a mutant virus is present that can evade an existing immune response. interestingly, a swarm of mutant viruses may possess a molecular memory that retains genetic mutants that demonstrated a selective advantage during previous cycles of viral replication [ , ] . thus, when the viral swarm encounters an exigent circumstance, such as infection of a host with an existing immune response or with a physiologic disturbance like fever [ ] , mutants already are present that were successful previously in a similar circumstance. viral mutants that are of low virulence would seem best fit for adaptation to the host and persistence in nature. those viruses would have few adverse effects on the host, allow host survival, and may have a prolonged period of viral shedding. in the case of bvdv, most viral isolates are of low virulence and noncytopathic in biotype. this is consistent with a virus that is well adapted to its host. however, low virulence and host survival may be selected against in a process that favors viral mutants that are more ''fit'' for replication in the host [ ] . viral mutants that replicate to a higher titer than the parent virus would have a competitive advantage and soon dominate the mutant swarm. this process could favor emergence of virulent viruses that exhibit enhanced viral replication, which in turn, could lead to extensive tissue damage and a burst of viral shedding in high numbers. the release of high numbers of virus improves the chance of viral transmission, making virulence a positive trait and giving virulent virus a competitive advantage over less virulent viruses [ ] . in the case of bvdv, this might explain the periodic emergence of virulent bvdv that produce large outbreaks of disease [ ] . recent studies have shown that bvdv exists as a quasispecies. those studies have focused on the untranslated region of the viral rna. this region of the viral genome is relatively conserved, due to its important role as a ribosomal entry site for production of viral protein, as the site for initiation of replication of virion rna, and for its role in encapsulation of the viral rna in viral progeny [ ] [ ] [ ] [ ] [ ] [ ] . however, altered base sequence in this region of the viral genome has been identified after passage of the virus in cell culture, and has been detected in viral rna that was extracted from tissues of an infected animal [ , ] . the ability to mutate rapidly allows a virus like bvdv to quickly produce mutants that are better fit to replicate in the host. as an example, mutations were created in the untranslated region of viral rna that impaired viral replication. the created mutations were ''repaired'' within a few rounds of viral replication in cell culture [ ] . the repair was done by natural selection of spontaneous mutants that were better fit for replication than the parent virus. populations of genetic variants of bvdv also have been identified within individual pi cattle [ ] . the genetic variants had different amino acid sequences in the immunologically important e envelope glycoprotein, an important target of viral neutralizing antibody. variation in amino acid sequence of the e protein may benefit a virus during an acute infection by allowing escape from the immune response. however, antigenic variation in viral proteins in the pi animal likely would be detrimental to the virus. this is because immunotolerance in a pi animal is specific to the persistent bvdv; hence, antigenic variation would likely trigger an immune response against that population of mutant virus. there would be selective pressure to maintain antigenic continuity during persistent infection. however, the detection of genetic variants in pi cattle suggests that those animals may enhance the diversity of bvdv by serving as a source of viral variants that can infect other cattle. also, this may explain the presence of viral neutralizing antibodies that are occasionally found in pi cattle with no known exposure to bvdv from an outside source. even though viral variants can be detected in a pi animal, the swarm of viral mutants as a whole maintains a stable master, or consensus, sequence over time. this has been shown for the relatively stable untranslated region of the viral rna and for the highly variable e envelope glycoprotein [ , ] . similarly, the master sequence remains relatively stable in individual animals and in groups of animals during outbreaks of acute disease [ ] . this relative stability allows use of molecular epidemiology to track a specific virus involved in a series of disease outbreaks. it also suggests that the genetic diversity and multiple viral genotypes found among bvdv in a large geographic area like north america is due to the gradual accumulation of mutations in bvdv of several different origins, as opposed to extremely rapid evolution of a single bvdv. although there is a tendency to maintain the master sequence of a virus under neutral conditions, the immune response of the infected host creates a nonneutral condition, and may select viral variants. this has been seen on farms that harbor multiple pi cattle, which likely originated from a single outbreak of acute infection in immunocompetent pregnant cattle. comparison of the bvdv from those animals showed that the viral isolates were similar; however, antigenic differences could be detected among the viral isolates [ ] . the selection of the antigenic variants likely occurred during the acute infection of the dams of those pi cattle and resulted in transplacental transmission of slightly different bvdv to a group of fetuses. in addition to having a high frequency of point mutations, rna viruses also have a propensity for recombination [ , , ] . this allows rna viruses to exchange segments of their genomes and potentially create new viral species. recombination in bvdv rna has been shown to be either homologous, involving self-viral rna, or heterologous, involving nonselfviral rna or host cell rna [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in bvdv, recombination of rna has not been shown to create new viral species (a new viral genotype), but it can cause a switch in viral biotype. two biotypes of bvdv (cytopathic and noncytopathic) exist in nature. the viral biotypes are characterized by their ability to cause cytopathic effect and cell death in cultured cells. cytopathic bvdv induce cytoplasmic vacuolation and death of susceptible cultured cells within a few days of infection. noncytopathic bvdv establish an inapparent persistent infection in cultured cells. noncytopathic bvdv is more prevalent in nature, and serves as the parent virus from which cytopathic bvdv arise after homologous or heterologous recombination in the noncytopathic viral rna. the recombination usually occurs in the genomic region encoding the ns - nonstructural protein of the noncytopathic bvdv and results in the insertion of either self or foreign rna into the ns - coding region. the genome of the resulting cytopathic bvdv is essentially identical to that of the parent noncytopathic bvdv except for the insert of additional rna. the recombination event causes the large ns - protein of noncytopathic virus to split into two smaller proteins that are termed ns- and ns- . the ns- protein is considered a molecular marker of cytopathic virus and the cause of cytopathic effect [ ] . reversion of cytopathic bvdv back to the noncytopathic biotype also occurs. the resulting noncytopathic bvdv usually lose the ability to express of the ns- protein, but some noncytopathic bvdv have been identified that retain expression of the ns- protein without causing cytopathic effect in cultured cells [ , ] . the high frequency of mutation, propensity for recombination, and selective pressure from immune responses stimulated by natural infection or vaccination has led to the creation of a large assortment of genetic and antigenic variants of bvdv. the genetic variants can be grouped based on the homology of aligned nucleic acid sequences from various segments of the viral genome [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the segments of the viral genome used most frequently for phylogenetic analyses (typing or grouping) of bvdv are the untranslated region and the immediately adjacent region that encodes the n pro viral protein. the array of bvdv form genotypes, subgenotypes within genotypes, and isolates within subgenotypes. current viral taxonomy places bvdv in the genus pestivirus in the family flaviviridae. pestiviruses segregate into at least five (possibly six) viral genotypes [ , ] . those genotypes are classical swine fever virus, bovine viral diarrhea virus type , bovine viral diarrhea virus type , border disease virus, and a genotype represented by a single viral isolate termed giraffe- . the viral genotypes are about % similar to each other in their base sequence. subgenotypes within a genotype are designated by a number followed by a lower case letter (bvdv type a, b, c, etc.) . subgenotypes are about % to % similar to each other. each subgenotype includes a group of viral isolates that usually are +% similar to each other. currently, subgenotypes of bvdv type and two subgenotypes of bvdv type have been identified [ , , ] . recent phylogenetic surveys suggest that there are regional differences in the distribution of viral genotypes and subgenotypes [ , [ ] [ ] [ ] [ ] . the regional distribution of viral genotypes and subgenotypes likely reflects historic routes for movement of cattle, vaccine usage over time, and geographic isolation of cattle populations. as the result of different selective pressures and management practices, bvdv has evolved into the array of genotypes and subgenotypes present today. there is some linkage of viral genotypes and subgenotypes with clinical manifestations of disease including thrombocytopenia, reproductive failure, or pneumonia [ , , , [ ] [ ] [ ] . also, regional bias may occur as to the viral genotype or subgenotype involved with the various disease forms that are seen after infection with bvdv. the genetic diversity seen among bvdv results in extensive antigenic diversity. monoclonal antibodies raised against a diverse array of bvdv identify a fairly large number of epitopes (single antigenic sites) on the immunologically important viral proteins. the large number of epitopes allows use of panels of monoclonal antibodies to differentiate bvdv based on their antigenic profile [ , , , ] . most field isolates of bvdv show unique patterns of monoclonal antibody binding when reacted with a large panel of monoclonal antibodies raised against several different viruses. in fact, bvdvs that are antigenically alike in monoclonal antibody assays are difficult to find. viruses are readily segregated into genotypes by patterns of monoclonal antibody binding. similarly, segregation of bvdv into genotypes can be done using convalescent serum or postvaccinal serum in viral neutralization assays [ ] [ ] [ ] [ ] [ ] [ ] . separation of viruses into subgenotypes using polyclonal serum and viral neutralization assays has proven difficult. even though antigenic differences likely exist between subgenotypes, the variable antibody response that occurs among cattle after infection or vaccination makes separating viruses into subgenotypes using polyclonal antibody uncertain. in contrast to antibody raised in response to viral infection, polyclonal antibody raised in cattle, sheep, or mice against the e envelope glycoprotein expressed in either baculovirus or vaccinia virus appears to be extremely virus specific, and may be able to separate viral isolates into different subgenotypes [ , ] . in summary, bvdv exists as an antigenically diverse array of viruses that manage to retain some antigenic similarity with each other and with other pestiviruses. thus, all bvdv are serologically related, but the strength of that relationship will vary. the clinical outcome following infection with bvdv is complex and dependent on multiple factors that are agent, host, and environmentally related. host factors that can influence the clinical outcome of infection include whether the host is immunotolerant or immunocompetent to bvdv, immune status (passive from colostral antibodies or active from exposure or vaccination), pregnancy status in females, gestational age of the fetus at the time of infection, level of environmental stress at the time of infection, and concurrent infection with other pathogens. it is well established that variation in virulence exists between different bvdv isolates. however, the basis for clinical variation at the virus level is not understood. it is important to realize that despite wide genetic and antigenic diversity of bvdv isolates, most viral isolates are capable of inducing some common clinical syndromes. all noncytopathic bvdv isolates appear capable of infecting the fetus resulting in abortion, congenital defects, or the development of immunotolerance and subsequent persistent infection (it should be noted that persistent infection has not been observed with cytopathic isolates of bvdv). the virus is known to have an affinity for cells involved in immunity, and is capable of inducing some degree of immunosuppression. the majority of both type and type bvdv isolates are of low virulence and induce subclinical to very mild disease [ ] . most animals infected with bvdv undergo subclinical infections that result in mild fever, leukopenia, and development of serum-neutralizing antibodies. subclinical infections explain the positive serum neutralization titers to bvdv that is found in the majority of unvaccinated cattle. it has been previously estimated that % to % of bvdv infections occur without manifestation of clinical signs [ ] . when bvdv infections result in clinical disease, it has historically been referred to as bvd. most clinical presentations of bvdv infection are mild, consisting of lethargy, anorexia, fever, diarrhea, and decreased milk production in lactating cows. beginning in , an atypical form of bvdv infection, referred to as severe bvd, was recognized in canada [ , ] . the disease had a peracute course, caused high morbidity, and resulted in a substantial number of deaths in all age groups. this new form of bvdv infection killed approximately % of veal calves in certain regions of canada [ ] . clinical disease in the canadian outbreak was characterized by fever, pneumonia, and sudden death in all age groups of cattle [ ] . viral isolates obtained from these severe acute outbreaks were genotype bvdv. acute bvdv infections in cattle also can cause a hemorrhagic syndrome [ , ] . these infections are characterized by severe thrombocytopenia, bloody diarrhea, epistaxis, hemorrhages on mucosal surfaces, hyphema, bleeding from injection sites, pyrexia, leukopenia, and death [ ] . thus far, only noncytopathic type bvdv has been associated with the hemorrhagic syndrome [ , ] . although type bvdv has been associated with many of the documented outbreaks of severe bvd and hemorrhagic syndromes, it should be emphasized that type bvdv isolates are capable of resulting in severe disease [ ] . outbreaks of severe clinical disease consisting of diarrhea, rapid dehydration, and death have been observed in association with isolation of type bvdv (dr. kenny brock, personnel communication). additionally, type bvdv isolates of low virulence are common, and are likely to predominate over virulent type isolates [ ] . regardless of viral genotype, there is a continuum of virulence among the different bvdv isolates that ranges from causing subclinical to mild disease to isolates that cause severe, life-threatening clinical syndromes. it is well established that acute bvdv infection can result in immunosuppression [ ] . bvdv-induced immunosuppression increases the host's susceptibility to other pathogens, and may enhance the pathogenicity of coinfecting pathogen. concurrent stress on the host at the time of bvdv infection is undoubtedly additive to the viral-induced immunosuppression. synergistic effects of bvdv infection have been demonstrated with mannheimia haemolytica, bovine herpesvirus- , and bovine respiratory syncytial virus. bovine viral diarrhea virus infections also have been associated with concurrent salmonellosis, escherichia coli, bovine papular stomatitis, rotavirus, and coronavirus infections. comparative experimental studies demonstrate that differences in the effect of bvdv isolates on cells of the immune system can be significant [ ] [ ] [ ] [ ] . in calves experimentally inoculated with either a low virulence type virus, a low virulence type virus ( ) or a high virulence type virus ( ), a corresponding %, %, and % drop in white blood cell count was observed between day of infection and day postinfection [ ] . these differences are most important when combined with other disease exposures such as those that may occur in a commingle feedlot environment. the role of bvdv in the bovine respiratory disease has been reviewed recently [ ] . in the united states, bvdv has been reported as the most common virus isolated from outbreaks of bovine respiratory disease. experimentally, it has been difficult to reproduce respiratory disease with bvdv alone, but synergistic effects have been documented between bvdv and m haemolytica [ ] , bovine herpesvirus- [ ] , and bovine respiratory syncytial virus [ ] . experimental studies have suggest that some bvdv isolates have more pulmonary tropism and are more likely to be associated with bovine respiratory disease than others [ , , ] . the reproductive consequences of bvdv are reviewed elsewhere in this publication. in brief, bvdv infections have been associated with infertility, early embryonic deaths, a variety of congenital defects, and fetal infection with seroconversion. most importantly, fetal infection between and days of gestation can result in the development of immunotolerance to the virus and the subsequent birth of calves that are pi with bvdv. cattle pi with bvdv serve as the major virus reservoir and source of virus transmission within and between farms. differences in reproductive outcomes are most dependent on time of infection. differences in the ability of individual bvdv isolates to cause reproductive failure have not been well documented, although it has been speculated that differences exist [ ] . review of diagnostic laboratory data by evermann supports this conclusion by finding that type bvdv isolates were more commonly associated with persistent infections, congenital defects, and weak calves, while type bvdv isolates were more commonly found in aborted fetuses [ ] . experimental studies provide evidence that different bvdv isolates have different fetal tissue tropisms, and this difference may result in different fetal pathologies and clinical outcomes [ , ] . in a dose titration study comparing the ability of a type and type isolate to cross the placenta and infect the fetus, fetal infection occurred in four of four heifers challenged with , , , and ccid /dose of type virus while occurring in four of four, four of our, three of four, and zero of four heifers challenged with , , , and ccid /dose of type virus, respectively [ ] . results form this study suggested that some bvdv isolates might be more likely to cross the placenta than others, although the reason for this is unknown. mucosal disease (md) is a unique clinical syndrome that occurs in cattle pi with bvdv [ ] . md occurs when cattle that are immunotolerant to, and pi with a ncp biotype of bvdv, become infected with a cp biotype of bvdv that shares close homology with the persistently infecting noncytopathic virus. thus, not every combination of ncp and cp virus will result in md. it is believed that the cp-bvdv most commonly arises de novo from the ncp, persistently infecting bvdv by molecular rearrangement. external sources of the cp virus can occur as demonstrated by the documented occurrence of md following the use of modified-live bvdv vaccines and experimental studies where md was produced by superinfection with cp-bvdv. cytopathic and noncytopathic biotypes are represented in type and type genotypes, and md has been documented to occur in both genotypes. differences between viral genotypes in md presentation has not been documented. both organism and immune response detection methods are used to diagnose bvdv. diagnostics target viral antigens (immunoperoxidase microtiter assay, antigen elisa, immunohistochemistry, fluorescent antibody), genomic material (pcr, in situ hybridization) or bvdv specific antibodies (virus neutralization, antibody elisa). these assays have varying risks for failure when used to detect an organism with the capability of having a diverse genetic and antigenic makeup, such as bvdv. antigen detection assays rely on either monoclonal or polyclonal antibodies to detect bvdv specific antigens. polyclonal antibodies derived from hyperimmunized swine or calves are generally broadly reactive as they contain antibodies directed against multiple epitopes, many of which are conserved among viruses. monoclonal antibodies are specific for one epitope, and if that epitope varies between viruses, binding of the monoclonal antibody can fail. most antigen detection assays use polyclonal antibodies or a pool of monoclonal antibodies to provide the broadest reactivity and capability of detecting a diverse population of bvdv isolates. pcr detects and amplifies genetic sequences that are unique to the organism of interest. the accuracy of pcr is dependent on the ability of pcr primers to specifically bind to target genetic material unique to the organism of interest. the difficulty that can arise with pcr is identifying genetic material that is unique to the organism of interest yet stable enough that it does not change significantly over time. many pcr methodologies have been reported for detecting bvdv [ ] [ ] [ ] [ ] [ ] . primers have been designed that are capable of detecting a wide variety of field samples [ ] [ ] [ ] [ ] . diagnostic pcr primers primarily have been directed against the untranslated region of bvdv where nucleotide homology can be as high as % between isolates [ ] , thus allowing for high epidemiologic sensitivity. assays to detect virus neutralizing (vn) antibodies also are affected by bvdv diversity. as a result of nonstandard assay procedures, neutralizing bvdv antibody titers reported by different laboratories can vary significantly [ ] . a significant variable that can differ from laboratory to laboratory is the reference virus used in the neutralizing assay. in a study by vaughn, animal diagnostic laboratories were asked to run bvdv vn antibodies on split serum samples collected from calves [ ] . the average of the calves reported by each lab ranged from : to : . it was concluded that different bvdv reference strains being used in the vn assays likely account for some of the observed lab-to-lab variation. vn antibody titers may be dramatically different depending on the viral genotype with which the animals are exposed to. in a study using seroconversion in unvaccinated heifers as an indicator for circulating bvdv, it was observed that type vn antibody titers were always highest if the actual virus circulating on the farm was type bvdv (table ) [ ] . the same observation was made for type vn antibodies and type virus. therefore, using a vn assay designed to detect type antibodies (assay using a type reference virus) in a herd where type virus is circulating may yield significantly different results than a vn assay using a type diversity among bvdv is a suspected cause of vaccination failure [ ] [ ] [ ] [ ] [ ] [ ] . however, several studies have shown that a bvdv type immunization induces clinical protection against a type challenge [ , [ ] [ ] [ ] [ ] [ ] . although protecting cattle against clinical disease is important, it may not be sufficient in terms of controlling reproductive failure. a key component in controlling bvdv is preventing fetal infections that result in the birth of calves pi with the virus. preventing fetal infection and the subsequent sequela involves controlling virus exposure and enhancing bvdv specific immunity in susceptible dams. an important issue is the ability of immunity developed against one virus strain to crossprotect against heterologous bvdv strains effectively enough to prevent fetal infection. several field studies suggest that immunologic protection against heterologous bvdv challenge may be incomplete with respect to fetal protection [ ] [ ] [ ] [ ] . early vaccines were developed with little knowledge of their ability to provide fetal protection. currently, efficacy data on fetal protection is not required for approval of vaccines for bvdv in the united states [ ] . experimental studies attempting to address fetal protection are limited, and have focused primarily on immunity developed following vaccination. results of vaccine fetal protection studies have been mixed, and are often dependent on the challenge model (see article on reproductive consequences of bvdv for further details). most trials have involved killed vaccines, and efficacy has ranged from % to %. in studies evaluating the fetal protection efficacy of a modified-live vaccine, cortese and brock demonstrated % and % fetal protection in heifers immunized one time with a commercially available type modified-live bvdv vaccine and challenged at days in gestation with type or type bvdv, respectively [ , ] . except for different challenge viruses, these studies were conducted similarly, suggesting that the vaccine was less likely to stimulate a fetal protective immunity against type viruses compared with type viruses. other studies have not fully evaluated protection against multiple viruses. the potential consequences of bvdv genetic and antigenic diversity are far ranging. the complexity of clinical presentations associated with bvdv likely arises from factors encoded by the virus genome. more importantly, prevention and control of bvdv may be complicated by diagnostic and immunization failure resulting from virus diversity. evolutionary pressures will continue to drive further diversity, making control of bvdv challenging. current and the potential for future bvdv strain diversity should be considered when designing bvdv control programs both at the individual farm and national herd level. rapid evolution of rna genomes quantitation of relative fitness and great adaptability of clonal populations of rna viruses rna virus mutations and fitness for survival mutation rates among rna viruses genetic variability: the key problem in the prevention and therapy of rna-based virus infections quasispecies structure and persistence of rna viruses genetic ''budget'' of viruses and the cost to the infected host: a theory on the relationship between the genetic capacity of viruses, immune evasion, persistence and disease memory in viral quasispecies viruses at the edge of adaptation contingent neutrality in competing viral populations exponential 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isolates. international symposium: bovine viral diarrhea virus, a year review the pathogenesis of mucosal disease bovine viral diarrhea virus infection: rapid diagnosis by the polymerase chain reaction application of the polymerase chain reaction to the detection of bovine viral diarrhea virus infections in cattle detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the untranslated region detection of bovine viral diarrhea virus by taqman reverse transcription polymerase chain reaction reverse transcription combined with polymerase chain reaction as a detection method for pestiviral infections comparison of virus isolation and reverse transcription polymerase chain reaction assay for detection of bovine viral diarrhea virus in bulk milk tank samples use of polymerase chain reaction to simultaneously detect and type bovine viral diarrhoea viruses isolated from clinical specimens nested reverse transcriptase-polymerase chain reaction (rt-pcr) for typing ruminant pestiviruses: bovine viral diarrhea viruses and border disease virus comparison of rt-pcr assay and virus isolation in cell cultures for the detection of bovine viral diarrhoea virus (bvdv) in field samples molecular biology of bovine viral diarrhea virus and its interactions with the host sn titer response comparison amoung fourteen diagnostic laboratories for antibodies protecting against ibr, bvd and brsv virus following the adminastration of modified live vaccine to weaning aged calves serologic evaluation of five unvaccinated heifers to detect herds that have cattle persistently infected with bovine viral diarrhea virus experimental exposure of vaccinated and non-vaccinated preganant cattle to isoates of bovine viral diarrhoea virus (bvdv) the efficacy of an experimental inactivated bvd-md vaccine serologic detection and practical consequences of antigenic diversity among bovine viral diarrhea viruses in a vaccinated herd investigation of bovine viral diarrhea virus infections in a range beef cattle herd a case report: evidence for type bovine viral diarrhea virus (bvdv)-associated disease in beef herds vaccinated with a modified-live type bvdv vaccine fetal exposure to bovine viral diarrhea virus despite vaccination in a wisconsin diary herd clinical and immunologic responses of vaccinated and unvaccinated calves to infection with a virulent type-ii isolate of bovine viral diarrhea virus assessment of protection from systemic infection or disease afforded by low to intermediate titers of passively acquired neutralizing antibody against bovine viral diarrhea virus in calves cross-protective efficacy of a bovine viral diarrhea virus (bvdv) type vaccine against bvdv type challenge an inactivated bovine virus diarrhoea virus (bvdv) type vaccine affords clinical protection against bvdv type evaluation of a modified live virus type- a bovine viral diarrhea virus vaccine (singer strain) against a type- (strain ) challenge united states code of federal regulations. animal and animal products protection of pregnant cattle and their fetuses against infection with bovine viral diarrhea virus type by use of a modified-live virus vaccine experimental fetal challenge using type ii bovine viral diarrhea virus in cattle vaccinated with modified-live virus vaccine key: cord- -salby fu authors: bujarski, jozef j. title: genetic recombination in plant-infecting messenger-sense rna viruses: overview and research perspectives date: - - journal: front plant sci doi: . /fpls. . sha: doc_id: cord_uid: salby fu rna recombination is one of the driving forces of genetic variability in (+)-strand rna viruses. various types of rna–rna crossovers were described including crosses between the same or different viral rnas or between viral and cellular rnas. likewise, a variety of molecular mechanisms are known to support rna recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among rna fragments of viral and/or cellular origin. such mechanisms as rna decay or rna interference are responsible for rna fragmentation and trans-esterification reactions which are likely accountable for ligation of rna fragments. numerous host factors were found to affect the profiles of viral rna recombinants and significant differences in recombination frequency were observed among various rna viruses. comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. many questions remain to be answered by forthcoming rna recombination research. ( ) how various factors modulate the ability of viral replicase to switch templates, ( ) what is the intracellular location of rna–rna template switchings, ( ) mechanisms and factors responsible for non-replicative rna recombination, ( ) mechanisms of integration of rna viral sequences with cellular genomic dna, and ( ) what is the role of rna splicing and ribozyme activity. from an evolutionary stand point, it is not known how rna viruses parasitize new host species via recombination, nor is it obvious what the contribution of rna recombination is among other rna modification pathways. we do not understand why the frequency of rna recombination varies so much among rna viruses and the status of rna recombination as a form of sex is not well documented. plus-stranded rna viruses include some of the most dangerous pathogens for animals and humans. moreover, a vast majority of plant viruses are (+) rna viruses. rna viruses demonstrate a large level of variability in their genetic information, due to either mutations, rna-rna crossovers (rna recombination), or reassortment. rna recombination was demonstrated for many rna virus species, whether under natural or experimental conditions. similar to genetic recombination in dna-based organisms, viral rna recombination is defined as the process of swapping rna fragments among rna molecules. if crossovers occur amongst the same rna templates in a homologous fashion, the exchanges are functionally equivalent to dna meiotic crossing-over. in some viruses, the frequency of homologous crossing-over is very high and practically every replicated viral rna molecule can be considered as chimerical in nature, as we have demonstrated for brome mosaic virus (bmv) rnas (urbanowicz et al., ) . a variety of events have been described that contribute to the formation of rna recombinants (figure ) . such events include crossovers between viruses belonging to the same or to different taxonomic groups, between viruses infecting different hosts, or from adopting genetic material from the host. numerous questions about molecular mechanisms of rna recombination remain unanswered. this review attempts to summarize the most important venues of rna recombination research, their challenges and future directions in order to draw more accurate models for this important rna virus phenomenon. since this issue of frontiers concerns plant pathology, most of the material discusses rna recombination in plant viruses. however, the less advanced aspects of plant recombination studies have been illustrated with examples taken from animal/human rna viruses in order to show mutual possibilities for model research. the generally accepted mechanism of rna recombination is currently explained by a copy-choice model where the viral rna polymerase (rdrp) complex in mrna viruses [reverse transcriptase (rt) in retroviruses] changes templates during synthesis of the nascent strand (galetto et al., ) . this swapping process generates recombinant rna molecules of mixed ancestry. although we begin to understand the nature of these processes, many questions are waiting for an answer. one group of questions revolves around the features that define the sites of crossovers. among the factors known to promote replicase to switch are sequence homologies between recombination substrates along with secondary structures at the crossover sites, as demonstrated with the bmv and other systems (figlerowicz and bujarski, ; nagy et al., b) . also, the transcriptional activity seems to promote template switching. for instance, an efficient recombination hot spot has been mapped within the intercistronic region of bmv rna , the site carrying the promoter of transcription of subgenomic rna (wierzchoslawski et al., ) . it is unknown what exactly facilitates crossovers at such sites. possibilities include a snatching process of already bound rdrp complex to the promoter site, the premature termination of rna synthesis and the replicase detachmentreattachment, or the effect of other bound viral and/or host factors (sztuba-solinska et al., ) . these mechanisms may depend upon the type of template-switching process (whether the crosses occur internally or near the ends of the rna templates) and on the involvement of crossover sequences in other processes, e.g., as a promoter of rna replication or transcription. template switching was found to occur between related but also between unrelated rna templates, generating legitimate (homologous) and illegitimate (nonhomologous) recombinants, respectively (nagy and simon, ) . since the latter involves sequences with little similarity, other factors must be important. some data indicate that switches depend upon sequence composition, with the au-rich regions promoting the rdrp detachment (nagy et al., a) and upon secondary structures (galetto et al., ) , along with protein or rna binding activity. switching may also depend upon the processivity (a measure of the average number of nucleotides copied per template association-disassociation cycle) features of the rdrp enzyme (breyer and matthews, ) . a mandatory replicase breaking site is the end of any rna template. end-to-end switching has been reported based upon in vitro results with rdrp enzymes from bovine viral diarrhea virus (bvdv), bmv, cucumber mosaic virus (cmv), and cowpea chlorotic mottle virus (ccmv) (kim and kao, ) . it is, however, not known how exactly such switches occur and whether the molecular mechanism is common among polymerases of different rna viruses. the strength of binding of the rdrp complex may play a key role during rna template detachment-reattachment. with an increasing number of available rna polymerase crystal structures, more is evident about the elements involved in rnareplicase interactions. for instance, removal of a β-hairpin loop from the hcv rdrp protein increased de novo rna synthesis and promoted rna binding (mosley et al., ) . the rna copying fidelity might be a matter of a nanosecond timescale complex dynamic in the rdrp enzyme that determines rna binding, nucleotide binding or catalysis (moustafa et al., ) and thus needs to be experimentally determined. the use of engineered replicase variants in rna recombination assays will shed new light onto the molecular details of template switching mechanisms. another, not well answered question is how rna template substrates come together in order to facilitate the switch. one possibility is that secondary structure regions can hybridize in trans bringing the two rna templates into a local interaction. such data are available, for instance, based upon limited observations in bmv (nagy and bujarski, ; dzianott et al., ) or analogously, during switches between dimeric rnas (within kissing loops) during reverse transcription inside the human immunodeficiency virus type- (hiv- ) virions , an atypical (+) sense rna virus. yet, other data reveal that (+) rna viruses are replicating in membranous structures called spherules or replication factories (laliberté and sanfaçon, ) . such host membrane-derived replication vesicles have limited loading capacity, but they may carry up to several positive and negative strand rna molecules (den boon et al., ) . recent advances reveal the assembly of replicase complexes within replication factories via highly orchestrated interactions between viral proteins, viral genomic rnas, and co-opted host factors (mine and okuno, ) . such a micro-environment may secure tight packaging and thus the closeness of internalized viral rna molecules. from the formal stand point then, one may consider rna recombination switches in (+) rna viruses inside replication factories as analogous to the switches that occur, e.g., during reverse transcription inside the hiv- virions. recently, we have demonstrated the participation of coat protein (cp) during bmv rna recombination . the nucleotide changes in cis-acting rna motifs and the amino acid replacements within the corresponding cp binding sites-both debilitated the bmv rna recombination. cp molecules likely mediate rna crosses via dimerization/oligomerization of bound cp subunits. indeed, the presence of bmv cp molecules has been demonstrated to be inside replication vesicles (bamunusinghe et al., ) . another untested possibility predicts that a bound cp functions as a road block catalyzing the detachment of the replicase complex. the cp may also affect the properties of viral replicase. for instance, it has been shown recently that norovirus rna synthesis was enhanced by co-expressed structural protein vp (subba-reddy et al., ). besides replicative copy-choice, the non-replicative mechanisms of viral rna recombination have been described, mainly for animal/human rna viruses, with almost no research focusing plant viruses. one of the best characterized non-replicative processes is demonstrated in the poliovirus where viable viruses were rescued in cells co-transfected with different pairs of viral rna fragments (gmyl et al., ) . it is likely the recombinants may have resulted from transesterification reactions with the end structures similar to known ribozymes via intermediary formation of , -cyclic phosphate. indeed, in vitro data show that the transesterification reactions in the bacteriophage qbeta rna are guided by secondary structures that direct the attack of a hydroxyl onto the phosphodiester bonds (chetverin et al., ) . later observations revealed enormous variability of the poliovirus genome and some variants may have been introduced by genetic errors due to non-replicative mechanisms (agol, ) . more recent results with partially-complementary rna-oligonucleotides demonstrated the spontaneous formation of novel rna molecules via , -phosphodiester bonds (lutay et al., ) . these data show that viral rna recombination can occur without participation of the rna polymerase enzyme. the exact mechanisms of these non-replicative events are not completely understood and require further studies. in contrast to poliovirus and other picornaviruses, bacteriophage qbeta demonstrates low levels of recombination frequency. by using a cell-free system, chetverin et al. ( ) have shown a high yield of primer-extension recombination with poliovirus replicase, but a low yield with qbeta replicase. thus, rna recombination by poliovirus vs. qbeta rdrps must be mechanistically different. although both utilize transesterification reactions, the precise molecular bases for rna swappings used by each of these enzymes are likely dissimilar reflecting different biochemical adaptations to the needs of individual viruses. it would be interesting to confirm experimentally the proposed transesterification models. among other examples of non-replicative recombination in mrna viruses, the co-transfections of replicating and nonreplicating rubella virus (rub) rna transcripts containing nonoverlapping deletions did restore the infectious virus (adams et al., ) . both, homologous and nonhomologous rna recombinants emerged. the mechanism seemed to involve end-to-end replicase switching after initiation of minus-strand synthesis. however, the details of such mechanisms have not yet been confirmed. another example of that sort involves recombination between bvdv and cellular rnas, which can occur in the absence of viral replicase (see section "recombination between viral and cellular rnas"). analogous studies in the area of plant virology remain to be performed. an important subject of rna recombination research is the role of host factors. while the involvement of viral rdrp proteins has been studied extensively, knowledge of the functions host components play is limited (nagy and pogany, ) . one study was done with a model system of tomato bushy stunt virus (tbsv) that can recombine in yeast cells. the authors screened a yeast knockout library to identify over thirty different host genes suppressing or accelerating the tbsv rna recombination (serviene et al., ; nagy, ) . an interesting example is the gene pmr which encodes an ion pump (pmr p) controlling the mn + concentration which may consequently affect the ability of tbsv replicase to change rna binding/template switching events. also stress signals, e.g., salt stress, affect viral recombination indirectly, by changing the concentration of recombination-essential proteins. future studies are required to understand the interrelated network of cellular factors that define the final outcome of tbsv rna recombinants, not only in model yeast cells, but also in natural tbsv plant hosts. moreover, these studies are limited to only one specific tbsv rna experimental recombination system, and it is unclear if other rna recombination events within the tbsv rna follow similar mechanistic pathways. in the copy choice mechanism, recombinant rnas are formed due to switching of viral replicase among rna templates. the switching properties likely depend on the co-recruited host factors. in bmv, a variety of host factors were found to be employed by the replicase complex (noueiry and ahlquist, ) . many of these factors facilitate the complex assembly, but some regulate viral gene expression or recruitment of bmv rnas to the membrane replication factories. yet, other factors modify lipid composition of the endoplasmic reticulum membrane which activates the replication complex. many of these factors can potentially affect the co-recruitment of rna recombination substrates and/or bmv replicase switching properties during recombination. bmv rna recombination was reported to occur in yeast cells (garcia-ruiz and ahlquist, ) , but a systematic identification of host factors participating in bmv rna recombination remains to be done. it will be interesting to find out whether these factors parallel those in the above tombusvirus recombination system. this data will broaden our knowledge about host pathways enabling rna viruses to recombine their genetic information. as such, it will contribute to predictions made on the stability of the rna viral genome in various hosts. the functions of recruited host proteins and host membranes in different (+) rna virus systems are now being progressively elucidated. comparison among three plant rna virus replication systems (tbsv, bmv, and dianthoviruses) reveals general patterns within the stepwise process of viral replicase complex assembly which requires concerted involvement of protein-protein, rna-protein, and protein-lipid interactions (mine and okuno, ) . however, each of these three plant virus systems recruits its own array of specific host factors. this suggests that each rna virus has significantly unique ways of adapting to the cellular environment in order to assemble a functional rna replication complex. this further suggests specific requirements are needed for rna recombination in each individual rna virus and therefore the recombination characteristics may significantly differ with each other among rna viruses. crystal structure studies help to reveal the complex and individual nature of viral replicases. examples being the structure of q{beta} phage polymerase, determined by takeshita and tomita ( ) , or the analysis of the crystal structure of tomato mosaic virus helicase as a component of the viral replicase complex (nishikiori et al., ) . rna recombination events between viral and cellular rnas have been observed for both plant and animal rna viruses. one example is rna recombination between the bvdv, a member of the pestivirus genus, and cellular rna sequences. it occurs at the presence yet also in the absence of an active viral rdrp enzyme, implying that the mechanisms must be different from replicative template switching events (becher and tautz, ) . the case of bvdv recombination has practical implications because the recombinant virus is lethal to its host. normally, the virus is persistent, limiting the efficiency of rna replication due to the dependency of a viral protease on limiting amounts of a cellular cofactor. in general, the uptake of a variety of cellular protein coding sequences at various positions in the pestiviral genomes has been reported, demonstrating that pestiviruses can gain access to the rna pool of their hosts via rna recombination. the example of bvdv shows us not only that recombination events with cellular rnas cannot be excluded for other viruses, but also that the recombinant rnas can be retro-transcribed and occasionally integrated into the host genome. the exact molecular mechanisms of the crossover events with cellular rnas remain to be elucidated, as well as what factors target the crossover sites both to viral and to cellular rnas. besides bvdv, hiv- is known to recombine effectively with host rnas, e.g., with host trnas after introducing its strong secondary structure elements into the hiv rna (konstantinova et al., ) . hiv- is capable of acquiring new genetic material, especially to the rt-encoding orf (van der hoek et al., ; berkhout, ) . information about similar recombinant crosses with host rnas in plant rna viruses remains very limited, and their mechanisms are waiting to be elucidated. one recombination process that was addressed with plant viruses has been the events between an invading virus and the transgene mrnas in transgenic plants (aaziz and tepfer, ) . one such example being recombination between two strains of cmv where one strain was expressed as a transgene while the other strain infected the transgenic plant (turturo et al., ) . this research group has also described recombination between related viruses (cmv and tomato aspermy virus tav), with the population of recombinants being similar to each other in transgenic and in nontransgenic plants, suggesting similar molecular mechanisms of recombination (jacquemond, ) . in general, this demonstrates that transgene viral mrnas enter the same pathway as do natural viral rnas, most likely operating in the cytoplasm. rna recombination between viral and micro (mi)rnas has not yet been reported. however, given the fact that this would be a useful source of already adapted elements to be acquired by the virus in order to secure the in-trans host-gene regulation, the lack of commonality of such an acquisition is surprising. since (+) rna viruses operate in the cytoplasm, as the mirnas do, there are likely either structural and/or functional constrains against such snatching events. future studies will certainly bring further insight to this question. recently, a reverse scenario was observed. nonretroviral rna sequences of bornaviruses and other (−) strand rna viruses were integrated into the host genome, including the human genome (belyi et al., ; horie et al., ) . also, mrna viruses were described to leave their sequences in the cellular dna of infected hosts (crochu et al., ; anne and sela, ; maori et al., ; zemer et al., ; geuking et al., ). these results demonstrate that rna viruses can serve as a source of genetic innovation for their hosts. the rt activity encoded by retrotransposons is most likely responsible for reverse transcription and integration, yet further molecular studies are needed. the above examples illustrate that the cytoplasmic rna processing mechanisms are able to cross paths with viral replication pathways inside the cell. despite diverse examples of viral rnas recombining with host rna sequences (and vice versa), many unanswered questions remain to be addressed. they include, but are not limited to, the sub-cellular location of recombination events, the role and availability of host rna degradome for recombination, or the link between the elements of rna degradation pathways and viral rna recombination. the molecular mechanisms of such crossover events are not well understood, especially whether template-switching or re-ligation processes are involved. more data, especially from plant rna virus systems are required to assess the general nature of these processes in plant vs. animal/human tissues. host rnas undergo extensive degradation and turnover, as do viral rnas (lloyd, ) . the participation of rna decay pathways in viral rna recombination has been studied in tbsv by the nagy group (jiang et al., ; jaag et al., ) . by testing eight known endoribonucleases, the authors have shown that mutations in the components of rnase mrp debilitated the production of endoribonucleolytically cleaved tbsv rna in yeast. also, by knocking down the rnase nme or silencing the xrn p exoribonuclease in nicotiana benthamiana, the production of cleaved tbsv rnas was debilitated, but recombination increased, suggesting the role of rna intermediates in recombination (jaag and nagy, ). similar effects promoting rna recombination were observed in yeast for xrn p exoribonuclease (serviene et al., ) . it is noteworthy that deletions of the host met p/hal p bisphosphate- -nucleotidase (a known inhibitor of the xmn p ribonuclease) or the inhibition of this nucleotidase with licl or nacl, also increased the frequency of tbsv rna recombination in yeast (jaag and nagy, ) . this shows that besides host factors, the salt stress can also affect viral rna recombination. whether other environmental conditions can influence viral rna recombination needs further studies. in contrary to rna decay enzymes, we observed debilitating effects of the host rna interference gene knockouts on bmv rna recombination in arabidopsis thaliana, and that bmv rna fragments have recombined with bmv rna progeny (dzianott et al., ) . it appeared that rna silencing (rnai) pathways participated in the rearrangement of genomic bmv rnas. therefore, bmv rnas can recombine via several mechanisms including template-switching events along with rnaibased sequence swapping. similarly, the promoting role of rnai in viral rna recombination was reported for mycovirus infection in chestnut blight fungus cells (sun et al., ; nuss, ) . these two examples show that the rnai mechanisms can function as antiviral tools, but also that rna silencing can promote additional variability to the viral rna genome. further studies are needed to determine the formation of viral rna recombinants from rnai-induced degradation products. the biological diversity within both plant and animal rna viruses is one of the largest found in all other forms of nature. rna recombination is a main contributor to the ever evolving rna viral genome. comparative analyses of rna viral sequences allow for the development of evolutionary models that demonstrate the associated adaptive phenotypic changes along with detecting the co-evolving sites within viral genomes (pond et al., ) . the wide imprints of rna recombination were found within natural populations of plant viruses. rna recombination seems to be particularly frequent among members of the family potyviridae, the largest family of plant rna viruses. frequent recombinational footprints were detected within the orfs of both their structural and nonstructural proteins (bousalem et al., ; visser and bellstedt, ; yamasaki et al., ) . phylogenetic surveys indicate not only intraspecies and intragenus, but also intergenous recombination crossover's footprints in potyviridae (desbiez and lecoq, ; valli et al., ) , supporting their apparent modular evolution. recombination with host rna was also detected, likely via retrotransposable elements (tanne and sela, ) demonstrating that, like animal viruses, plant viruses can expand their coding capacity via recombination with the host's messenger rna pool (chare and holmes, ) . also, the populations of plant viruses with genomes producing sgrnas, e.g., closteroviridae, luteoviridae, or viruses with multipartite genomes, e.g., bromoviridae, seem to accumulate recombinants readily. evolutionary pathways were proposed for the emergence of members of luteoviridae (domier et al., and moreno et al., ) . luteoviruses have mastered the process of modular swap (pagán and holmes, ) and the reconstructed phylogeny reveals their sequence evolution by intrafamilial as well as extrafamilial rna recombination (moonan et al., ) . the most frequent swaps map to the junction between the cp and rdrp orfs . in addition, some luteoviruses were found to recombine with host (chloroplast) sequences (mayo and jolly, ) . one extreme example of interspecies recombination is in circoviruses that arose by recombinants between plant dna nanoviruses and mammalian rna caliciviruses. in this case, the likely mediator has been a retrovirus that retro-transcribed the rna into dna (davidson and silva, ) . although likely, such events have not been experimentally confirmed and further research is required. among animal viruses, coronaviruses are highly recombinogenic (woo et al., ) and natural rna recombinant variants were described for flaviviruses (gonzález-candelas et al., ) . by having one of the highest recombination rates among all viruses, retroviruses generate polymorphic sequences that increase their chances for survival under changing selection pressures (delviks-frankenberry et al., ) . besides retroviruses, picornaviruses are naturally highly recombinogenic (lukashev, ) . in fact, rna recombination is their key genetic feature maintaining the global pool of variants from which the recombination snapshots generate new recombinant forms of picornaviruses. for instance, a model describing recombination between poliovirus and coxsackie virus was presented to illustrate the effects on viral emergence and evolution (combelas et al., ) . this and other studies reveal multiple mechanisms leading to genetic variability of polioviruses (savolainen-kopra and blomqvist, ), with significant contribution of homologous recombination events that fix advantageous mutations or remove deleterious ones. however, further research is required to understand the detailed evolution mechanisms of polioviruses. the evolutionary genetics of emerging plant rna viruses was studied by elena et al. ( ) . apparently, devastating virus epidemics can spread from new plant virus variants that acquired new virulence factors. this study shows a multifaceted picture of virus emergence. changes in ecological conditions bring together the reservoir viruses and their crop hosts, often as a result of interplay among the environment, genetic plasticity, and the required host factors. the stochastic processes contribute to the beginning of viral emergence in a new host species, followed by the adaptation phase. also, vectors impose strong bottlenecks during host-to-host transmissions. the reservoir population seems to be the most important determinant of viral emergence, but little is known about viruses of wild plant species that work as reservoirs. for all the mentioned rna virus systems, of either plants or animals, detailed roles during virus evolution of rna secondary structures, the function of sequence similarity or the impact of rna co-packaging during rna recombination, are all not well understood. inaccuracies of viral rna replication, damage from environmental factors, and attacks by rna-modifying enzymes, all can contribute to rna genome corruption and thus generate a question of how rna viruses maintain their genetic integrity (barr and fearns, ) . it seems that viral rdrps are sufficiently flexible to accommodate alternative initiation mechanisms, enabling terminal repair, terminal transferase activity, and recombinational crosses in the case of damaged key terminal sequences. among a variety of mechanisms to protect rna viral genome integrity, recombination allows for exchange of sequences between rna templates, protecting not only their entire genome, but also their vulnerable termini. a typical example of efficient terminal crossover exchanges is seen within the noncoding region of bmv rnas (bujarski and kaesberg, ). the differences in replicase architecture might affect the predilection of a particular virus for rna recombination. the molecular aspects of the theory on "adaptable" viral rdrps have not been elucidated and structural studies will contribute to the answers. rna recombination research concerns both virus evolution (where the most important subject is the detection of recombination imprints among natural viral rna sequences) and the mechanism of recombination (by using the experimental systems of enhanced recombination frequency). as regards to the evolutionary studies, various computer programs are used for massive comparisons of viral sequences in order to reveal the recombination footprints. the examples of such software include, but are not limited to, topali, recco, gard, rdp, geneconv, chimaera, maxchi, bootscan, siscan, phylpro, diplomo, simplot, lard, and seq. these programs can identify the recombination sites among different viral strains, different viral species, and even between the virus and the host (chare and holmes, ) . with advent of next-generation massive sequencing, the genetic diversity of viral rna genomes can be characterized through the mapping of polymorphisms and measurement of mutation frequencies as well as by detection of recombination events to a single-nucleotide resolution (routh et al., ) . such approach is very sensitive and unbiased, and it can identify hundreds of thousands of recombination events, allowing for a detailed description of rna crossover profiles. the detection of recombination events in the laboratory is challenging because rna-rna crossovers apparently are rare events and thus the main effort is to elaborate on experimental systems of engineered rna templates of increased recombination activity. the efficient recovery of recombinants in mixed infections could be achieved by using temperature sensitive mutants, a long-term method used for animal rna viruses (hirst, ; pringle, ) . in whole plants, an important problem is that most recombinants are not competitive with the parental types and therefore disappear. one way to increase recombination rate is by using viral mutants bearing sequence modifications at their utrs, which decreases the replication abilities of parental molecules, as was used to detect the bmv recombinants (bujarski and kaesberg, ) . another approach utilizes viral rnas bearing silent markers or via mixed infections with two low-competing viral strains (e.g., as the used by us mixed infection with both type and fescue strains of bmv). other plant virus recombination systems employ mixtures of two parental rnas with one component carrying a deleterious mutation, e.g., satellite and genomic rnas of tcv (zhang et al., ) , or defective interfering and genomic rnas of tbsv, cucumber necrosis virus (cnv) (white and morris, ) , and potato virus x (pvx) (draghici and varrelmann, ) . all these types of recombination systems can be used in cell-free extracts (utilizing viral rdrp preparations), in single-cell (protoplast) hosts, in whole plant hosts, and even in yeast. some of the systems make use of transient expression vectors by agro-infiltrating plant leaves (kwon and rao, ) . with these systems in hand, virologists can address such aspects of the rna recombination process as the essential role of rna sequence and structure, especially the role of rna motifs, the function of viral replicase (rdrp) and other viraland/or host-encoded proteins, or the mutual host-virus effects in short-term virus evolution. the main analytical effort in the recombination experiments is to identify rna recombination products and to map the location of cross sites. usually, viral rnas are extracted and amplified by rt-pcr and the resulting cdna products are cloned followed by sequencing and/or restriction digestion of a large number of clones. this way the crossovers are detected and mapped within the sequence markers, providing information about both frequency and distribution of recombination events. proper controls are required to guard against rt-pcr generated recombinants. genetic rna recombination is a major driving force for rna virus diversity. by understanding the factors and the mechanisms that affect recombination, one can ultimately develop better means for controlling rna virus infections. in this review i have described the current status of rna virus recombination research and its future directions. i have also noted its progress over the last several years emphasizing on some future research venues. evidently, there is still much to be learned about the mechanistic details of rna recombination. for example, it is not yet clear how various factors modulate the ability of viral replicase to switch templates, such as the role of rna template structures, the molecular and structural features of replicase proteins, or the functions of other viral and host factors during cross-over events. also, the intracellular location of the rna-rna template switching has not been confirmed. besides copy-choice, rna viruses can recombine with non-replicative rnas. it is not exactly known what mechanism is responsible for ligation of viral rna fragments, or where inside the cell this process occurs. rna viruses were found to recombine with cellular rnas, but again where in the cell and what factors enable such events, is not well known. and the opposite, the exact steps and the molecular mechanisms of the rna viral sequence integration with the cellular dna have not been untangled. amongst other questions, not much is known about how splicing or active ribozymes can contribute to the rna virus recombination (edgell et al., ) . from the evolutionary stand point, rna recombination seems to play a key function during virus speciation and emergence, but its shared contribution that parallels other rna modification pathways has not yet been assessed. we do not fully understand how rna viruses achieve their high potential of parasitizing new host species via recombination (domingo, ) . the entire population of rna variants that are present in reservoir hosts can now be determined with the tools of next-generation sequencing so that the role of recombinants can be more precisely evaluated (beerenwinkel et al., ) . the frequency of rna crossing-over varies among rna virus species and there is little evidence that recombination was favored by natural selection. because of this and since recombination rates follow the patterns of rna genome organization, simon-loriere and holmes ( ) postulate that rna recombination is a by-product of viral genome arrangement acting on selected aspects of the virus life cycle. thus, according to the authors, rna recombination does not seem to function as an obligatory form of sex in rna viruses. yet further studies are required, especially since muller's ratchet effects were observed in rna viruses (turner, ) and the chimeric nature of viral rnas due to frequent homologous rna swaps was determined, e.g., in bmv (urbanowicz et al., ) . despite the above deficiencies, the so far accumulated knowledge about viral rna recombination has already found some practical applications. for example, measures could be taken to reduce recombination while designing the antiviral resistance in transgenic plants with artificial micro rnas (fahim and larkin, ) or with double stranded rna-expressing transgenes (zhang et al., ) . also, the potential instability and recovery of the wild-type virus via recombination can be reduced during construction of plant rna viral vectors (nagyová and subr, ) . the author thanks margaret bujarska for valuable comments on this manuscript. jozef j. bujarski was supported through a grant from the national science foundation (mcb- ) and through the plant molecular biology center at northern illinois university. the author apologizes to any author that has been omitted in this review for either space reasons or due to the nature of composition of this article. recombination in rna viruses and in virus-resistant transgenic plants analysis of intermolecular rna-rna recombination by rubella virus 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determinants of junction site selection in rna virus recombinants and defective interfering rnas dissecting the requirement for subgenomic promoter sequences by rna recombination of brome mosaic virus in vivo: evidence for functional separation of transcription and recombination coronavirus diversity, phylogeny and interspecies jumping complete genomic rna sequences presence of hepatitis c virus dna sequences in the dna of infected patients recombination between satellite and genomic rnas of turnip crinkle virus robust rnai-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -p wmqmvj authors: kim, kiwook; song, yeon han; park, joo-hyun; park, hye kyeong; kim, su young; jung, hun; lee, sung-soon; koo, hyeon-kyoung title: rhinovirus associated severe respiratory failure in immunocompetent adult patient date: - - journal: tuberc respir dis (seoul) doi: . /trd. . . . sha: doc_id: cord_uid: p wmqmvj rhinovirus infection is typically associated with the common cold and has rarely been reported as a cause of severe pneumonia in immunocompetent adults. a -year-old previous healthy woman, who consumed half a bottle of alcohol daily, presented with respiratory failure after one week of upper respiratory infection symptoms. radiography revealed bilateral, diffuse ground glass opacity with patchy consolidation in the whole lung field; bronchoalveolar lavage fluid analysis indicated that rhinovirus was the causative organism. after five days of conservative support, the symptoms and radiographic findings began to improve. we report this rare case of rhinovirus pneumonia in an otherwise healthy host along with a review of references. rhinovirus infection is typically associated with the common cold , and exacerbation of asthma symptoms . rhinovirus infection can extend to lower respiratory tract in children [ ] [ ] [ ] or immunocompromised hosts , , but is generally not concerned as singular cause of severe pneumonia, especially in immunocompetent adults. although polymerase chain reaction (pcr) methods have enhanced the detection of respiratory viral infection, pcr cannot help distinguish between with patchy consolidations in the whole lung field, but pleural effusion was not present (figure ). laboratory tests revealed a white cell count of , /mm (differential count: . % neutrophils, . % lymphocytes, and % eosinophil), hemoglobin concentration . g/dl, platelet count , /mm , erythrocyte sedimentation rate mm/hr, and c-reactive protein level . mg/dl. analysis of arterial blood gases indi-cated a pao of . mm hg, paco . mm hg, hco . mmol/l, and sao . % without acidosis. the patient' s liver and renal function tests were within normal range. high-flow o therapy and broad-spectrum antibiotics with oseltamivir (tamiflu, roche, utley, nj, usa) were started empirically, and bronchoscopy was performed immediately. total cell counts of bronchoalveolar lavage (bal) fluid were cells/mm , www.e-trd.org including % neutrophils, % mononuclear cells, % eosinophil, and % macrophages. cultures for common bacteria, acid-fast bacilli, and fungi were all negative. pcr for pneumocystis jirovecii, and mycobacterium tuberculosis were also negative. however, pcr of bal fluid indicated the presence of rhinovirus, which was not detected following pcr of the nasopharyngeal specimen. mycoplasma antibody and urinary pneumococcal antigen test were negative. serologic test for venereal disease research laboratory test, hepatitis b surface antigen, hepatitis b, c antibodies, and human immunodeficiency virus, as well as for autoantibodies such as anti-nuclear antibody, rheumatoid factor, and anti-neutrophil cytoplasmic antibody were all negative. clinical symptoms and infiltration on chest x-ray began to improve after five days ( figure b) , and the patient was discharged from hospital after three weeks. although technical advances have allowed for increased detection of viral pneumonia, viral infections other than influenza are generally not considered as causes of severe respiratory tract infection in immunocompetent hosts, because viral clearance usually occurs rapidly in healthy individuals. however, the outbreak of severe acute respiratory syndrome, avian influenza a (h n ) virus, and the pandemic influenza a (h n ) virus raised the importance of lower respiratory tract viral infections. additionally, new respiratory viruses, such as human metapneumovirus, coronavirus, and human bocavirus were found to be causes of severe viral pneumonia. the use of molecular diagnostic assays has enhanced the detection of respiratory virus infection; however, the identification of virus does not necessarily mean that it is a lower respiratory tract infection pathogen [ ] [ ] [ ] . therefore, it is still believed that severe viral pneumonia caused by frequently exposed rhinovirus could hardly occurs in immunocompetent adults. according to choi et al. , viruses are frequently detected in the airway of pneumonia patients who require intensive care unit admission, and may cause severe forms of pneumonia. in this study, rhinovirus was identified in . % of respiratory specimens evaluated, but most of patients were the elderly individuals who had underlying comorbidities including structural lung disease, diabetes mellitus, solid cancer, or hematologic malignancies. on the contrary, our relatively young immunocompetent patient suffered severe rhinovirus pneumonia without bacterial co-infection, which was confirmed by bal fluid analysis, and not by the nasopharyngeal specimen. two possible explanations exist for the occurrence of this extremely rare case. first, despite prior indications, severe rhinovirus pneumonia may actually occur in an immunocompetent host. second, chronic alcoholic ingestion could be a major immune modulator for respiratory viral infection, apart from alcoholic liver damage. some previous reports revealed the influence of alcohol on innate immunity in the respiratory system [ ] [ ] [ ] , but the unfavorable impact could play a more critical role in viral defense mechanisms than expected. in conclusion, although the clinical significance of habitual alcoholic ingestion in viral pneumonia requires further evaluation, viral etiology should be considered in cases of severe pneumonia even though in immunocompetent adults, especially if alcoholics. no potential conflict of interest relevant to this article was reported. virological studies in natural common colds in sheffield in patterns of illness in rhinovirus infections of military personnel respiratory viruses and exacerbations of asthma in adults perez-brena p. role of rhinovirus in hospitalized infants with respiratory tract infections in spain rhinovirus associated with severe lower respiratory tract infections in children rhinovirus and the lower respiratory tract two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus rhinovirus outbreaks in long-term care facilities incidence and characteristics of viral community-acquired pneumonia in adults viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation viral pneumonia viral infection in patients with severe pneumonia requiring intensive care unit admission the alcoholic lung: epidemiology, pathophysiology, and potential therapies chronic ethanol ingestion increases superoxide production and nadph oxidase expression in the lung inhibition of tlr -and tlr -induced type i ifn induction by alcohol is different from its effects on inflammatory cytokine production in monocytes key: cord- -u ud vmq authors: lussi, carmela; schweizer, matthias title: what can pestiviral endonucleases teach us about innate immunotolerance? date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: u ud vmq pestiviruses including bovine viral diarrhea virus (bvdv), border disease virus (bdv) and classical swine fever virus (csfv), occur worldwide and are important pathogens of livestock. a large part of their success can be attributed to the induction of central immunotolerance including b- and t-cells upon fetal infection leading to the generation of persistently infected (pi) animals. in the past few years, it became evident that evasion of innate immunity is a central element to induce and maintain persistent infection. hence, the viral non-structural protease n(pro) heads the transcription factor irf- for proteasomal degradation, whereas an extracellularly secreted, soluble form of the envelope glycoprotein e(rns) degrades immunostimulatory viral single- and double-stranded rna, which makes this rnase unique among viral endoribonucleases. we propose that these pestiviral interferon (ifn) antagonists maintain a state of innate immunotolerance mainly pertaining its viral nucleic acids, in contrast to the well-established immunotolerance of the adaptive immune system, which is mainly targeted at proteins. in particular, the unique extension of ‘self’ to include the viral genome by degrading immunostimulatory viral rna by e(rns) is reminiscent of various host nucleases that are important to prevent inappropriate ifn activation by the host’s own nucleic acids in autoimmune diseases such as aicardi-goutières syndrome or systemic lupus erythematosus. this mechanism of “innate tolerance” might thus provide a new facet to the role of extracellular rnases in the sustained prevention of the body’s own immunostimulatory rna to act as a danger-associated molecular pattern that is relevant across various species. viruses are never able to propagate and survive on their own, i.e., they are totally dependent on a host that they can infect. consequentially, the field of virology is intimately linked to immunology as, during the long time of co-evolution, the hosts have acquired a vast array of antiviral defense mechanisms and vice versa. bovine viral diarrhea virus (bvdv) is probably one of the most wide-spread viruses, at least among the "terrestrial viruses". in this mini review, we will focus on the interplay of bvdv, especially its viral rna, with the innate immune defense of the host animals, because this is the key element to explain the long-term survival of this virus in its host population. finally, we hypothesize that the survival strategy of bvdv to induce adaptive and, likewise, innate immunotolerance might provide a new viewpoint for the way the host handle its own, potentially immunostimulatory, self nucleic acids to prevent them from inappropriate chronic activation of the interferon (ifn) system. the pestiviral mechanisms to avoid detection and, thus, to behave identical as the body's own rna, might well be analogous to the mechanisms of its host not to recognize own structures, the latter being of paramount importance to prevent autoimmune reactions. bovine viral diarrhea virus (bvdv), including the two species (also called 'genotypes') bvdv-i and bvdv-ii, classical swine fever virus (csfv), border disease virus (bdv) of sheep, and several tentative species belong to the genus pestivirus in the family flaviviridae [ ] . bvdv is a cattle pathogen of major importance with a worldwide distribution. its viral life cycle was recently described in a number of excellent reviews [ ] [ ] [ ] [ ] [ ] and, thus, is only briefly summarized here. pestiviruses are single-stranded (ss) rna viruses with an envelope containing three viral glycoproteins, i.e., e rns , e , and e . the genome with positive polarity encodes for a single large open reading frame (orf). by virtue of a large variety of possible mutations, bvdv exists as a cytopathic (cp) and a noncytopathic (ncp) biotype, defined by their effect on cultured cells. upon attachment of the virus particle to the cell surface, the virus enters its host cell via clathrin-mediated endocytosis. fusion of the viral with the endosomal membrane is then initiated by acidification of the organelle. cap-independent translation is mediated by an internal ribosomal entry site (ires) and the polyprotein is then further processed by cellular and viral proteases into at least structural and non-structural viral proteins. virus assembly most likely occurs in intracellular vesicles and exocytosis of mature particles occurs in a non-lytic way, at least for the ncp biotype. infection of cattle with either biotype results in transient viremia and infected animals show no disease signs, mild diarrhea, fever, and coughing, but severe thrombocytopenia and hemorrhages have also been reported [ ] . upon resolution of infection, the animals will be protected from re-infections. by contrast, infection of pregnant cows within the first days of gestation with an ncp, but not cp, biotype of bvdv may result in the birth of persistently infected (pi) calves. the clinical symptoms of such pi animals vary considerably and range from unapparent infection to severe growth retardation. gastrointestinal and/or lung diseases are frequently described, with lung-centered pathology observed mainly in young calves and mucosal pathology predominantly in older animals ( [ ] , and references therein). these pi calves are highly susceptible to secondary infections with other pathogens, and are at risk of developing fatal mucosal disease (md). the latter can occur at any time during the life of the pi animal if mutations or recombination with viral or cellular rna with the persisting ncp strain lead to the generation of an antigenically homologous cp biotype (for reviews, see refs. [ , , ] ). however, the development of such a cp biotype in these pi animals is rather an evolutionary misfortune as the cp strain will be eliminated with the death of its host animal [ ] . it is exclusively the ncp biotype of pestiviruses that is transmitted in the long term from pi animals to naïve pregnant host's in order to produce new pi calves [ ] . hence, persistent infection at the level of the single animal is responsible for viral persistence in the host population. consequently, it's the pi animals that are specifically searched for and eliminated in order to eradicate bvdv in regional and national control programs [ , [ ] [ ] [ ] . as positive-sense rna viruses, pestivirus replication occurs via a semi-conservative model [ ] using a double-stranded (ds) rna template to synthesize plus-strand, genomic viral rna. accordingly, replicative forms (rf) and replicative intermediates (ri) were detected in bvdv-infected cells, and it was estimated that the ri contain - nascent strands per template [ ] [ ] [ ] . thus, pestiviral replication involves the formation of dsrna intermediates in the cytosol of infected cells as seen with a variety of rna and dna viruses [ ] . this could be confirmed in bvdv-and csfv-infected cultured cells by immunofluorescence or immunoelectron microscopy and flow cytometry using a dsrna-specific monoclonal antibody [ ] [ ] [ ] . in line with the rather unrestrained replication of the cp biotype of pestiviruses [ ] , the amount of plus-and minus-strand viral rna and, thus, also of dsrna is up to two order of magnitude higher in cells infected with cp than with ncp viruses ( [ , , ] , and references therein). as dsrna is a potent pathogen-associated molecular pattern (pamp) [ ] , it comes of no surprise that pestiviruses are able to induce a type-i ifn response. however, ifn induction strongly depends on the virus strain, its biotype, virulence, and the cell type being infected. thus, infection of many cell types by pestiviruses of the cp, but not the ncp, biotype induces ifn type-i synthesis in vitro (for review, see ref. [ ] ), whereas in calf testicle cells [ ] and porcine plasmacytoid dendritic cells (pdc) [ ] , ifn type-i was induced by ncp bvdv and ncp csfv, respectively. notwithstanding, the amount of dsrna present in cells infected with pestiviruses of the ncp biotype is basically able to induce ifn expression in most cell types, which become obvious as mutant ncp strains lacking the ifn antagonist n pro (see below) replicate to similar or only partially reduced levels as its wt parent strains but readily induce ifn synthesis [ ] [ ] [ ] . thus, it can be conceived that the threshold for ifn induction, e.g., the amount of trigger required to induce an innate immune response, and the effectiveness of the pestiviral ifn antagonists varies between different cell types. the pattern recognition receptors (prr) that sense the pestiviral infection are much less well characterized. viruses are mostly recognized by virtue of their genome (for reviews, see e.g., refs. [ ] [ ] [ ] ), and thus, the cytosolic rlrs (rig-i-like receptors, such as rig-i, mda- and lgp ) and the toll-like receptors (tlrs) in the endolysosomal compartments were obvious candidates. transfection of total rna extracts isolated from cp bvdv-infected cells into uninfected mdbk cells induced ifn synthesis in a triphosphate-independent manner [ ] , which points to mda- as possible prr. however, as rig-i is not strictly dependent on a triphosphate moiety [ ] , other receptors in addition to mda- could not be excluded. accordingly, hüsser et al., using lentivirusmediated transduction of short hairpin rna, nicely demonstrated that csfv is sensed by mda- , rig-i and tlr- in porcine pk- cells [ ] . in addition, ifn-a secretion in pdcs induced by csfv infection or by cell-cell contact with csfv-infected cells was severely reduced by an oligodeoxynucleotide inhibitor of tlr [ ] . activation of pdcs by csfv infection required replication of the virus, as uv inactivation or neutralization of the virus suspension with neutralizing antibody completely abrogated ifn-a release, whereas ifn expression upon cell-dependent rna transfer was independent on infectious virus particles [ , ] . these results show that cytosolic and endolysosomally localized prrs are able to detect the presence of pestiviral rna. rlrs located in the cytosol most probably detect replicative double-stranded intermediates of various length formed during viral replication in productively infected cells. however, it remains unknown how this dsrna gets access to tlr- containing compartments. on the one hand, dsrna might be liberated from infected cells that undergo spontaneous or virus-induced apoptosis and then becomes endocytosed by neighboring cells [ ] . on the other hand, viral rna present in the cytosol might be shuttled to endosomes by the formation of autophagosomes as shown for vsv and activation of tlr- in mouse pdcs [ ] . but the fact that pestiviral replication was purported to be even enhanced by autophagy in pk- or mdbk cells [ , ] argues against the latter possibility. finally, virus replication complexes might be already formed in membranous vesicles as described, e. g., for corona-or hepatitis c viruses [ , ] , but such large membrane arrangement were not observed in pestivirus infected cells [ ] . notwithstanding, dsrna was localized inside the lumen and outer membranes of multivesicular bodies (mvbs) in mdbk cells infected with the pestivirus strain giraffe- , but whether these vesicles represent autophagosomes that hide the dsrna from detection by the innate immune system followed by disposal in lysosomes, or whether they are true sites for viral replication is currently unknown [ ] . finally, viral ssrna represents a pamp on its own as shown by the activation of tlr- in porcine pdcs [ ] . this indicates that viral replication is not required to generate a danger signal, but viral rna synthesis might be required in order to produce a sufficient amount of trigger molecules. the exact structure of the ssrna molecule required to activate tlr- / is not yet known, but au-or gu-rich regions, or inosine-containing immunostimulatory ssrna were reported to effectively activate tlr- [ , ] . interestingly, inosine incorporation seems to increase the rna's secondary structure that enables its recognition by tlr- [ ] , further demonstrating that highly structured ssrna in addition to dsrna is a tlr- agonist [ ] . this is confirmed by the fact that in vitro transcribed ssrna of the bvd viral genome possess highly structured regions resistant to serum rnases and to rnase a (preferentially ssrnases) that are able to induce activation of tlr- [ , ] . the -und -utrs and long-range interactions between these two regions might be especially immunostimulatory [ , ] , but most regions within the bvd viral genome seem to possess high-ordered structures able to induce tlr activation [ ] . in summary, various single-and double-stranded intermediates of viral rna replication and viral genomic ssrna or fragments thereof that were released by premature decay of extracellular or endosomal virus particles represent immunostimulatory nucleic acids. based on their different localizations, a variety of prrs in different compartments, e.g., rlrs in the cytosol and tlrs in endolysosomes, might become activated by pestiviruses. infection of the fetus at an early stage of development as described above effectively bypasses the adaptive immune system by establishing self-tolerance including b-as well as t-cells. in addition, maternal neutralizing antibodies cannot cross the ruminant epitheliochorial placenta, further protecting the virus from a humoral immune response within the fetus. however, in order to succeed in establishing persistent infection, bvdv still requires to cope with the innate immune system already active from the outset. thus, the interaction of ncp bvdv with its host bypasses the adaptive immunity by inducing central immunotolerance, as well as evades innate immunity, with the ifn system as one of the most important antiviral defense systems of the host. the n-terminal non-structural autoprotease n pro and the envelope glycoprotein e rns are two ifn antagonists expressed by pestiviruses that are unique to this genus within the flavivirus family. in the past few years, it became more and more evident that both antagonists are required in a non-redundant way to successfully establish persistent fetal infection [ ] . n pro expressed in virus infected cells is responsible for polyubiquitinylation and proteasomal degradation of the transcription factor irf- in an as yet unknown manner but without requiring its proteolytic activity. by contrast, secreted e rns prevents the activation of the host's tlrs also in non-infected cells by endonucleolytic degradation of viral rna prior to their activation of the corresponding prrs (for reviews, see refs. [ , , , , ] ). collectively, it appears that n pro and e rns effectively reduce or at least delay ifn induction. the fact that the highly replicating cp biotype of pestiviruses similarly express these two ifn antagonistic proteins indicate that there exists a delicate balance between the level of pamps and the capacity to limit their effects on the innate immune response of the host. with pestiviruses exhibiting a rather broad cell tropism, expression of n pro as the very first protein enables an efficient and fast inhibition of dsrna-induced responses in all cells containing replicating virus. in addition, the rather unspecific cell and host tropism of soluble e rns (see below), which is even active, e.g., in canine or human cells [ ] , prevents tlr activation in a large variety of uninfected cells within the host animal. nevertheless, despite the availability of such effective inhibitory mechanisms, both biotypes of bvdv induce the expression of ifn in vivo after infection of adult animals [ ] [ ] [ ] . by contrast, only ncp bvdv strains are immunologically silent in fetuses and pi animals [ , ] . the latter fact might still be debated as chronic up-regulation of type-i and type-ii interferon was reported in bovine fetuses infected early in utero [ ] . indeed, % of pi animals but only % of non-pi control animals expressed mx protein in pbmcs ex vivo. however, there was no correlation with the amount of viral rna or e rns protein and only a week positive correlation with the infectious virus titer in the plasma of the pi animals (t.t.h. pham blume and m. schweizer, unpublished observation). this indicates that it is rather the increased susceptibility of pi animals to secondary infections [ ] than the persisting virus itself that is the cause for the increased activation of the ifn system in these animals. whether this increased susceptibility is related to the selective immunosuppression elicited by the expression of the ifn antagonists and whether it is more distinctive for pathogens exploiting these pathways remains to be shown. thus, the precise adjustment of inhibitory and stimulatory triggers of the innate immune system and their spatiotemporal control in vivo are not yet sufficiently characterized. notwithstanding, it appears that the evasion of the ifn response is the central element for ruminant pestiviruses to induce persistent infections. in the next paragraphs, we will specifically describe the mechanisms of e rns contributing to the establishment and maintenance of innate immunotolerance in pi animals, and put it into relation to other host and viral nucleases that are involved in the depletion of immunostimulatory self and nonself nucleic acids. e rns , initially termed e , was first detected at the surface of pestiviral particles and shortly thereafter also in the supernatant of virus infected cells [ , ] . accordingly, e rns was detected in vivo with concentrations up to ng/ml in the serum of pi animals [ ] . both, secreted and structural e rns are mostly found as disulfidelinked homodimers of around kda [ ] , with carbohydrates contributing approximately half of the apparent molecular weight [ ] . e rns contains nine highly conserved cysteine residues that form four intramolecular disulfide bonds [ ] . the c-terminal cysteine at the position (c ) forms an intermolecular disulfide bond between two e rns monomers and a substitution of this residue results in a loss of the dimeric status [ ] . to that effect, viruses encoding monomeric e rns are not restricted in their replication in vitro but are attenuated in vivo [ , ] . as envelope glycoprotein, e rns plays a role in virus attachment through interactions with the cell surface glycosaminoglycans (gags) [ , ] . binding of e rns to gags involves a cluster of basic residues ( -kklenksk- ) near the c-terminus, with the lysine residues at positions and being critical for binding [ ] . nonetheless, pseudotyped particles containing only e and e of csfv were still able to mediate virus entry [ ] . in addition to its contribution to virus attachment, the c-terminus is folded into an amphipathic helix that anchors e rns in plane into the membrane of the viral envelope [ , ] . detailed analyses depicted that the amphipathic helix lies with a slight tilt within the membrane just underneath the lipid head domain [ , ] . to ensure a sufficient number of protein molecules for the production of new virus particles, a large number of the e rns proteins remains within the infected cells in a not yet defined part of the endoplasmic reticulum (er) by a specific retention signal located within the cterminal amphipathic helix [ ] . in summary, the c-terminus of e rns fulfills different tasks: it anchors the protein into the viral envelope, it attaches soluble e rns to the cell surface via its gagbinding sites and it helps to maintain an appropriate ratio of cellassociated and soluble e rns . in , it was reported that, in addition to its function as envelope glycoprotein, e rns resembles ribonucleases of the rnase t family and indeed degrades preferentially ssrna ( [ ] ; for review, see refs. [ , , ] ). thereby, e rns , but not rnase-inactive mutants, potently inhibit ifn expression induced by the addition of extracellular synthetic or viral ss-or dsrna [ , , ] . according to x-ray structure analyses, however, e rns is only able to bind ssbut not dsrna in the active site [ ] . hence, the mechanism of e rns to degrade dsrna remains to be investigated, but we propose that it might act as a nicking endoribonuclease targeting the two strains of dsrna individually ( [ ] , and lussi et al., in preparation). these in vitro results are also applicable in vivo, as bvdv and csfv mutant viruses encoding for an e rns protein lacking its rnase activity are severely attenuated [ , ] . owing to the gag-binding site within the amphipathic helix, e rns rather unspecifically binds to cell surfaces, and is thus able to act as ifn antagonist in cells of various species, e. g., caprine, ovine, canine, or human cells. mutant proteins lacking amino acid residues of the c-terminus, including the gag-binding site, were severely limited in their inhibition of dsrna-induced ifn expression [ ] . furthermore, binding to the cell surface was followed by an energy-dependent uptake via clathrin-mediated endocytosis, which strongly indicates that e rns cleaves its substrate in an intracellular compartment [ ] (fig. b) . this is corroborated by the fact that rnase-active e rns effectively inhibited activation of tlr- by extracellularly added dsrna or viral ssrna that is resistant to serum rnases of the host (fig. a) , and of tlr- by virus-infected cells in contact with pdcs [ , ] . by contrast, activation of tlr- by r was not inhibited by e rns [ ] further indicating that it does not inhibit any downstream signaling but rather degrades the viral pamp prior to activation of tlrs (fig. ) . as extracellular dsrna was reported to be delivered to endosomal andby an unknown wayto cytosolic prrs, it cannot formally be excluded that rlrs are activated as well [ ] . but e rns potently inhibits ifn induction by extracellular dsrna, and it can thus be postulated that the dsrna might well be degraded within an endolysosomal compartment prior to any transfer to the cytosol. accordingly, ifn induction was unimpeded in e rnsexpressing cells upon lipofectin-mediated transfection of poly (ic), whereas the effect of dsrna added to the medium was completely blocked [ ] . an endosomal location of e rns is also in agreement with its preference for a slightly acidic milieu. e rns is active in a broad ph range from about - . with an optimum around a ph value of , measured in a mm sodium-or tris-acetate buffer [ ] . by contrast, e rns degraded poly(ic) but not in vitro transcribed dsrna in cell culture medium (ph . - . ) with the latter being cut at a ph value of . [ ] . however, by comparing the activity of e rns at ph . and . in tris-acetate buffer versus cell culture medium (mem), we demonstrated that the activity of e rns is much lower in hepesbuffered mem irrespective of the ph tested ( table ), implying that there might be an unknown inhibitory factor in the cell culture medium different from the buffer substance itself. based on the fact that gag-binding of e rns is required prior to its uptake by endocytosis, we were able to effectively prevent or reverse binding of e rns to cell membranes by heparin treatment (fig. a) . furthermore, even after removal of all extracellular e rns by heparin after one hour of incubation, the viral rnase was still able to inhibit dsrna-induced ifn expression in bovine turbinate cells up to - days later [ ] . finally, complexation of dsrna with the human cathelicidin ll- completely protected the nucleic acid from degradation by e rns in vitro. nonetheless, ifn induction by these dsrna-ll- complexes was inhibited in e rnstreated cells, which suggests that the rna dissociates from ll- in order to the nucleic acid being accessible to degradation by the viral rnase within the corresponding compartment (fig. b ). this is in complete accordance with e rns being active at ph values as low as . and with the fact that poly(ic) dissociates from ll- at low ph values, e.g., upon endosomal acidification [ ] , which is a further indication for the endolysosomal localization of e rns . the exact location of e rns inside the various cell types, and the mechanism of how it specifically encounters its targets, i.e., ss-and dsrna, remain to be established. fig. . pestiviral e rns inhibits ss-and dsrna-induced type-i interferon (ifn) synthesis. viral ss-and dsrna, e.g., from dying infected cells, are potent pathogen-associated molecular pattern (pamp) that are sensed by the corresponding pattern-recognition receptor (prr) such as tlr- and tlr- . both toll-like receptors are located in endolysosomal compartments and, once bound by their substrates, they induce downstream signaling that leads to ifn expression. as many regions of the bvd viral genome are resistant to degradation by extracellular serum rnases, they effectively induce the cell's innate immune response (a). by contrast, soluble e rns is taken up by clathrinmediated endocytosis that enables this rnase to effectively degrade the viral pamps prior to tlr activation (b). the annotation of the elements in the figure is depicted in the panel on the right. a number of viruses express ribonucleases, which play crucial roles in viral replication and in virus-host interactions. these viruses comprise negative-strand rna virus of the orthomyxo-, arena-, or bunyavirus families, plus-strand rna viruses such as nidoviruses, or dna viruses such as herpesviruses. the role of these virus-encoded exo-and endoribonucleases, e.g., in proof-reading, mrna cap snatching, protein synthesis shutoff, or rna interference were described in a number of excellent reviews [ , , [ ] [ ] [ ] . in the following, we therefore only describe few examples for the role of viral rnases in evasion of the host's innate immune response. viruses in the order nidovirales, including corona, arteri-and roniviruses, encode for a large number of ifn antagonists within their extraordinary large rna genome [ ] . among them, the exonuclease exon within non-structural protein (nsp) , the endoribonuclease endou encoded by nsp , and possibly the nsp b of the arterivirus porcine reproductive and respiratory syndrome virus (prrsv) possess rnase activity. the latter seems to be conserved only in prrs viruses, whereas nsp in coronaviruses was reported to lead to host translational shut off by degrading host mrnas through a yet unknown host endonuclease, but its precise role and conservation among the nidoviruses remain to be table degradation of in vitro transcribed pestiviral ss-and dsrna [ ] by e rns in various buffer systems at ph . or . , with rating from very strong degradation (+++; green) to no degradation (red). trisac: tris-acetate buffer; mem: minimum essential medium; n.d.: not done. fig. . pestiviral e rns inhibit viral rna-induced ifn synthesis in endosomal compartments. blocking e rns from entering the cell by heparin treatment prevents this viral rnase to inhibit ss-and dsrna-induced ifn expression (a). by contrast, despite protection from rnase degradation by complexation of nucleic acids, e.g., by ll- , ifn expression induced by complexed viral rna is nevertheless inhibited by e rns , as endosomal acidification leads to separation of ll- from the rna that makes it immediately amenable for degradation by e rns also at low ph values (b). the annotation of the elements in the figure is depicted in the panel on the right. clarified [ , , ] . the exoribonuclease exon within the nterminal part of nsp in coronaviruses (that also harbors an n methyltransferase activity at the c-terminus) is responsible for 'proof-reading' during replication of these large nidoviruses, but additionally, a role for exon in degrading rna to avoid their recognition by the host's prrs was suggested [ ] . finally, the endonuclease nsp (nsp in arteriviruses) cleaves ss-and dsrna preferably at uridine residues [ , , ] . the substrate of the rnase activity in nsp is not yet known, but it might be conceivable that nsp degrades viral rna to avoid its recognition as pamp by cellular prrs [ ] . alternatively, nsp of the arterivirus prrsv was reported to degrade mavs mrna [ ] , but this is still debatable as this conclusion was presumably drawn from experiments using single protein overexpression and the original data were not yet published. akin to pesti-and coronaviruses, arenavirus infections are linked to suppression of the host innate immune system. the nucleoprotein (np) of the prototypic arenavirus lymphocytic choriomeningitis virus (lcmv) was shown to be able to inhibit an ifn type i response by blocking irf- nuclear translocation [ ] . this observation was later extended to other arenaviruses, including lassa virus, and it was demonstrated that a - exoribonuclease activity in the c-term of np is essential for the ifn suppression. determination of the crystal structure of np of lassa and tacaribe virus and biochemical analyses confirmed that the structure of np contains an exonuclease domain of the dedd family that is able to cleave -triphosphate dsrna templates in vitro. collectively, there is strong evidence that the exonuclease activity of np is conserved in all arenaviruses and is required to prevent activation of rig-i by the degradation of viral pamps ( [ , ] , and references therein). almost every virus encodes for at least one mechanism to prevent its nucleic acid being recognized by the host and thereby activating the host's ifn response [ ] . overall, however, there is only few experimental evidence that viral nucleases indeed degrade their viral pamps to evade the host's innate immune defense. by contrast, there are several instances of host nucleases that use a similar strategy to degrade own danger molecules that might participate in autoimmune pathologies as exemplified in the following section. inappropriate activation of type-i interferon goes along with many autoinflammatory and autoimmune diseases, and there is strong evidence that dysregulation of various host pathways that are required to contain immunostimulatory self nucleic acids play a causative role (for recent reviews, see e.g., refs. [ ] [ ] [ ] [ ] [ ] [ ] ). in the following, a few examples are briefly described to illustrate the role of ifns and of nucleases regulating ifn synthesis in autoimmune diseases. in systemic lupus erythematosus (sle), circulating ifn-a levels correlate with disease severity. the recognition of self-dna or self-rna by tlrs followed by an ifn-dependent auto-amplification loop seems to be a major mechanism of disease pathogenesis. thus, in sle, pdcs are continuously activated by immune complexes (ic) comprising self-nucleic acids (e.g., from apoptotic or necrotic cell material) and autoantibodies to self-rna, -dna or nucleoproteins followed by fc receptor mediated uptake, which ultimately leads to the secretion of large amounts of ifn type-i in a tlr- / or tlr- -dependent manner. the constant ifn production, the ifn-dependent maturation of myeloid dendritic cells followed by stimulation of autoreactive t-cells and the differentiation of bcells into autoantibody-secreting plasma cells further aggravate the disease symptoms [ , [ ] [ ] [ ] . anti-inflammatory treatment of sle by glucocorticoids, which are thought to act via inhibition of nf-kb, shows only limited success in relieving sle disease symptoms, as the continuous activation of tlrs in pdcs by selfnucleic acids also activate nf-kb that enhances pdc survival and, thus, continued ifn secretion is not abrogated [ ] . in addition to autoantibodies, the antimicrobial cathelicidin peptide ll- and high-mobility group box protein (hmgb , a nuclear dna-binding protein released from necrotic or cytokine-stimulated cells) might protect the self-nucleic acids from degradation by host nucleases, and facilitate their uptake by pdcs and b-cells. interestingly, ll- which is produced by keratinocytes and neutrophils after skin injuryis similarly overexpressed in psoriatic skin lesions, and its ability to complex self-rna and self-dna, to enhance its retention in the endosomes of plasmacytoid and of myeloid dendritic cells, and to trigger tlr-dependent ifn synthesis might be responsible for the observed breaking of self-tolerance [ , , ] . finally, there is genetic evidence obtained from studies with various knockout mice and genome-wide association studies that support the role of innate and adaptive immune responses in the development of sle, such as pathways involved in removal of apoptotic bodies and ics (including dnase i and trex- (dnase iii)), prr activation and ifn expression, and interference with t-and bcell signaling [ ] . remarkably, mice with the y-linked autoimmune accelerating (yaa) locus show enhanced sensitivity to develop lupus depending on the background of the mice, which was attributed to a x to y chromosomal translocation resulting in the duplication of the gene encoding for tlr- . the latter observation might also relate to the fact that women are around times more often affected by sle than men, which might be caused by incomplete x chromosome inactivation leading to insufficient tlr- dosage compensation [ ] [ ] [ ] . aicardi-goutières syndrome (ags) is a genetically determined, autosomal recessive disease of progressive encephalopathy of early childhood, very similar to congenital viral infections. some children develop early-onset sle or a cutaneous form thereof, familial chilblain lupus. ags is caused by mutations in trex- , various components of the rnase h complex, samhd , adar- and mda- (for review, see e.g., [ , ] ). trex- is the main - dna exonuclease in mammalian cells and might be involved in the disposal of dna from endogenous retroelements. recently, it was reported that trex- exerts in addition rna exonuclease activity on ssrna and on rna/dna hybrids and both, dna and rna exonuclease activity, are lost by the mutations found in ags patients [ ] , which in the end leads to spontaneous activation of the cytosolic dna sensor cyclic gmp-amp synthase (cgas) followed by ifn expression [ ] . rnase h is composed of three different subunits performing endonuclease activity cleaving rna in rna/dna hybrids or it cleaves the phosphodiester bond of individual ribonucleotides in dna duplexes. the precise role of samhd in ags is not yet known. the wild-type enzyme is a triphosphohydrolase, which reduces the cellular dntp pool, and is an rnase, whose endogenous rna substrate still needs to be identified. in any case, spontaneous ifn expression in samhd knockout mice was reported to involve intracellular rna and dna sensors, as additional knockout of the adapter proteins mavs or sting abolished ifn production [ ] . finally, adar- deaminates adenosine to inosine in dsrna and was reported to suppress ifn signaling, possibly by marking endogenous dsrna as self avoiding recognition by mda- [ ] . analogously, the mutations found in mda- in ags lead to a gain-of-function phenotype with increased affinity to dsrna and enhanced ifn expression. combined, mutations that cause an aberrant nucleic aciddependent signaling and increased ifn expression were described in all of these autoimmune disorders that finally lead to disease pathology. the "ifn signature" is a hallmark of all these "type i interferonopathies" [ ] , and retroelements, which make up half of the human genome, seem to be an important source of endogenous ligands for the host prrs. finally, there is a strong overlap between the control of self-nucleic acids and the antiviral innate immune response, further highlighting the need for a tight regulation. during viral infections, the adaptive immune system is primarily responsible for the detection of nonself proteins. as viruses lack the elements of metabolism required for independent multiplication and, therefore, depend on the enzymes of their host cells to synthesize their proteins, viruses are largely recognized by their dna and rna genomes. thus, it is the innate immune system that is in charge of detecting viral nucleic acids, which makes it a formidable task for the host to separate foreign from body's own as nucleic acids are not pathogen-specific by default. the difficulty of differentiation of self from infectious nonself nucleic acids becomes especially apparent when considering that around % of our own genome consists of retroviral sequences [ , ] . to specifically detect nonself nucleic acids by the corresponding prrs, a number of strict controls are required in order to avoid autoimmune reactions. for instance, nucleic acid-sensing tlrs are mostly confined to the endolysosomal compartment and their activation is ph dependent; modification of host nucleic acids, e.g., methylation of nucleosides or incorporation of pseudouridine, prevents their recognition by prrs or even negatively regulates tlr activation; or sequestration might render rna or dna invisible to the host. finally, if all else fails, improper activation of the innate defense is prevented by degradation of immunostimulatory nucleic acids by host rnases or dnases [ , ] . thus, quite some knowledge was gained on the role of host nucleases in preventing autoimmune diseases (compare section . ), but most of these are localized intracellularly. by contrast, despite extracellular rnases were mentioned many times in the literature to play a role in eliminating free extracellular rna (for instance in ref. [ ] ), a specific role of these rnases in the elimination of immunostimulatory self rna has not unequivocally been demonstrated. one reason for this might be the large variety of endogenous rnases found in the serum, e.g., rnases of the rnase a and t family [ ] [ ] [ ] , which might prevent the establishment of single-gene knockout mice with a clear phenotype. in order to survive in the host population, we propose the hypothesis that ruminant pestiviruses induce persistence in its host animals by completely pretending to be part of the body's own, which is a clear advantage for the survival of the host and for successful virus transmission. as a result of the early fetal infection, bvd viral proteins already become part of the host's own with regard to the adaptive immune system by inducing central immunotolerance. as there is no or only limited long-term innate immune memory [ ] , maintaining tolerance to self nucleic acids is an enduring challenge for any host. as the pestiviral genome appears to be at least partially resistant to the host's extracellular rnases, the host's safeguard mechanism as described above fails to prevent tlr activation by misdirected viral ss-and dsrna. thus, the extracellularly secreted viral endonuclease e rns might be regarded as an extension of the host's rnase substrate specificity to avoid inappropriate activation of the innate immune system by immunostimulatory viral rna (fig. ) . with its capability of being endocytosed into endolysosomal compartments and with its rnase being active over a broad ph range, e rns is able to efficiently degrade viral pamps at all relevant compartments. even in the case of extracellularly sequestered immunogenic rnas that are protected from degradation by rnases, e rns effectively prevents them from stimulating tlrs as they are required to dissociate prior to activation of tlrs [ ] , which immediately exposes them to the pestiviral rnase (fig. ) . consequently, the virus in persistently infected animals is entirely tolerated by the host similarly to its own immunostimulatory nucleic acids without inducing overt disease [ ] . similarly, overexpression of tlr- in transgenic mice lead to a lupus-like disease phenotype, which could be partially reversed by additionally overexpressing secreted bovine rnase a [ ] . the host's ifn response is the prime antiviral defense system by inducing direct innate immune reactions and by shaping adaptive immunity. thus, the survival strategy of bvdv consists of being non-cytopathogenic and producing less dsrna than its cp counterpart, and expressing the ifn antagonists n pro as the first protein in order to reduce or even avoid ifn production in infected cells and e rns to degrade immunostimulatory viral rna before they might activate the host's prrs. notably, both pestiviral ifn antagonists are not only required to constantly maintain innate immunotolerance during persistent infections, but they also play an important role in acute infections [ ] . thus, rnase-inactive mutants of pestiviruses are attenuated upon acute infections [ , ] , and the evasion of the host's ifn response by n pro and e rns upon acute infection with csfv is also important for the virulence of the virus and the severity of immunopathology caused by the infection [ ] . thus, the pestiviral ifn antagonist, on the one hand, extend the host's specificity to tolerate self nucleic acids to its own viral rna during persistence and, on the other hand, participate in the evasion of the host's ifn response during acute, transient infections, further illustrating the dichotomy of immunotolerance and the antiviral immune response. this model might well shed new lights on fundamental questions on the innate tolerance to self nucleic acids and the specific detection of viral nonself rna. these aspects are highly relevant also for the prevention of chronic ifn induction and autoimmunity induced by "self-rnas" that might be fundamental beyond the mechanism of an animal disease [ ] . finally, the mechanism of the soluble pestiviral endoribonuclease e rns during persistent infection to support the virus in its strategy to pretend to be part of the body's own might be analogous to the role of the host's own extracellular nucleases to continuously maintain innate immunotolerance to its own immunostimulatory nucleic acids. matthias schweizer received his m.sc. in biochemistry in from the department of biochemistry at the university of zurich, switzerland and his phd in at the institute of biochemistry i at the swiss federal institute of technology (eth) in zurich, switzerland. the doctoral studies were on the oxidative regulation of mitochondrial ca + homeostasis and included a stay at tcom, university of north texas, fort worth (tx). after postdoctoral work at the eth and the institute of veterinary virology (ivv) at the university of bern, he received his "habilitation" (postdoctoral lecture qualification) in virology at the vetsuisse faculty of the university of bern, switzerland. since , he is head of the research group "virus infections of ruminants" and deputy head of the virology section of the institute of virology and immunology (ivi) of the federal food safety and veterinary office in cooperation with the vetsuisse faculty of the university of bern. his main interests are the interaction of ruminant pestiviruses with their host cells to induce persistence, the evolution of bvdv, and in applied research projects to further understand transmission and pathogenesis of pestiviral infections. carmela lussi is a phd student in the group of dr. matthias schweizer at the institute of virology and immunology (ivi) of the federal food safety and veterinary office in cooperation with the vetsuisse faculty of the university of bern. after receiving the bachelor of science in biology, she obtained her master of science in molecular life sciences with further specification in microbiology and immunology in at the faculty of science of the university of bern. within her phd studies, carmela lussi focusses on the role of pestiviral protein e rns in innate immunotolerance. virus taxonomy molecular biology of bovine viral diarrhea virus bovine viral diarrhea virus: global status molecular biology of pestiviruses the molecular biology of pestiviruses bovine viral diarrhea virus infections: manifestations of infection and recent advances in understanding pathogenesis and control clinical appearance and pathology of cattle persistently infected with bovine viral diarrhoea virus of different genetic subgroups rna recombination in pestiviruses-cellular rna sequences in viral genomes highlight the role of host factors for viral persistence and lethal disease cytopathic bovine viral diarrhea viruses (bvdv): emerging pestiviruses doomed to extinction bovine virusdiarrhöe (bvd): von der biologie zur bekämpfung pestivirus control programs: how far have we come and where are we going? anim bvdv control and eradication in europe-an 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and interferons interferons and viruses: an evolutionary arms race of molecular interactions microbial sensing by toll-like receptors and intracellular nucleic acid sensors, cold spring harbor perspect viral rna detection by rig-i-like receptors identification of the role of rig-i, mda- and tlr in sensing rna viruses in porcine epithelial cells using lentivirus-driven rna interference efficient sensing of infected cells in absence of virus particles by plasmacytoid dendritic cells is blocked by the viral ribonuclease e rns fc gamma rii-dependent sensitisation of natural interferonproducing cells for viral infection and interferon-alpha responses extracellular dsrna: its function and mechanism of cellular uptake autophagydependent viral recognition by plasmacytoid dendritic cells autophagy during early stages contributes to bovine viral diarrhea virus replication in mdbk cells autophagy enhances the replication of classical swine fever virus in vitro to sense or not to sense viral rna-essentials of coronavirus innate immune evasion flaviviridae replication organelles: oh what a tangled web we weave identification of rna sequence motifs stimulating sequence-specific tlr -dependent immune responses inosine-mediated modulation of rna sensing by toll-like receptor (tlr ) and tlr inosine-containing rna is a novel innate immune recognition element and reduces rsv infection beyond dsrna: toll-like receptor signalling in rna-induced immune responses pestiviral e rns blocks tlr- -dependent ifn synthesis by ll complexed rna conserved rna secondary structures and long-range interactions in hepatitis c viruses the untranslated regions of classic swine fever virus rna trigger apoptosis bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting e rns rnase and n pro protease pestiviruses: how to outmaneuver your hosts charleston, type i and iii interferon production in response to rna viruses prolonged activity of the pestiviral rnase e rns as an interferon antagonist after uptake by clathrinmediated endocytosis alpha/beta and gamma interferons are induced by infection with noncytopathic bovine viral diarrhea virus in vivo in vitro and in vivo detection of mx gene products in bovine cells following stimulation with alpha/beta interferon and viruses acute non-cytopathic bovine viral diarrhea virus infection induces pronounced type i interferon response in pregnant cows and fetuses establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type i interferon innate and adaptive immune responses to in utero infection with bovine viral diarrhea virus processing of the envelope glycoproteins of pestiviruses a second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus hog cholera virus: molecular composition of virions from a pestivirus a structural model of pestivirus e rns based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide mutation of cysteine of pestivirus e rns rnase prevents homodimer formation and leads to attenuation of classical swine fever virus dimerisation of glycoprotein e rns of classical swine fever virus is not essential for viral replication and infection passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein e-rns interactions of bovine viral diarrhoea virus glycoprotein e rns with cell surface glycosaminoglycans identification of the glycosaminoglycan-binding site on the glycoprotein e rns of bovine viral diarrhoea virus by site-directed mutagenesis characterization of classical swine fever virus entry by using pseudotyped viruses: e and e are sufficient to mediate viral entry the carboxy-terminal sequence of the pestivirus glycoprotein e rns represents an unusual type of membrane anchor the pestivirus glycoprotein e rns is anchored in plane in the membrane via an amphipathic helix structure of the membrane anchor of pestivirus glycoprotein e rns , a long tilted amphipathic helix lipid binding of the amphipathic helix serving as membrane anchor of pestivirus glycoprotein e rns a new type of intracellular retention signal identified in a pestivirus structural glycoprotein identification of a structural glycoprotein of an rna virus as a ribonuclease viral rnase involvement in strategies of infection role for bovine viral diarrhea virus e rns glycoprotein in the control of activation of beta interferon by double-stranded rna crystal structure of the pestivirus envelope glycoprotein e rns and mechanistic analysis of its ribonuclease activity the pestiviral ifn antagonist e rns cleaves dsrna as nicking endoribonuclease recovery of virulent and rnase-negative attenuated type bovine viral diarrhea viruses from infectious cdna clones mutations abrogating the rnase activity in glycoprotein e rns of the pestivirus classical swine fever virus lead to virus attenuation dsrna and the innate antiviral immune response rnase of classical swine fever virus: biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies ll- peptide enhancement of signal transduction by toll-like receptor is regulated by ph emerging roles for rna degradation in viral replication and antiviral defense virus-encoded endonucleases: expected and novel functions nidovirus ribonucleases. structures and functions in viral replication coronavirus virulence genes with main focus on sars-cov envelope gene interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus structures of arenaviral nucleoproteins with triphosphate dsrna reveal a unique mechanism of immune suppression exonuclease domain of the lassa virus nucleoprotein is critical to avoid rig-i signaling and to inhibit the innate immune response importance of nucleic acid recognition in inflammation and autoimmunity type i interferonopathies: mendelian type i interferon upregulation type i interferonopathiesan expanding disease spectrum of immunodysregulation nucleic acid-sensing tlrs and autoimmunity: novel insights from structural and cell biology rna degradation in antiviral immunity and autoimmunity nucleic acidsensing receptors: rheostats of autoimmunity and autoinflammation advances in understanding the role of type i interferons in systemic lupus erythematosus systemic lupus erythematosus-a disease with a dysregulated type i interferon system type i interferons: crucial participants in disease amplification in autoimmunity tlr recognition of self nucleic acids hampers glucocorticoid activity in lupus nucleic acids and endosomal pattern recognition: how to tell friend from foe? front little peptide, big effects: the role of ll- in inflammation and autoimmune disease genetics of systemic lupus erythematosus: immune responses and end organ resistance to damage tlrdependent and tlr-independent pathways of type i interferon induction in systemic autoimmunity x chromosome reactivation perturbs intracellular self/not-self discrimination sexx matters in immunity aicardi-goutières syndrome and the type i interferonopathies human dna exonuclease trex is also an exoribonuclease that acts on single-stranded rna trex deficiency triggers cell-autonomous immunity in a cgasdependent manner spontaneous type i ifn response in samhd -deficient mice requires both, functional intracellular rna and dna sensing pathways rna editing by adar marks dsrna as "self the enemy within: endogenous retroelements and autoimmune disease the potential role of retroviruses in autoimmunity plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases the mammalian secreted rnases: mechanisms of action in host defence t family ribonucleases: ancient enzymes with diverse roles the eight human canonical ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins innate immune memory: a paradigm shift in understanding host defense two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases increased ribonuclease expression reduces inflammation and prolongs survival in tlr transgenic mice immune responses against classical swine fever virus: between ignorance and lunacy, front innate immunity: quo vadis? many thanks to fabienne wyss for performing the experiments shown in table , to giuseppe bertoni and eveline kindler for critically reading the manuscript, and to all the colleagues and students that were involved in our studies over the recent years. we are indebted to all of them. our work was supported over the years by internal funds of the institute of veterinary virology (now institute of virology and immunology) of the vetsuisse faculty university of bern, the federal food safety and veterinary office fsvo, and the swiss national science foundation. key: cord- -nreqluol authors: heise, m.t. title: viral pathogenesis date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: nreqluol viral pathogenesis describes the processes by which viral infections cause diseases and involves virus–host interactions at the cellular and systemic level that determine whether a virus will cause a disease, what form that disease takes, and how severe the disease will be. though the pathogenesis of each virus is unique, there are several common points in the virus life cycle that are shared between all pathogenic viruses, and by considering these common aspects of the virus-induced disease process, we can explore some general concepts in viral pathogenesis while illustrating some of the virus specific processes that shape disease outcomes. viral pathogenesis is a term that generally describes the processes by which viral infection results in a disease. however, viruses can range from small rna viruses (e.g., flaviviruses such as dengue virus) to large dna viruses (e.g., herpesviruses and poxviruses), all of which interact with the host in unique ways to drive the virus-induced disease process. these virus specific disease outcomes are driven by fundamental differences in viral replication cycles, modes of transmission, tissue tropism, interactions with the host immune response, as well as a multitude of other variables. furthermore, due to differences in a wide range of factors, including elements such as host genetic variation, host immune status, viral dose, or route of inoculation, infection with the same virus often results in varied disease outcomes in different individuals, where some individuals may develop no disease at all, while others are symptomatic and may develop serious or life threatening disease. therefore, it is difficult to provide a general overview of viral pathogenesis that accurately encompasses the full range of virus-induced disease processes. however, while the pathogenesis of each virus and its associated disease(s) has unique aspects, it is also true that there are several common stages in the viral life cycle/disease process that are shared between all pathogenic viruses, and consideration of these common processes can be used to illustrate several key concepts in viral pathogenesis. for example, since viruses are obligate intracellular pathogens that are not capable of reproducing themselves outside of a permissive host cell, a virus must successfully gain entry to a target cell and propagate itself to cause a disease. whether a virus can accomplish this task depends on interactions with key host molecules, such as cell surface receptors, which determine whether the virus can successfully infect and reproduce itself within its target cells. therefore, for the purposes of this overview, we will consider several common virus/host interactions, including: ( ) factors that affect viral tissue tropism and host range, ( ) viral immune evasion, and ( ) viral effects on target cells or tissues. by focusing on these key steps in the viral life cycle/disease process, we can discuss some general concepts that illustrate how these interactions impact viral pathogenesis, while also emphasizing the virus specific aspects of these interactions that result in each virusinduced disease having unique attributes. as noted above, viruses cannot replicate outside of a permissive host cell, and gaining access to and being able to replicate within these cells represents a key part of any virus's life cycle. furthermore, the induction of disease is usually dependent upon the effects of the virus on cells or organ systems that it infects, such as direct killing of essential host cells (e.g., neurons) by the virus (see below). therefore, viruses that cannot gain access to and replicate within permissive host cells are generally not able to cause disease. furthermore, viruses that cannot gain access to the specific tissue that is associated with a disease, such as the central nervous system for viruses that cause encephalitis, are also less likely to cause severe diseases. though there are multiple virus-host interactions that determine whether a virus can successfully replicate within a target cell, for the purposes of brevity, we will consider specific examples of stages in the viral life cycle where interactions with the host determine whether the virus can successfully replicate in target cells, and how these interactions promote the virusinduced disease process figure . viral entry into host cells usually involves interactions between molecules on the surface of the virus and specific cell surface receptors, which allow the virus to bind to the host cell and initiate the viral entry process. in some cases, viruses interact with a single cell surface molecule, which mediates viral binding to the cell and also facilitates viral cell entry. for example, several rhinoviruses bind to icam- (cd ), and these interactions promote viral infection of the cell (reviewed in rossmann et al., ) . in contrast other viruses, such as herpes simplex virus, interact with cell surface molecules such as heparin sulfate to facilitate viral attachment to the cell, and then engage specific host receptor proteins on the cell surface that mediate viral entry into the cell (reviewed in spear, ) . viral interactions with these receptors can have a significant impact upon several aspects of viral pathogenesis, including determining the cell or tissue tropism of a virus or even whether a virus can efficiently infect and cause disease in a specific host species. the importance of virus-receptor interactions in disease pathogenesis is nicely illustrated by interactions between human immunodeficiency virus (hiv) and two host molecules, cd and ccr , which mediate hiv binding and entry. hiv infection requires interactions between the viral gp protein and cd (sweet et al., ) , a host protein expressed on a subset of t cells (cd positive helper t cells) and a limited number of other cell types, such as macrophages. since hiv infection is dependent on interactions with cd , cd positive helper t cells are the major target of hiv replication, and viral replication in these cells results in the progressive loss of cd t cells over the course of the hiv infection. cd t cells play an essential role in regulating the immune response, and hiv-mediated killing of these cells ultimately leads to immune suppression that leaves hiv infected individuals susceptible to lethal opportunistic infections that would normally be controlled by persons with fully functional immune systems. therefore, viral interactions with cd and subsequent viral tropism for these cells, directly contributes to disease pathogenesis during hiv infection. in addition to the importance of cd in determining hiv tropism, a second hiv/receptor interaction further illustrates the importance of receptors in driving viral pathogenesis. following hiv gp binding to the cd molecule, the gp molecule undergoes a conformational change, which allows gp to interact with one of two coreceptor molecules, ccr or cxcr , that then leads to viral fusion (alkhatib et al., ; feng et al., ) . the importance of these interactions is illustrated by the fact that a small subset of humans has a nonfunctional form of ccr , and these individuals are highly resistant to hiv infection (liu et al., ; dean et al., ; huang et al., ) . in other words, if the virus cannot get into its appropriate target cells through interactions with ccr , it cannot efficiently infect these individuals and cause disease. in addition to affecting disease pathogenesis by determining viral cell tropism, virus-receptor interactions can also broadly impact disease pathogenesis by determining whether a virus will efficiently infect a new host. this is particularly important for zoonotic viruses, which are viruses that naturally reside in animals, but which jump to humans and cause diseases. to successfully make the transition from its natural animal host to humans, a zoonotic virus must either interact with receptors that are highly conserved between species, or the virus must change (mutate) in a way that allows it to adapt to efficiently interact with receptors in the new host. an example of the first situation is provided by sindbis virus, a mosquitoborne virus that must efficiently infect both mosquitos and vertebrate hosts. recent studies have identified natural resistance-associated macrophage protein (nramp), as a receptor for sindbis virus in both mosquitoes and vertebrate cells (e.g., humans) (rose et al., ) . nramp is highly conserved between species, and it is likely that by interacting with this highly conserved receptor protein, sindbis virus is able to readily replicate in both mosquitoes and vertebrates. in contrast to situations where a virus interacts with a highly conserved receptor, there are also situations where the virus receptor is significantly different between species. therefore, for the virus to successfully make the transition between the original animal host and humans, it is likely that the virus will have to adapt to more efficient use of the receptor in the new species. an example of this type of interaction is provided by the sars coronavirus (sars-cov), which caused an outbreak of severe acute respiratory disease in - . sars-cov infects cells through interactions between the viral spike (s) protein and angiotensin converting enzyme- (ace ) (li et al., ) . however, sars-cov is thought to normally reside in bats (lau et al., ) and the bat-derived virus does not efficiently interact with human ace . however, studies suggest that mutations within the receptor binding domain of sars led to more efficient interactions with the human ace molecule, and that viruses with these adaptive mutations were better able to infect human cells (reviewed in bolles et al., ; graham and baric, ) . this is supported by the finding that introducing the region of the s protein that binds to human ace into the bat sars virus allows that virus to bind to human ace and efficiently infect human cells (becker et al., ) . these results further reinforce the idea that virus receptor interactions play a crucial role in determining whether the virus can efficiently infect the host and ultimately cause disease. though receptor interactions represent a crucial component of virus-host interactions and viral pathogenesis, a number of other factors can also determine which tissues a virus infects. most viruses encode their own replication machinery, however they are still dependent upon the host cell for a number of functions, including processes that promote viral entry or the translation and assembly of viral proteins. recently, the field of virology has become very interested in identifying 'proviral' factors, which are host molecules that promote efficient viral replication. identification of these proviral factors not only enhances our knowledge of how viruses interact with the host cell, but also may identify host pathways that could be targeted to inhibit viral replication and develop new therapies. many proviral factors are components of generally important cellular processes, such as the host translation machinery or cellular protein transport pathways, which are likely to be important for broad classes of viral pathogens. however, there are instances where host factors interact with specific viruses to enhance viral replication in specific cell types, thereby affecting both viral cell tropism and disease pathogenesis. in the most extreme examples, viral replication, and hence the ability to cause disease, would be severely compromised by the absence of a specific proviral factor. an excellent example of a virusspecific proviral factor is provided by hepatitis c virus (hcv) interactions with mir- . hcv is a positive stranded rna virus that infects the liver and causes chronic disease in a significant fraction of infected individuals, putting these individuals at risk for the development of chronic liver failure and the development of hepatocellular carcinoma (reviewed in cabibbo et al., ) . though hcv may be able to replicate in cell types such as b and t cells, the major site of hcv replication are hepatocytes within the liver. a key determinant of hcv's tropism for hepatocytes is the host microrna mir- . micrornas are small ( - ) nucleotide host rnas, which regulate a number of biological processes within the host (reviewed in szabo et al., ) . in the context of hcv infection, mir- is expressed specifically within the liver and its expression enhances hcv rna levels in hepatocyte cell lines (jopling et al., ) . though the mechanisms of mir- 's actions on hcv are not completely understood, mir- interacts with rna structures in the end of the viral genome, and these interactions are essential for mir- 's effects on hcv (jopling et al., ) . the importance of these interactions for hcv replication and disease pathogenesis is illustrated by recent studies in a chimpanzee model of hcv where administration of a mir- specific antagonist resulted in decreased viral loads within the liver and a reduction in hcv associated disease signs within the liver (lanford et al., ) . therefore, hcv interactions with mir- affect viral replication and disease pathogenesis within the liver, illustrating the potential importance of viral interactions with proviral host factors in promoting viral replication and driving the pathogenesis of virus-induced diseases. it is important to note that in addition to proviral factors, there are also classes of host proteins that can act as restriction factors that actively inhibit a virus's ability to replicate and cause disease. these types of host restriction factors have been extensively studied in the context of retro/lentivirus infection and an excellent example of this type of factor is provided by trim a. trim a is part of a class of tripartite motif containing host proteins, a large number of which have been shown to exhibit antiviral or immune regulatory functions (reviewed in mcnab et al., ) . studies looking at restriction factors that limit the ability of hiv to replicate in nonhuman primate cells found that the rhesus macaque trim a molecule strongly interacts with the hiv capsid to block viral infection at an early stage in the viral replication process (stremlau et al., ) . in contrast, the human trim a molecule, which interferes with retroviruses and lentiviruses such as equine infectious anemia virus, interacts less efficiently with hiv (stremlau et al., ) . efficient interactions between trim and hiv appear to protect macaques from hiv replication and disease, while less efficient interactions between human trim a and hiv in part explain the enhanced susceptibility of humans to hiv infection. therefore, the presence or absence of appropriate restriction factors can have a major impact on host susceptibility to virusinduced disease, where the presence of a strong restriction factor would inhibit viral replication and prevent or limit virusinduced disease. in addition to trim a, other factors that mediate host range restriction during lentivirus/retrovirus infection include apobec and tetherin (reviewed in luban, ) . the host innate and adaptive immune systems play a major role in protecting from virus-induced disease by limiting or preventing viral replication. the type i interferon system and other components of the innate immune response are rapidly activated in response to viral infection and play a crucial role in limiting viral replication and spread within the host. likewise, components of the adaptive immune system, such as cytotoxic cd positive t cells and antibody, are involved in clearing virus from infected tissues and can provide long-term immunity to prevent reinfection. the complexity of the immune systems and its interactions with each viral pathogen is such that a comprehensive overview of virus-host immune interactions is beyond the scope of this article. however, though there are aspects of the virus-host immune interaction that are unique to each pathogenic virus, every viral pathogen must successfully avoid or actively antagonize the host immune response to infect, replicate, and disseminate within the host. in fact, viruses with defects in blocking the host immune response are often attenuated in their ability to cause disease. therefore, immune evasion represents a common theme in viral pathogenesis that we will explore further. while different viruses employ a wide range of strategies to avoid/antagonize aspects of the innate and adaptive immune system, we will focus specifically on viral interactions with the innate immune system and the interferon response in particular to illustrate the importance of these interactions on viral pathogenesis. though multiple arms of the innate immune system contribute to viral control, including components such as the complement cascade, natural killer cells, and proinflammatory cytokines, several lines of evidence suggest that the type i interferon system plays an essential role in the pathogenesis of most viral pathogens. most, if not all of the pathogenic viruses affecting humans either avoid or actively antagonize some aspect of the type i interferon response, and viruses that are defective for avoiding/antagonizing the type i interferon system are often attenuated for replication and disease in immunocompetent animals (garcia-sastre et al., ; talon et al., ; leib et al., ; bouloy et al., ) . furthermore, animals lacking a functional type i interferon system exhibit enhanced sensitivity to a wide array of viral pathogens (leib et al., ; ryman et al., ; white et al., ; bouloy et al., ; schilte et al., ) , which suggests that type i interferon (ifn) plays an essential role in limiting viral replication and protecting from disease. the type i interferons are a group of cytokines consisting of a several related alpha interferon molecules and a single interferon beta protein, that are induced in response to stimuli associated with viral infections, such as double stranded rna, and which bind to a common type i interferon receptor complex. signaling via the type i interferon receptor leads to the induction of hundreds of interferon-stimulated genes (isgs). though the function of many of these isgs still needs to be elucidated, it is clear that many of these molecules have antiviral activity against one or more viral pathogens (schoggins and rice, ) , while some isg molecules modulate other aspects of the host immune response, including antigen presentation (schoggins and rice, ) . therefore, the type i interferon system limits viral replication and dissemination by inducing an antiviral state within host cells, and then promotes viral clearance by modulating other aspects of the host immune response, including host antibody and t cell responses. for the purposes of this overview, we will briefly summarize several key aspects of the type i interferon response to illustrate how viruses can avoid or antagonize this response. the production of type i interferon can be induced by signaling through several different pattern recognition molecules, including certain toll-like receptors and cytoplasmic nucleic acid sensors, such as rig-i. these pattern recognition molecules recognize pathogen associated molecular patterns (pamps), which are molecules that have signatures commonly associated with viral infection, such as double stranded rna. readers who are interested in more detailed discussion of the pattern recognition molecules that regulate the type i ifn response are directed to the following reviews (kawai and akira, ; nakhaei et al., ) . though simplified for this overview, following interactions with a viral pamp, each pattern recognition receptor will activate a signaling cascade that ultimately converges on a set of transcription factors (irf or irf ), which upon activation, transit to the nucleus to induce type i ifn transcription and subsequent production of type i interferon. type i interferons are secreted from the cell, and can then act in an autocrine (on the cell that produced the interferon) or a paracrine (affecting the surrounding cells) manner. the type i interferon receptor consists of two subunits ifnar and ifnar , which upon interferon binding, dimerize at the cell surface. the receptors are associated with two protein tyrosine kinases, janus activated kinase (jak ) and tyrosine kinase (tyk ), that are brought together during receptor dimerization. jak and tyk then undergo auto-and/ or transphosphorylation (de weerd et al., ; de weerd and nguyen, ) , which leads to their activation, and they in turn phosphorylate tyrosine residues present on the receptor tails that serve as docking sites for signal transducers and activators of transcription (stat) factors. jak and tyk phosphorylate stat and stat , which then form heterodimers and interact with interferon regulatory factor (irf ), where this complex localizes to the nucleus and binds promoters containing ifn-stimulated response elements to drive expression of isgs, which mediate direct antiviral effector functions and modulate the host immune response (kawai and akira, ; nakhaei et al., ; deweerd, ) . importantly, though there are instances of viruses avoiding or antagonizing specific antiviral effector molecules (daffis et al., ) , the majority of known viral interferon evasion strategies are directed at either avoiding recognition by host pattern recognition molecules or targeting the signaling pathways associated with either type i interferon induction or interferon receptor signaling. therefore, we will briefly discuss a few examples of interferon antagonism to illustrate the importance of these interactions in viral pathogenesis. a number of viruses antagonize type i interferon induction either by targeting specific components of the interferon induction pathways or by broadly inhibiting de novo rna synthesis/protein translation in the infected cell, which effectively blocks the production of type i interferon. one strategy for avoiding type i interferon induction is to shield viral pamps (e.g., viral rna) from recognition by host rna sensors, such as rig-i and mda . a number of viruses encode rna binding proteins that have been shown to inhibit type i ifn induction. for example, the ns protein of influenza a virus, which interferes with type i ifn induction at several stages in the induction process, exhibits potent antagonist activity against type i interferon induction, and this antagonism is in part due to rna binding activity by ns (donelan et al., ) . another example of this type of strategy is provided by the nucleocapsid protein (n) of the sars coronavirus, which also inhibits type i interferon induction at an early stage in the rna recognition process, and this inhibitory activity is dependent upon the rna binding activity of the n protein (lu et al., ) . in addition to blocking host recognition, a wide array of viruses encode proteins that block specific components of the type i interferon signaling cascade. for example, the influenza a virus ns protein, in addition to shielding the viral rna from recognition, prevents rig-i activation and interferon induction in response to influenza infection by blocking the function of trim , a ubiquitin ligase that regulates the activation of the rna sensor, rig-i (gack et al., ) , while a second viral protein, pb -f interferes with type i interferon induction through interactions with mavs, an adaptor molecule that is essential for rig-i-mediated type i interferon induction (conenello et al., ; varga et al., ) . the fact that influenza virus employs multiple strategies to block type i interferon induction argues that viral interactions with the type i interferon system are particularly important in regulating viral replication, and this is further supported by the fact that viruses with mutations in either ns or pb -f induce higher levels of type i interferon and are subsequently attenuated in their ability to cause disease (conenello et al., ; donelan et al., ) . therefore, viruses that are defective in their ability to antagonize the host type i interferon system are often unable to replicate and spread efficiently within the host, illustrating the importance of viral immune evasion strategies in determining whether a virus will be pathogenic ( figure ) . in addition to blocking type i interferon induction, a number of viruses also interfere with type i interferon receptor signaling, since this effectively blocks the induction of antiviral isgs. for example, the type i interferon signaling pathway is targeted at multiple stages by members of the flavivirus family, with multiple proteins from the same virus often targeting different steps of the pathway (reviewed in diamond, ) , again illustrating the importance of interferon antagonism for viral success. as is the case with antagonism of type i interferon induction, viral interactions with the type i interferon signaling pathways are likely to have a major impact on virus-induced disease. studies with reovirus have shown the induction of virus-induced myocarditis is associated with suppression of type i interferon receptor signaling, where the viral m gene from a myocarditic virus exhibits enhanced ability to interfere with the irf component of the isgf signaling complex (zurney et al., ) . these results suggest that viral antagonism on interferon receptor signaling can have a major impact on the pathogenesis of virus-induced disease, and that differences in the efficiency of interferon antagonism between related viruses can have significant impacts on what types of disease those viruses cause. as noted above, viruses are obligate intracellular pathogens that cannot reproduce themselves outside of host cells. therefore, the types of cells that a virus targets and what effect the virus infection has on a target cell plays a major role in determining whether a virus will induce disease and if so, what type of disease the virus causes. most simply, some viruses are cytopathic, in that the virus infection results in direct killing of the host cell, while other viruses are noncytopathic and do not directly kill the infected cell. however, while some viruses cause disease due to their targeting and killing of essential cell types, such as neurons, the mechanisms leading to virusinduced disease are often complex and we will discuss some examples of different interactions between viruses and host cells or the host immune response that affects disease outcomes (figure ). immune killing of host cell transformation tumor development in the presence of a robust type i interferon response, and the absence of effective viral interferon antagonism, type i interferon will induce an antiviral state that limits viral replication and spread, thereby limiting virusinduced disease. (b) if the virus effectively interferes with the type i interferon response, interferon will be prevented from inducing a robust antiviral state within the host, and the virus is able to replicate to higher levels, will spread more efficiently, and may cause more severe disease. a number of viruses are directly cytotoxic and productive viral replication leads to direct cell killing. therefore, if the cell type that these viruses replicate in is essential, such as neurons, virus-induced killing of these cells can directly lead to disease. a number of viruses, including alphaviruses such as sindbis virus and venezuelan equine encephalitis virus (vee), cause direct killing of host cells. while the mechanisms leading to cell death are not completely worked out, it is generally accepted that these viruses induce apoptotic cell death in infected cells griffin et al., ) . since these viruses exhibit strong tropism for neurons, viral replication within the central nervous system leads to neuron death and degradation of neurologic function. though there is evidence that the host immune response exacerbates virus induced disease during both sindbis virus and vee infection (rowell and griffin, ; kimura and griffin, ; charles et al., ) , in the case of vee, mice lacking a functional adaptive immune system still succumb to virus-induced disease (charles et al., ) , suggesting the direct cell killing by the virus contributes to disease pathogenesis. the host immune systems plays a crucial role by protecting from virus-induced disease, however there are clear instances where an overactive or inappropriate host immune response contributes to the pathogenesis of virus-induced disease. this is perhaps most clearly illustrated in situations where a virus is noncytopathic and does not cause the direct death of infected host cells, yet viral infection still results in tissue destruction and disease. hepatitis b virus (hbv), which causes serious acute and chronic hepatitis in infected humans, nicely represents this situation and also illustrated both the importance of the host immune system in promoting viral clearance and the potential for ineffective or overly active immune responses to potentiate virus-induced disease. in most immunocompetent individuals, hbv infection causes acute hepatitis, where the host immune response plays an essential role in viral clearance, but in the process, also causes some liver injury (chisari et al., ) . hbv is noncytopathic for hepatocytes and during the early stages of hbv infection there are no signs of liver disease, even though the virus is replicating to high levels within the liver (chisari et al., ) . it is only after the host adaptive immune response has started to clear the virus from the liver that signs of liver injury are evidence, which suggests that though beneficial in clearing the virus, aspects of this immune response are pathologic. through the use of transgenic mouse models and hbv infection of chimpanzees, it has been shown the virus specific cd t cells are responsible for both the clearance of the virus from the liver and are the mediators of the acute liver injury associated with hbv infection (thimme et al., ) . immune interactions also play a major role in the pathogenesis of chronic hbv infection, where chronic disease is associated with a weak hbv specific cd t cell response that is unable to clear the infection (chisari et al., ) . furthermore, evidence from hbv transgenic mouse models suggests that suboptimal cd t cells responses that fail to clear hbv infection may be associated with chronic liver inflammation and damage that ultimately leads to the development of hepatocellular carcinoma (chisari et al., ) . immune pathology is not restricted to infection by noncytopathic viruses, and overactive or inappropriate immune responses are thought to contribute to disease even when the virus itself is capable of causing direct cell killing. as noted above, even though sindbis virus and vee are thought to directly contribute to virus-induced neurologic disease by killing neurons, it is also clear that components of the host adaptive immune response can exacerbate the disease process with these viruses (charles et al., ; rowell and griffin, ; kimura and griffin, ) . this concept is also illustrated by ross river virus (rrv), another alphavirus that is associated with severe arthralgia and myalgia in infected humans. studies in humans and mouse models have shown that rrv replicates to high levels in joint and muscle tissues and that viral replication within these tissues leads to the development of arthritis and myositis hazelton et al., ; morrison et al., ) . further analysis found that even though rrv is cytopathic, depletion of macrophages significantly reduced the severity of virus-induced disease, suggesting that components of the inflammatory response, rather than direct virus-induced killing, are responsible for virus-induced disease (lidbury et al., ) . furthermore, activation of specific components of the host complement cascade, including mannose binding lectin and the c component of complement, are associated with severe rrv-induced disease in humans and mice lacking either of these factors are highly resistant to virus-induced disease (morrison et al., ; gunn et al., ) . it is also important to remember that while the above examples focus on instances where components of the immune response directly contribute to cell killing in virally infected tissues, immune-mediated pathology may not always be restricted to direct effects on virally infected cells or within virally infected tissues. a number of chronic viral infections, including hepatitis c virus (hcv) infection, are associated with the development of immune complexes, where aggregates of virus and antibodies precipitate within small blood vessels and lead to the development of inflammation (vasculitis) (reviewed in lauletta et al., ) . therefore, while generally beneficial, there are instances where overactive or inappropriately activated components of the antiviral host immune response directly contribute to the pathogenesis of virusinduced disease. since there is significant person to person variation in immune function, variation in the magnitude and composition of the host immune response may play a major role in determining whether an individual mounts an immunopathologic immune response and thereby develops disease. not all pathologic interactions between viruses and their target cells/tissues involve direct cell killing and tissue damage, and virus-induced cancer is a serious consequence of some viral infections. while cancers associated with some viral infections are due to indirect effects such as immune suppression associated with hiv infection or chronic inflammation associated with hbv or hcv infection (see above), there are also examples of situations where viral infection directly promotes tumor development. several gamma herpes viruses, including the human gamma herpes viruses, epstein barr virus (ebv) and kaposi sarcoma-associated herpesvirus (kshv), are associated with lymphoproliferative disorders and cancer in humans. both of these viruses encode a number of proteins, such as ebv ebna and lmp or kshv lana- and v-cyclin that are associated with cellular transformation and cancer (reviewed in cesarman, ) . likewise, human papilloma viruses (hpv), which can cause several types of cancer, including cervical cancer, encode several proteins, including viral e and e proteins, that interfere with cell cycle progression checkpoints and promote cellular transformation (korzeniewski et al., ) . though infection with any of these viruses has the potential to cause cancer, as is the case with most aspects of viral pathogenesis, there are multiple additional factors that determine whether an individual is at risk for development of disease. in the case of ebv, since the viral proteins are recognized by the host immune response, individuals with healthy immune systems are at much lower risk of developing ebv associated cancers (reviewed in cesarman, ) . likewise, in the case of hpv infection, there are multiple hpv genotypes and the risk of developing hpv associated cervical carcinoma is much greater with certain high risk hpvs, such as hpv or (korzeniewski et al., ) . therefore, like other aspects of viral pathogenesis, a complex series of virus-host interactions determines whether infection with cancer associated viruses ultimately results in disease development. viral pathogenesis represents a complex series of interactions between viruses and the host that determine whether the virus will successfully establish infection within the host and if so, whether this infection will result in virus-induced disease. as discussed above, though the pathogenesis of each virus is unique, there are several stages in the viral life cycle that are shared by all pathogenic viruses which illustrate common themes in viral pathogenesis. however, it is important to remember, that within each of these common stages, there is tremendous variation in how each virus interacts with the host, and that these complex interactions are ultimately what determine whether a viral infection results in disease. isolation of ross river virus from epidemic polyarthritis patients in australia cc ckr : a rantes, mip- alpha, mip- beta receptor as a fusion cofactor for macrophage-tropic hiv- synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice sars-cov and emergent coronaviruses: viral determinants of interspecies transmission genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss causes of and prevention strategies for hepatocellular carcinoma gammaherpesvirus and lymphoproliferative disorders in immunocompromised patients immunopathogenesis and immune modulation of venezuelan equine encephalitis virus-induced disease in 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phenotype of wild-type and mutant herpes simplex viruses in vivo angiotensin-converting enzyme is a functional receptor for the sars coronavirus macrophage-induced muscle pathology results in morbidity and mortality for ross river virus-infected mice homozygous defect in hiv- coreceptor accounts for resistance of some multiply-exposed individuals to hiv- infection sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism trim and the regulation of hiv- infectivity tripartite-motif proteins and innate immune regulation complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease characterization of ross river virus tropism and virus-induced inflammation in a mouse model of viral arthritis and myositis rig-i-like receptors: sensing and responding to rna virus infection natural resistanceassociated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts picornavirus-receptor interactions contribution of t cells to mortality in neurovirulent sindbis virus encephalomyelitis alpha/ beta interferon protects adult mice from fatal sindbis virus infection and is an important determinant of cell and tissue tropism type i ifn controls chikungunya virus via its action on nonhematopoietic cells interferon-stimulated genes and their antiviral effector functions herpes simplex virus: receptors and ligands for cell entry the cytoplasmic body component trim alpha restricts hiv- infection in old world monkeys species-specific variation in the b . (spry) domain of trim alpha determines the potency of human immunodeficiency virus restriction cd : its structure, role in immune function and aids pathogenesis, and potential as a pharmacological target microrna silencing and the development of novel therapies for liver disease influenza a and b viruses expressing altered ns proteins: a vaccine approach cd (þ) t cells mediate viral clearance and disease pathogenesis during acute hepatitis b virus infection neurovirulent strains of alphavirus induce apoptosis in bcl- -expressing cells: role of a single amino acid change in the e glycoprotein the influenza virus protein pb -f inhibits the induction of type i interferon at the level of the mavs adaptor protein role of alpha/beta interferon in venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the ' untranslated region reovirus mu protein inhibits interferon signaling through a novel mechanism involving nuclear accumulation of interferon regulatory factor key: cord- -f ab authors: barr, j.n.; fearns, r. title: genetic instability of rna viruses date: - - journal: genome stability doi: . /b - - - - . - sha: doc_id: cord_uid: f ab despite having very limited coding capacity, rna viruses are able to withstand challenge of antiviral drugs, cause epidemics in previously exposed human populations, and, in some cases, infect multiple host species. they are able to achieve this by virtue of their ability to multiply very rapidly, coupled with their extraordinary degree of genetic heterogeneity. rna viruses exist not as single genotypes, but as a swarm of related variants, and this genomic diversity is an essential feature of their biology. rna viruses have a variety of mechanisms that act in combination to determine their genetic heterogeneity. these include polymerase fidelity, error-mitigation mechanisms, genomic recombination, and different modes of genome replication. rna viruses can vary in their ability to tolerate mutations, or “genetic robustness,” and several factors contribute to this. finally, there is evidence that some rna viruses exist close to a threshold where polymerase error rate has evolved to maximize the possible sequence space available, while avoiding the accumulation of a lethal load of deleterious mutations. we speculate that different viruses have evolved different error rates to complement the different “life-styles” they possess. viruses are enormously successful. they have been identified in organisms within all domains of life. despite decades of scientific effort to combat viruses that cause disease in humans and economically important crops and animals, there are relatively few cases in which we have succeeded. viruses have shown they are able to adapt and multiply to overcome almost any obstacle that is imposed on them. this remarkable adaptability can be attributed to their extremely high replication rate and their propensity for mutation. this is particularly true of the viruses that have rna genomes: the riboviruses and retroviruses. this chapter will focus on these rna viruses and on the exciting research that has provided valuable insight into how rna viruses benefit from their genetic variability. in the first two sections of the chapter, two fundamental concepts are introduced: the intimate relationship between rna viruses and their hosts, and the idea that viruses behave as quasispecies. having introduced these concepts, the remainder of the chapter discusses the viral and host mechanisms that govern rna virus genetic variability and the ability of viruses to withstand mutation. we then discuss evidence that at least some rna viruses have a replication fidelity that is poised to maximize genome sequence space without incurring catastrophic lethal mutations and describe how this can be exploited to control viral infections. throughout the chapter, we attempt to convey the diversity of rna virus biology and mutation frequency and we conclude by speculating that each rna virus has evolved an error rate that complements its genome replication strategy and mode of transmission. rna viruses are very simple entities with small genomes that vary in length from about to kb, depending on the virus. thus, they have very limited coding capacity and so, similarly to dna viruses, they are obligate intracellular parasites, depending on a host cell to provide energy generating systems, ribo-and deoxyribonucleotides, cellular translation machinery, trnas and amino acids to translate their mrnas, cellular enzymes to posttranslationally modify their proteins, and cellular structures such as membranes, vesicular compartments, and/or cytoskeleton networks to act as a scaffold for assembling and transporting components required to make virus particles. there are many rna viruses and they vary enormously in their genome structures and mechanisms of replication. however, in its most distilled and generic form, the rna virus infection cycle consists of the steps shown in fig. first a protein on the surface of the virus particle attaches to a receptor molecule on a host cell enabling the viral genome to be delivered into the cell. the genome is expressed to produce viral proteins and replicated multiple times to produce progeny genomes. the progeny genomes are packaged with the proteins that make up the virus particle and are released to infect new cells. thus, viruses multiply by a process of genome replication, expression, and assembly, rather than division, and a cell infected by a single infectious virus particle could release thousands of progeny virions in a matter of hours. this enables viruses to multiply very rapidly and to achieve large population sizes. because viruses depend on a host cell to be able to replicate, their ability to multiply is heavily influenced by the biology of each cell that they encounter, such as the nature and density of surface molecules that can act as viral receptors, the cell's metabolic rate and availability of macromolecules, as well as the cell's innate antiviral defenses that have evolved to suppress viral replication. in addition to being able to replicate within a single cell type, most viruses require the capacity to replicate and spread within a multicellular host organism, which has tissues with varied cellular characteristics, physiological and anatomical constraints, and an adaptive immune response. while some viruses might only require the ability to infect one tissue to be successfully maintained in the environment, some viruses need to infect and multiply in different tissues to be spread to a new host and complete their transmission cycles. for example, measles virus initially infects alveolar macrophages and dendritic cells in the lung. it is then transferred to t-and b-lymphocytes and is amplified and spread systemically throughout the body. infected lymphocytes can then transfer the virus to the basolateral surface of lung epithelial cells by attaching to an epithelial cell receptor. the virus multiplies further in the lung epithelium and is spread to new hosts by coughing and sneezing [ , ] . thus, measles virus requires the ability to infect multiple cell types to complete its transmission cycle. viruses must also be capable of replicating within populations of hosts whose immune responses are shaped by different histories of virus exposure and some viruses even require the ability to replicate in different host species. for example, west nile virus transmission is dependent on the virus being able to replicate efficiently in both mosquitos and birds. in mosquitos the virus multiples in the salivary glands and is transmitted to birds when the mosquito takes a blood meal. the virus is amplified in virus. an rna virus particle, or virion, consists of an rna genome (blue) surrounded by a protein coat or capsid (black). some viruses also have a lipid envelope studded with viral proteins surrounding the capsid (not shown). the virus particle attaches to a receptor on the surface of a susceptible host cell ( ), and becomes internalized ( ) . the viral genome codes for viral proteins (black shapes) ( ) and is replicated via a replication intermediate (red) ( ) . newly synthesized genomes and proteins assemble together ( ) and newly made virus particles are released ( ) . birds and can be transferred to further mosquitos [ ] . because rna viruses need to replicate in these highly variable and dynamic environments, they need to be highly adaptable to maintain their existence. this adaptability is conferred by their genetic heterogeneity. in the late s it was discovered that the nucleotide sequences of rna bacteriophage, qβ, are highly heterogeneous [ ] , and this observation has since been extended to all rna viruses. accurate quantification of rna virus mutation rates is challenging, but they have been estimated at − to − per nucleotide per round of copying [ , ] . this equates to approximately one mutation per genome replication event, which is a considerably higher rate than that of bacteria, estimated at one mutation per genome replication events [ ] . in addition to point mutations, recombination between viral genomes can occur in high frequency in some rna viruses, resulting in replacement of different regions of genome sequence. any particular rna virus population is always in flux, with new mutations arising and deleterious mutations being lost through selection. the high mutation rate of rna viruses, coupled with their very high levels of replication and the large population sizes that they can achieve means that rna viruses exist as a swarm of variants rather than as a single genotype entity. thus, rna viruses are a genetic paradox: they are in one sense very simple entities, having very limited genetic information, but on the other hand, they are genetically complex, having the capability to access millions of sequence combinations. adding to this complexity, there is evidence for some rna viruses that they can exist as quasispecies in which the related genome sequences can complement each other and function cooperatively [ , ] . thus, when a virus spreads from cell to cell and host to host, it is the properties of a swarm of genetically related but distinct viruses that enables this to occur, not the properties of a single, isogenic virus. as described in detail later in this chapter, rna viruses require a high mutation rate to enable them to survive the varied environments that they encounter in the course of their transmission cycle. interestingly, they also have evolved genome sequences that have a bias that allows them to rapidly adapt. however, there is also evidence that at least some have a mutation rate that is so high that they are poised at the edge of a threshold of viability, with small increases in mutation rate causing them to accumulate so many lethal mutations that they are extinguished. together, these findings suggest that rna viruses have evolved to have a specific mutation rate and mutation bias to enable them to survive in the particular environments in which they need to exist. there are several sources of genetic variability in rna viruses, some are inherent to the biology of the virus and others are consequences of the cellular environment. the viral mutation rate is the rate at which a viral genome acquires mutations per genome replication event and is determined by the viral polymerase and any proofreading activities that the virus encodes. the mutation frequency of a virus is the frequency with which mutations accumulate over a virus infection cycle and can be impacted by the mode of virus replication, and cellular factors. thus, to understand how viral genetic heterogeneity arises, it is helpful to have an appreciation of the mechanisms by which rna viruses replicate their genomes. rna viruses can be divided into different classes by virtue of their distinct genome structures and strategies of genome replication [ ] (fig. . ) . the riboviruses replicate their genomes via an rna intermediate synthesized by a viral rna-dependent rna polymerase (rdrp). riboviruses can have single or double-stranded rna genomes; those with single-stranded genomes can be further characterized by being either positive or negative stranded (ie, having a genome that is of the same sense, or the opposite sense to mrna, respectively). riboviruses can also have genomes contained within a single piece of rna, or a genome that is divided into multiple segments. another class of rna virus is the retroviruses. these viruses have an rna genome, which is reverse transcribed by the viral reverse transcriptase enzyme into double-stranded dna. the virusspecific double-stranded dna then integrates into the host genome and becomes a template for cellular rna polymerase ii, which synthesizes multiple copies of rna to generate the progeny viral genomes. it is important to appreciate that this classification system does not relate in any way to the tissues or hosts that a virus can infect, or the way in which it is transmitted to new hosts. for example, hepatitis c virus (hcv) and west nile virus are both positive-strand rna viruses, but they cause very different diseases and are spread in different ways. both rdrps and reverse transcriptases have the potential to introduce deletions, insertions, and nucleotide mismatches into the nucleic acid product [ ] [ ] [ ] . unlike dna-based life forms, most rna viruses have no mechanisms to identify and repair mismatches [ , ] and so polymerase error is not corrected. the error-prone nature of polymerase activity, coupled with the absence of a proofreading mechanism, is the key reason why rna virus genomes acquire mutations and exist as a swarm of genetic variants. although all rdrps and reverse transcriptases are capable of introducing mutations, they are not equally error prone. for example, the viral mutation rate inversely correlates with genome size, such that viruses with larger genomes have a lower per nucleotide mutation rate than those with small genomes [ ] . this is intuitively logical as a high mutation rate in a virus with a large genome would increase the chance of genomes acquiring a lethal mutation and so viruses with low fidelity polymerases could not be sustained. this suggests that viruses with larger genomes have evolved to limit their mutation rate and some rna viruses encode proteins that function to mitigate polymerase error, as described in the following. however, even when related viruses with similar genome lengths are compared, there are differences in polymerase fidelity [ , ] . for example, in a side-by-side comparison, using in vitro biochemical assays, the rdrp of coxsackievirus b is of higher fidelity than that of poliovirus, even though these are highly related viruses [ ] . in sum, these facts suggest that polymerase error rate is determined by selection pressures related to viral genome size and other facets of virus biology. the molecular mechanisms that govern polymerase fidelity have been elucidated by detailed enzyme kinetics studies of wild-type polymerases and by studying mutant versions of polymerase with altered fidelity [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these studies have shown that the error rate of the polymerase can be modulated by single amino acid substitutions in the enzyme, and that substitutions outside the active site can have an effect. thus, the structure of the polymerase is tuned to enable it to manifest a particular fidelity. in addition to controlling the rate of replication error, polymerase determinants can also influence what substitution mutations are introduced. in a landmark study, a novel sequencing approach was employed to identify low-frequency mutations that accrued in the poliovirus genome under relatively constant conditions [ ] . viral populations present at different times were analyzed to determine what mutations accumulated in this stable environment, where selection pressure was minimized. this analysis showed that transitions occurred more frequently than transversions, and within these categories there was variation: c-to-u and g-to-a transitions accumulated more frequently than u-to-c or a-to-g. thus, these studies indicate that there is directionality to the mutation pattern of the viral swarm. similar findings had been made with hiv [ ] and studies with west nile virus have shown that different polymerase variants have different mutational biases [ ] . thus, rna viruses do not incur substitutions randomly, but have a mutation bias that is likely governed by the molecular determinants of fidelity in the polymerase. this bias might play an important role in allowing the virus to generate a favorable spectrum of sequences following a genetic bottleneck. although the viral polymerase is typically the key viral factor that determines how faithfully the viral genome will be copied, it is not the only factor and there are examples of viruses in which other proteins can come into play to reduce polymerase error. as noted earlier, the genomes of all rna viruses are relatively small compared to those of the largest dna viruses, and it is thought that the high mutation rate of rna virus polymerases imposes an upper limit on genome size. however, there is a wide range of genome sizes within the rna viruses, with the largest being those of the coronaviruses, at up to kb. this is more than twofold longer than most other rna viruses. it has now become apparent that the reason why the coronaviruses can sustain this relatively large rna genome is that they have an rna proofreading activity [ , ] facilitated by an exonuclease that probably functions by removing incorrect insertions at the ′-end of the rna product during rna synthesis [ ] . interestingly, the activity of the exonuclease is significantly enhanced by an additional coronavirus protein [ , ] . the fact that a multipartite complex performs the polymerization and rna proofreading activities raises the intriguing possibility that coronavirus fidelity could be regulated. some of the nonsegmented, negative-strand rna viruses also have an additional protein that might function to limit rdrp error. the pneumovirus subfamily has genomes of approximately kb, and so they are in the midrange of rna virus genome sizes. these viruses encode a small protein called m - . it has been found that deletion of m - of human meta-pneumovirus results in increased accumulation of transitions, transversions, deletions, and insertions in the viral rna, suggesting that m - serves to increase the fidelity of the viral polymerase [ ] . the mechanism by which m - functions is not known, but it has no known enzyme activity and so it is unlikely to function as an exonuclease, but instead might serve to increase fidelity by altering rdrp structure. a deoxyuridine-triphosphatase (dutpase) enzyme is expressed by some, but not all, retroviruses [ , ] . this enzyme hydrolyzes dutp and maintains low dutp:ttp ratios, thus limiting misincorporation of deoxyuridine into viral dna. the viral dutpase has been shown to limit the mutation rate of feline immunodeficiency virus and caprine arthritis-encephalitis virus [ , ] . interestingly, the primate lentiviruses, including hiv, do not encode a dutpase, but might package a cellular dna repair enzyme, uracil dna glycosylate, into their virions to help limit the mutation rate [ , ] . in addition to the mutations that can be introduced when the polymerase selects an incorrect nucleotide during rna synthesis, genetic variation can also arise by recombination. recombination can occur when two or more viral genomes enter the same cell and a part of one genome is incorporated into the other. this can result in significant changes in genome composition with dramatic impact on virus biology. for example, there is evidence that recombination might have been a factor that enabled the emergence of sars coronavirus [ ] and it is a key factor in emergence of pandemic influenza viruses [ ] . however, while recombination can impact diversity, there is debate as to whether it has evolved as a means to generate variability, or is merely a consequence of viral genome replication [ ] . in this respect, it is interesting that even viruses with similar genome structures can undergo different rates of recombination, perhaps suggesting that recombination is also finely tuned by evolutionary pressure. there are three mechanisms by which rna viruses can recombine: templateswitching recombination, nonreplicative recombination, and re-assortment ( fig. . ) . template-switching, otherwise known as copy-choice recombination, can occur during the process of rna synthesis if the viral polymerase transfers from one template to another, while remaining attached to the nascent nucleic acid chain [ ] . this results in production of a mosaic genome. template switching tends to occur between sequences of close similarity to give rise to a homologous recombination event. nonhomologous recombination can also occur, but this typically results in defective genomes and is observed less frequently. viruses differ significantly in the rate with which they can recombine by template-switching [ ] . it can be highly frequent in retroviruses, particularly hiv, and also in coronaviruses. the high frequency of recombination in these viruses may be due to the replication strategies that they have. in the case of retroviruses, the reverse transcription process is highly complex and the reverse transcriptase must switch from one template to another during dna synthesis [ ] . likewise, in coronaviruses, transcription of the genome, to allow gene expression, requires the rdrp to transfer from one site to another on the genomic template [ ] . thus, the fact that the polymerases of these viruses have evolved to transfer to a different template sequence probably means they are more likely to do so during other aspects of rna synthesis. recombination can occur in other positive-strand rna viruses besides coronaviruses, and in double-stranded rna viruses, although the recombination frequency apparently varies between viruses. for example, it occurs frequently in the positive-stranded enteroviruses, such as poliovirus, but less so in the flaviviruses, such as hcv [ ] . template switch recombination is much less frequent in the negative-strand rna viruses [ ] , probably because their genomes are not naked rna, but rather are encapsidated, or buried, in protein called nucleoprotein [ ] . the polymerase transiently displaces nucleoprotein as it moves along the template and only recognizes rna sequences as the nucleoprotein is displaced. this probably prevents the rdrp from transferring from one genome to a similar sequence in another genome to yield functional recombinants. however, negative-strand rna viruses containing gene duplications have arisen naturally [ ] and defective interfering genomes, which contain promoters and partial genome sequences, but not complete genomes, are often detected. these findings suggest that the rdrps of the negative-strand rna viruses are capable of jumping from one sequence to another, or that nonreplicative recombination (described later) can come into play, but perhaps most products of these events are nonviable and so are not detected. in contrast to template switch recombination, nonreplicative recombination seems to be a relatively rare event that to date has only been described for a few positive-strand rna viruses. this mechanism was documented by recovery of viable viruses (or replicative templates) following cotransfection of cells with two viral rna fragments, each of which was unable to function in replication independently. the fragments recombined to form functional rnas [ ] [ ] [ ] [ ] . this mechanism of nonreplicative recombination might not involve a viral enzyme activity. instead, it seems that the two rna strands are joined together either by a transesterification reaction [ ] , or by ligation, presumably by cellular ligases [ ] [ ] [ ] . thus, rna genome fragments created by physical shearing, nuclease cleavage, or cryptic ribozyme activity have the potential to be joined to form a novel viral genome, which can be further refined by homologous recombination to remove duplicated sequence [ ] . re-assortment is a process that can occur during coinfection of a cell with viruses with segmented genomes [ ] . during re-assortment, a virus can exchange one of its own segments for that of another related virus. this process is well studied in influenza virus, in which it occurs frequently. influenza a virus has eight genome segments that all need to be packaged into a virion for that virus particle to be infectious. this process is not completely random; there are packaging signals in the rna segments and epistatic interactions enable the correct complement of segments to be incorporated into virions. there are many subtypes of influenza virus, but if the packaging signals of two viral subtypes coinfecting a cell are sufficiently similar, this enables a segment from one virus to be incorporated into another, resulting in release of virions with a new genome composition. because different viral subtypes have different antigenic properties, this process has significant impact on influenza virus epidemiology [ ] . as described earlier, the mutation frequency of a virus differs from the mutation rate, in that it refers to the accumulation of mutations over a virus infection cycle, for example, from the point of entry of a virus into a cell until release of infectious progeny. in addition to having different genome structures and nucleic acid intermediates, different rna viruses have different numbers of replication events per infection cycle and so this can impact on mutation frequency. retrovirus reverse transcriptase only copies the genome twice during an infection cycle: once to generate cdna from the rna template and a second time to synthesize the complementary dna strand to generate double-stranded dna. thus, these are the only two occasions in the retrovirus infection cycle where the viral polymerase can introduce mutations. the cellular rna polymerase ii enzyme is responsible for generating multiple copies of genome rna that become packaged into viral particles, and while there is the potential for error to be introduced by rna polymerase ii, cellular proofreading mechanisms come into play at this step and so the major source of mutation during retrovirus genome replication is the reverse transcriptase [ ] . in the riboviruses, the viral rdrp is responsible for all genome replication events and it copies the genome multiple times. thus, in this case, there are many more opportunities for mutations to be introduced by polymerase error. within the riboviruses, there are different modes of genome replication, referred to as a stamping machine or geometric modes, and the degree to which a virus employs one mode versus the other will affect mutation frequency [ ] . in stamping machine mode, the infecting genome template is used to make multiple progeny genomes, but these genomes are not used as templates until they have been delivered into another cell. it is thought that double-stranded rna viruses use this mode primarily. in contrast, in geometric replication, an incoming genome template acts as a template to make multiple complementary strands (or antigenomes), which in turn act as templates to make multiple genome sense strands, within the same infection cycle. in this case, there are many more opportunities for mismatch errors to be introduced than in the stamping machine mode. positive and negative sense rna viruses probably use a combination of both modes, but the exact contribution of each to the output virus is not well characterized, except in a few cases [ ] . the mutation rate of the viral polymerase, coupled with the replication mode that the virus employs (and extrinsic factors, described in the following text) will determine the extent of genetic variability of viruses released from an infected cell. the cellular environment can impact virus mutation rates and frequency. for example, dntp pool imbalances can affect retrovirus mutation rates [ ] , and it has been suggested that differences in substitution rates between rna viruses is a consequence of differences in virus rna synthesis rates in different cell types [ ] . in addition to these effects, there are also cellular factors that can result in increased mutation in rna viruses. adenosine-to-inosine modification by enzymes called adenosine deaminase acting on rna (adar) is the most common form of rna base modification that occurs in mammals. a-to-i conversion has important consequences in the coding potential of substrate rnas, as inosine is decoded as a g by polymerases during template copying. the a-to-i conversion in a dsrna duplex also has consequences to stability of rna secondary structures, as the a:i pairing is less stable than a canonical a:u pair. this can have important consequences for rnas that depend on their structure rather than sequence for their function [ ] . adar modification of cellular double-stranded rna was shown to prevent its recognition by the cytoplasmic sensor of nonself rnas that would otherwise lead to chronic activation of innate immune pathways [ ] . there is also evidence that adar can modify viral rnas. sequence analysis of rna virus genomes has revealed that they preferentially accumulate a-to-g transitions, which are characteristic hallmarks of adar activity. measles virus is a negative-stranded rna virus, responsible for an acute disease predominantly in infants, but in rare instances associated with a fatal latent infection of the cns known as subacute sclerosing panencephalitis (sspe). analysis of measles virus genomes from sspe victims has revealed abundant a-to-g transitions, suggesting a role for adar in establishment of sspe [ ] . consistent with an antiviral role for adar, measles virus infection of adar knock-out cell lines displayed increased cellular pathology, and similar findings were reported for other rna viruses, implicating adar as a cellular restriction factor for a wide range of negative-stranded rna viruses [ ] . direct evidence of adar modification of a viral rna genome comes from studies of hepatitis delta virus (hdv). hdv is the smallest of the rna viruses and encodes just two proteins, hdag-l and hdag-s, both of which are essential for virus viability. hdag-l and hdag-s share the same amino terminal open reading frame, but hdag-l possesses a carboxyl terminal extension that is accessed when the stop codon at the end of the hdag-s orf is bypassed. early during infection only the truncated hdag-s is expressed, but then at later times expression of hdag-l increases due to the sitespecific modification of the stop codon by adar [ ] . this editing event is highly specific and is promoted by the highly secondary structured hdv rna genome. this action by adar is clearly proviral, in that without the activity of adar, no infectious hdv particles would form. another family of cellular factors that can modify the sequence of viral genomes is the apobec family of enzymes. these comprise an extensive arm of the innate immune system [ ] . they are responsible for the modification by deamination of cytosine residues to uracil, which is an activity largely performed on single-stranded dna substrates, leading to the phenomenon of hypermutation. apobec activity can affect the retroviruses. hiv infection is blocked by apobec, unless it expresses the viral infectivity factor (vif). the mechanism for this blockade relies on the packaging of multiple apobec family members within hiv virions, which can act on the hiv genome once it has been copied by reverse transcriptase into a complementary dna. the effect of apobec activity can be the modification of up to % of susceptible cytosine residues, resulting in a drop in infectivity of up to -fold. together, the studies described earlier show that there is a range of viral and host factors that combine to alter mutation frequency. the question that arises is: how do rna viruses withstand mutation? the ability of a genome to withstand genetic or environmental perturbations without a change in phenotype is referred to as genetic robustness [ ] . the high mutation rate that rna viruses incur comes at a cost. it has been estimated that - % of virus genomes generated during infection are defective [ ] and so at an individual level, most viral genomes are not robust. this is not surprising: the small size of rna virus genomes limits their coding potential and so they have limited genetic redundancy. moreover, rna virus are highly compact, often containing overlapping reading frames, and nucleotide sequences that function at the rna level, for example, as cis-acting elements that enable genome replication, as well as at the protein level. however, robustness is influenced by the genetic background in which it operates and so in the case of rna viruses, genetic robustness is considered in the context of the viral swarm, rather than individual genotypes. rna viruses are not all equally robust, and even closely related viruses can exhibit different degrees of robustness [ ] . there are several factors that contribute toward this [ , ] which are described in the following paragraphs. robustness is conferred if a virus has an ability to more readily arrive at a new optimal or adapted genotype in the face of a changed environment, and the genetic composition of the viral swarm can facilitate this. because the majority of nucleotide changes in rna virus genomes are either strongly deleterious or lethal, the population is perpetually refined as deleterious genomes become purged through selection, leaving only mutations with phenotypically neutral or advantageous consequences to persist [ , ] . the neutral mutations can impact robustness. an explanation for this is that if the virus encounters a new environment, multiple nucleotide changes might need to occur for it to arrive at an optimal genotype. if some of these changes are already in place, then the jump to the new genotype is more likely to occur. this means that a population that includes a high proportion of neutral mutations will be more adaptable in the face of environmental change, as genomes with neutral mutations can act as stepping-stones toward reaching the new adapted genotype [ ] (fig. . ) . thus one viral determinant of robustness is their high mutation frequency, which results in a more extensive neutral network [ , [ ] [ ] [ ] . consequently, factors that affect mutation frequency, such as polymerase fidelity and replication mode, will also impact robustness. interestingly, there is evidence that some rna virus genomes have evolved to enable rapid adaptation. experiments in which synonymous mutations were introduced into rna virus genomes and fitness was assessed showed that the rna nucleotide sequence has an effect on fitness, independently of its effect on protein sequence [ ] . this could be due to effects on rna structures and cis-acting elements. however, experiments with poliovirus showed that this might not be the only explanation. in this case, a region of the poliovirus genome that does not contain cis-acting rna structures was recoded with synonymous mutations. the virus variant containing the synonymous mutations had reduced robustness and was attenuated in an animal model [ ] . this finding suggests that wild-type poliovirus occupies a sequence space that enables it to rapidly adapt to environmental pressure. another viral determinant of robustness relates to the ability of rna viruses to generate large numbers of genomes within individual infected cells. a consequence of the resulting polyploidy is that a genome containing a detrimental change can be complemented by the properties of another genome that is unaltered. this mechanism also has a downside in that it reduces the ability to purge poorly adapted genotypes, and thus their persistence in a population may lead to a reduction in its overall fitness. interestingly, the huge range in the extent of polyploidy that occurs throughout the infection cycle may allow different levels of robustness at different times of the virus life cycle, with more opportunity for complementation at later stages of infection when the copy number of viral genomes is at its highest. such a scenario may have important consequences for viruses that stimulate innate immunity early on in the infection cycle. the innate immune response poses a high adaptive requirement at a time when viral genome numbers are at their lowest. conversely, persistent viruses that maintain high copy numbers for extended periods of time without inducing cell death, such as hcv, may be particularly robust due to the wide range of genotypes contained with the massive population of persisting rnas. the presence of multiple genomes within the same cell can also enable recombination. recombination is another factor that influences robustness, as it can result in purging of multiple mutations from a genome in a single recombination or re-assortment event [ ] . rna virus robustness can also be impacted by host cell factors. the ability of chaperones to buffer mutations was first proposed for the groel molecular chaperone [ ] . subsequently it has been experimentally observed that chaperones, such as members of the heat shock protein and families, play important roles in the infection cycles of many rna viruses. it has been proposed that viruses have evolved the ability to interact with chaperones in order to buffer the effects of deleterious coding mutations that would otherwise prevent their correct folding [ , ] . this provision is particularly important as viruses depend on assembly of high-order multimers to build their capsids, a major component of the virions that are released. in these cases, a single misfolded protein has the potential to disrupt the function of the entire complex and so mechanisms that facilitate appropriate protein folding can have a significant impact. although there are a number of properties of rna viruses that contribute to genetic robustness, the role of robustness in the natural history of rna viruses is a controversial topic. a virus population with increased neutral genotypic diversity and thus high robustness can readily adapt to new environments due to its inherent diversity, and increased availability of adaptive pathways. this has important implications for viral pathogenesis and robustness has been shown to increase virulence in host organisms [ ] . however, it appears that the converse can also be true and under certain conditions the neutral network can be composed of genotypes that are unable to reach a high level of fitness in the new environment [ ] . this suggests that it may be difficult to make generalizations over how robustness shapes virus adaptability. as mentioned at the beginning of this chapter, genetic heterogeneity of rna viruses is such a key facet of their biology that it brings up the question of whether their high mutation rates have been selected for and are of evolutionary benefit. fidelity comes at the price of elongation efficiency [ ] . thus, it is possible that the high mutation rates of rna viruses are simply a consequence of polymerases that are under selective pressure to replicate genomes very rapidly to ensure efficient viral infection [ ] [ ] [ ] . according to this view, rna viruses have evolved a balance between rapid genome synthesis and error, such that the mutations that they incur are tolerable and on occasion advantageous, but are not necessary for virus survival. however, while genome synthesis rate is certainly an important factor in virus fitness [ ] ; for some viruses there is also evidence that the high mutation rate is beneficial and that rna virus polymerase fidelity is tuned, enabling the virus to maximize sequence space while avoiding the accumulation of so many deleterious mutations that the genomes become nonviable. this is the concept that rna viruses are "on the edge." in this example, the green mutation alone is deleterious, but is neutral or beneficial in combination with the red mutation. if the neutral network contains genomes with red mutations, it provides a stepping-stone to enable introduction of the green mutation. (c) a neutral network containing genomes that have different codons for the same amino acid can provide a stepping-stone to genomes containing different spectra of amino acids following a single nucleotide substitution. in this example, a neutral network contains genomes coding for leucine at a given position, but the genomes differ by coding for leucine with either uua or cua. this expands the range of amino acids that could arise following a single nucleotide change. as described earlier, most mutations that arise are deleterious and so there is a significant cost to having an error-prone polymerase. furthermore, while complementation between defective genomes can occur, enabling genetic robustness, it is also possible for defective genomes to have an antagonistic effect, for example, by expressing mutant proteins that function as dominant negatives. nonetheless, despite these disadvantages, it is possible to generate mutant viruses that have changes in the polymerase that result in its increased accuracy; these are known as high-fidelity mutants. elegant studies performed with a poliovirus high-fidelity mutant showed that efficient spread within a host requires a quasispecies, and an error-prone rdrp to generate it [ , ] . naturally, poliovirus replicates in the gut, but it can replicate in other tissues and spread to the spinal cord and brain. the ability to infect this variety of tissues requires poliovirus to overcome significant barriers to replication [ ] . experiments comparing the growth characteristics of wild type and a variant of poliovirus with a highfidelity rdrp showed that the high-fidelity variant could replicate relatively efficiently compared to the wild-type virus in a single multiplication cycle in cell culture [ , ] , and if introduced into mice intravenously, it could replicate efficiently in the spleen, kidney, and small intestine [ ] . thus, in this case, genome replication was not significantly delayed by the increased accuracy in rna synthesis. however, in contrast to wild-type poliovirus, this high-fidelity mutant virus could not efficiently spread to the central nervous system (cns), hence the % lethal dose (ld ) was increased -fold [ , ] . to examine if the defect in virus spread was due specifically to the mutation (perhaps this variant of the rdrp could not function in a neuronal environment), or to the lack of genome diversity within its population, vignuzzi and coworkers increased the diversity of the high-fidelity virus by treating it with mutagens. this had the dramatic effect of increasing the ability of the high-fidelity virus to replicate in the spinal cord and brain, and the ld was restored to the same level as wild-type poliovirus. this result showed that poliovirus spread to the cns is dependent on the virus being able to establish a highly diverse population. in addition, it was shown that coinfection of mice with wild-type and high-fidelity mutant virus enabled the high-fidelity virus to reach the brain [ ] . this indicates that different viral genotypes in the quasispecies can complement each other to facilitate infection spread. it is not known exactly how complementation functions in this case, but it is easy to imagine that one variant of a virus might be more efficient at subverting innate immune defenses (which could impact virus genomes within the same cell and neighboring cells), whereas another variant might express a capsid protein better adapted to bind to a new cell receptor. in its natural context, poliovirus is spread through ingestion of contaminated water and so there is no necessity for poliovirus to be able to spread to the cns to be able to complete its transmission cycle. however, these studies are important because they show that viruses can benefit from polymerase infidelity and a high mutation rate, particularly under conditions where they encounter a change in environment [ , ] . studies with a number of viruses indicate that these findings are widely applicable in rna virology and so it seems likely that rna viruses have evolved a high mutation rate that enables them to rapidly adapt to the dynamic and varied environments in which they exist. the studies described earlier show that rna viruses benefit by having an error-prone polymerase to enable them to adapt to new conditions. however, there is also a cost if the polymerase has mutations that decrease its fidelity, so that the error rate is increased. experiments performed with coxsackievirus b and poliovirus showed that low-fidelity mutants were able to replicate efficiently in cell culture when propagated at high multiplicity of infection (ie, when the population size was large), but were extinguished when the viruses were propagated under low multiplicity conditions, which mimics conditions when a virus first establishes infection in a host or when it has overcome a barrier, such as adaptation to a new host cell type. consistent with these findings, both the coxsackievirus b and poliovirus low-fidelity mutants were attenuated in vivo. in the case of the coxsackievirus b , low-fidelity mutants were unable to establish productive infection in the heart, the usual site for coxsackievirus b virus replication, and in the case of poliovirus they were unable to reach the cns [ , ] . comparison of the high-and low-fidelity poliovirus variants indicates how much latitude there is in the mutation rate for this virus. the high-fidelity rdrp had an approximately twofold decrease in nucleotide misincorporation rate, and the low-fidelity rdrp had a twofold increase [ ] . thus, the range in misincorporation rate that can lead to virus extinction in an animal host is not that substantial, even in a virus that is relatively genetically robust. this indicates that the fidelity of the polymerase, coupled with the impact that accuracy has on the rate of rna synthesis, is optimized to enable viruses to adapt to the many environments in which they need to exist while avoiding extinction [ ] . the propensity that rna viruses have for mutation seems to have opened this up as an avenue for host cell defense. pathogenic viruses and their hosts are engaged an epochal "arms race" in which the host evolves immune defenses to suppress virus infection and the virus in turn evolves countermeasures to disable host defenses. the existence of apo-bec and adar, cellular proteins that can increase virus mutation frequency, suggests that mammalian hosts have taken advantage of the high mutation rate of viruses and evolved mechanisms to induce further mutations in the viral genomes and push viruses toward extinction [ ] . conversely, primate lentiviruses have evolved vif, a protein that can target apo-bec for proteosomal degradation, indicating that these retroviruses have evolved a mechanism to counter this cellular defense [ , ] . likewise, the nonsegmented, negative-strand rna viruses, which are susceptible to adar, maintain their genomes encased in a ribonucleoprotein complex throughout the infection cycle, reducing the opportunity for them to adopt double-stranded rna structures, the substrate for adar. this perhaps prevents adar causing as much damage as it otherwise might. the high mutation rate of rna viruses has often been an impediment to drug and vaccine development as viruses can rapidly gain resistance to antiviral drugs and to the immune response elicited by vaccines. however, our increasing understanding of function and consequences of genetic variability has opened new avenues for controlling viral infection. as described earlier, small decreases in polymerase fidelity can have dramatic effects on viral infectivity. similarly, studies have shown that small increases in viral mutation rate caused by treatment with mutagenic compounds can result in significant decreases in viral fecundity [ , ] . thus, treatment with mutagens that increase the accumulation of mutations in the viral genome can lead directly to virus extinction, or can reduce virus infection to enable effective clearance with other inhibitors, given in combination, or by host immune responses [ , ] . the identification of high-fidelity mutant viruses that can infect animals has also suggested a means to exploit these mutants as vaccine candidates. live-attenuated virus vaccines can be highly effective, but have the disadvantage that they can potentially revert to a wild-type pathogenic phenotype. by engineering recombinant viruses with increased fidelity, it is possible to generate viruses that are attenuated, as described earlier, and that elicit protective immune responses, with reduced risk of reversion [ ] . the rna viruses are hugely diverse, not only in their genome structures and replication strategies, but also in their "lifestyles," which can differ significantly, even between closely related viruses. what has emerged from studies of virus genetics is that rna viruses are also highly divergent in terms of their polymerase fidelity, recombination rates, replication modes, and genetic robustness. we speculate that rna viruses have evolved such that there is an intricate balance between these factors that is tuned to match the "lifestyle" of each virus, enabling it to occupy the niche in which it exists. there is some evidence to support this idea. for example, a side-by-side comparison of influenza virus and hiv polymerase fidelity showed that influenza virus rdrp is much less error prone than hiv reverse transcriptase. this may be a reflection of the fact that the influenza virus rdrp performs many more genome replication events during an infection cycle than the hiv reverse transcriptase and needs to be less error prone to avoid having a mutation frequency that is too high [ ] . another example comes from studies of west nile virus. while the fidelity of the west nile virus rdrp has not been directly compared to that of other viruses, there is a greater difference in fidelity between the wild-type west nile virus rdrp and a high-fidelity mutant than has been found for most rdrps [ ] . this could suggest that west nile virus rdrp is naturally more error prone than most. this could be a necessary feature of west nile virus to enable it to cycle back and forth between mosquito and avian hosts. understandably, much of the work that has been performed so far has focused on viruses that are "model" viruses-those that are relatively easy to culture in vitro and replicate rapidly. however, a fuller understanding of how the factors that influence genetic diversity intertwine with virus biology will come from extending the work that has been performed so far and applying it to other viruses that have similar genome structures and replication strategies, but diverse lifestyles, such as west nile virus and hcv, or vesicular stomatitis virus and measles virus. research in this area will potentially be transformed by new sequencing techniques, such as cirseq, which can detect low-level genetic variants above the background of errors introduced during rna sequencing [ ] , and base-seq, a method for obtaining long stretches of sequence that can be used to identify haplotypes [ ] . ultimately, application of cutting-edge sequencing technologies, mathematical analyses, and virology studies to a range of viruses will enhance our understanding of the genesis and functional consequences of rna virus genetic instability. complementation the ability of the products of one viral genome to provide a function that cannot be performed by the products of another viral genome. copy-choice recombination a recombination event that occurs when the viral polymerase switches to another template while remaining attached to the nascent rna. also known as template switch recombination. epistatic mutation a phenomenon in which mutations have different effects in combination than individually. fidelity the accuracy with which the polymerase copies the template. a high-fidelity polymerase will make fewer errors than a low-fidelity polymerase. genetic robustness the degree to which a genome can withstand environmental or genetic perturbation. geometric mode a mode of genome replication in which the newly synthesized genomes become templates for further rounds of genome replication during the infection cycle. infection cycle the cycle of events by which an infectious virus particle infecting a cell results in release of virus progeny. in the virology field, this is often referred to as the virus replication cycle, but infection cycle was used here to avoid confusion with genome replication. lethal dose (ld ) the quantity of infectious virus that is required to cause death in % of inoculated hosts. live-attenuated virus vaccine a vaccine that consists of a live (infection-competent) virus that contains mutations that reduce the disease symptoms, usually by impairing its ability to efficiently complete its infection cycle. mutation rate the rate at which a viral genome acquires mutations per genome replication event. mutation frequency the frequency at which a viral genome acquires mutations per viral infectious cycle. this frequency could be affected by cellular factors and the mode of viral replication, as well as by polymerase fidelity. nonreplicative recombination a recombination event in which two rna fragments are joined together by either a trans-esterification reaction, or ligation by cellular ligases. persistent virus a virus that can infect a host and maintain the infection for extended periods of time. hiv and hcv are examples of persistent viruses. polyploidy the presence of multiple viral genomes within the same cell. quasispecies a collection of closely related viral genomes, genetically linked through mutation, that compete within a highly mutagenic environment, interact cooperatively, and collectively contribute to the population phenotype. re-assortment a recombination event that can occur with viruses with segmented genomes, in which a genome segment from one virus is packaged into a virus particle in place of a genome segment from another virus, thus producing a virus with a novel complement of genome segments. retrovirus a class of rna viruses that replicate their genomes via a double-stranded dna intermediate. reverse transcriptase a viral enzyme encoded by retroviruses that is responsible for generating a double-stranded dna copy of the viral rna genome. ribovirus rna viruses that replicate their genomes via an rna intermediate. rna-dependent rna polymerase a viral enzyme encoded by riboviruses that is responsible for generating the viral genome rna and the rna replication intermediates. rna virus a virus that carries a genome composed of rna in the virus particle. stamping machine mode a mode of genome replication in which the incoming genome is reiteratively used as a template to produce multiple copies of replication product. swarm a population of closely related viruses, connected through mutation, similarly to a quasispecies. we have used the term swarm in many instances here because a population of virus variants might not always fully fulfill the definition of quasispecies. synonymous mutation a nucleotide substitution that does not result in an amino acid change. template switch recombination a recombination event that occurs when the viral polymerase switches to another template while remaining attached to the nascent rna, also known as copy-choice recombination. transmission cycle the cycle of events by which a virus is transmitted from one host to another host in the same species. the pathogenesis of measles nectin is the epithelial cell receptor for measles virus the global ecology and epidemiology of west nile virus nucleotide sequence heterogeneity of an rna phage population rates of spontaneous mutation viral mutation rates viral quasispecies rna virus populations as quasispecies expression of animal virus genomes fidelity of hiv- reverse transcriptase the accuracy of reverse transcriptase from 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mutator strains are attenuated in vivo back to the future: revisiting hiv- lethal mutagenesis structure-activity relationships and design of viral mutagens and application to lethal mutagenesis hiv accessory proteins versus host restriction factors rna virus error catastrophe: direct molecular test by using ribavirin lethal mutagenesis of hiv with mutagenic nucleoside analogs viral error catastrophe by mutagenic nucleosides therapeutically targeting rna viruses via lethal mutagenesis engineering attenuated virus vaccines by controlling replication fidelity biochemical characterization of enzyme fidelity of influenza a virus rna polymerase complex base-seq: a method for obtaining long viral haplotypes from short sequence reads key: cord- -w pzdc authors: tsai, kevin; cullen, bryan r. title: epigenetic and epitranscriptomic regulation of viral replication date: - - journal: nat rev microbiol doi: . /s - - - sha: doc_id: cord_uid: w pzdc eukaryotic gene expression is regulated not only by genomic enhancers and promoters, but also by covalent modifications added to both chromatin and rnas. whereas cellular gene expression may be either enhanced or inhibited by specific epigenetic modifications deposited on histones (in particular, histone h ), these epigenetic modifications can also repress viral gene expression, potentially functioning as a potent antiviral innate immune response in dna virus-infected cells. however, viruses have evolved countermeasures that prevent the epigenetic silencing of their genes during lytic replication, and they can also take advantage of epigenetic silencing to establish latent infections. by contrast, the various covalent modifications added to rnas, termed epitranscriptomic modifications, can positively regulate mrna translation and/or stability, and both dna and rna viruses have evolved to utilize epitranscriptomic modifications as a means to maximize viral gene expression. as a consequence, both chromatin and rna modifications could serve as novel targets for the development of antivirals. in this review, we discuss how host epigenetic and epitranscriptomic processes regulate viral gene expression at the levels of chromatin and rna function, respectively, and explore how viruses modify, avoid or utilize these processes in order to regulate viral gene expression. although all human cells in a given individual contain the same genome, they differ widely in the genes they express and, hence, in their biological properties. it is therefore well established that gene expression is not determined simply by the sequence information that is encoded into genomic dna, but rather is subject to multiple levels of control. this review discusses two complementary regulatory mechanisms that act, respectively, at the dna and rna levels. in the case of the host-cell genome, the level of expression of specific genes is regulated not only by flanking promoters and enhancers but also by how accessible the genes embedded within cellular chromatin are to transcription factors. this accessibility in turn is regulated by processes that include dna methylation, histone remodelling, alternative histone variant usage and the deposition of modifications on histone tails, collectively referred to as epigenetic gene regulation , (box ). similarly, the function of the mrnas transcribed from a given gene is regulated not only by their nucleotide sequence but also by a range of covalent modifications that are added to individual nucleotides, the most prevalent of which is methylation of the n position of adenosine (m a) (refs , ). these rna modifications -which have been reported to regulate the stability, the translation and even the immunogenicity of rna molecules -are referred to as epitranscriptomic modifications. epigenetic and epitranscriptomic gene regulation likely initially evolved as means of regulating cell growth and differentiation. however, they are also relevant to several important diseases, including cancer, in which both processes have been reported to be dysregulated [ ] [ ] [ ] . their importance for the regulation of viral gene expression has only recently begun to emerge. as obligate intracellular parasites, viruses misappropriate parts of the host-cell machinery in order to allow the expression of viral genes. this dependence on cellular gene regulation pathways can, however, also lead to viral gene expression being subject to host repression. indeed, epigenetic repression has been proposed to function as a form of antiviral restriction used by host cells as an innate immune defence against dna viruses, in a tug of war that is only revealed when viral countermeasures are experimentally removed . alternatively, viruses may repurpose epigenetic repression in order to suppress their own gene expression, as a way to establish latent infections and prevent the production of immunogenic viral proteins . by contrast, epitranscriptomic modifications generally enhance the function of viral mrnas, and both dna and rna viruses have therefore evolved to maximize the level of these modifications on their transcripts. in this review, we discuss epigenetic repression mechanisms, including histone tail modifications and chromatin a dna-protein complex consisting of genomic dna wrapped around organizing proteins called histones (see box ) . the addition of a small chemical group called a methyl group (ch ), found as a chemical modification on dna, rna and proteins (in particular, histones). a viral life-cycle stage in which the viral genomic material persists long-term in the host cell with minimal viral gene expression and replication. alternative histone variants that are loaded onto viral dna upon entry into host-cell nuclei and how viruses avoid or even utilize this repression to enhance viral replication or persistence. we then explore the epitranscriptomic rna modifications, including methylation and acetylation, found on viral transcripts, and how they enhance viral gene expression or help evade innate immune detection. we highlight the importance of non-encoded regulatory information found on viral chromatin and rna and discuss how the regulatory pathways that are influenced by this information could serve as novel targets for antiviral therapies. epigenetic repression and its avoidance incoming viral dna molecules are sensed by target cells and are rapidly loaded with histones bearing heterochromatic marks, thus inhibiting viral gene expression. how cells distinguish viral dna from their own remains undefined, though epigenetic repression of foreign dna seems mainly to be orchestrated by proteins associated with the pro-myelocytic leukaemia nuclear bodies (pml-nbs) and by the innate immune dna sensor interferon-inducible protein (ifi ) , - . here we discuss how herpesviruses, hepatitis b virus (hbv) and retroviruses can be epigenetically repressed and how these viruses can disrupt or avoid repression in order to initiate productive infections. we also discuss how epigenetic repression can be misappropriated to establish viral latent infections. epigenetic suppression of viral dna is frequently associated with nuclear protein aggregates called pml-nbs or nuclear domain (nd ) ( fig. a ). pml-nbs consist of cage-like structures constructed of pml proteins, which contain other effector proteins that together appear as punctate foci when visualized by immunofluorescence microscopy [ ] [ ] [ ] . several dna viruses, including simian virus , adenovirus type , herpes simplex virus (hsv- ) and human cytomegalovirus (hcmv), localize to and establish viral replication sites adjacent to pml-nbs, suggesting that this structure is at least spatially, if not functionally, related to viral replication . through the recruitment of cofactors, pml-nbs can be associated with many cellular functions, including antiviral defence and transcriptional repression. the expression of the pml-nb components sp and pml increases in response to interferon signalling, and pml-nb components including pml, sp , daxx and atrx suppress viral replication , [ ] [ ] [ ] [ ] . daxx and sp also have transcriptional repressive functions, likely through recruiting co-repressors such as hp and hdac ii [ ] [ ] [ ] [ ] . moreover, daxx and atrx can load the histone variant h . onto heterochromatic regions , . all of the functions above implicate box | relationship of chromatin architecture and histone modifications to gene expression the genomic dna of cells is bound by basic proteins called histones, with every ~ bp of dna wrapped around a core octamer containing two molecules apiece of the histones h a, h b, h and h , to form chromatin (see the figure) . although chromatin can protect genome integrity, histones can also hinder the accessibility of genomic dna for transcription. this accessibility is a function of the degree of compaction of the histones bound to that genomic dna, with open regions being referred to as euchromatin, whereas tightly compacted regions of dna are referred to as heterochromatin. chromatin compaction constitutes a form of gene regulation not directly encoded in the dna nucleotide sequence itself; this is termed epigenetic gene regulation, as it is the molecular mechanism underlying epigenetic inheritance . epigenetic gene regulation can take the form of dna modifications, histone tail modifications, chromatin remodelling and alternative histone subunits (see the figure) . dna modifications mainly involve cpg methylation, in which a methyl group is added to deoxycytidine residues located in cg-rich genomic regions. cpg methylation is primarily associated with repressive heterochromatin. histone tail modifications -including methylations, acetylations, phosphorylations and ubiquitylations -are the best understood form of epigenetic regulation. examples have been found on all four canonical histones (h a, h b, h and h ). h and h acetylations are associated with active euchromatin, whereas methylations are more diverse in function. for example, h lysine and lysine tri-methylation (h k me and h k me , respectively) mark heterochromatin, whereas h k me marks are found on actively transcribed euchromatin . chromatin remodelling involves atp-dependent histone chaperones that may slide, evict or load histones, modulating the local density of histones on the chromatin. finally, chromatin accessibility can be modulated through the use of alternative histones, such as the h a histone variant in place of h , or the h . histone variant as a replacement for h . (ref. ). the canonical h histone variants h . and h . are deposited by the histone chaperone caf- during dna replication. however, histones can be displaced by transcription, and they need to be replaced by the histone chaperone complex hira, which deposits the replacement histone h variant h . . thus, h . previously was thought to mark transcriptionally active chromatin. however, h . later was found also to be loaded by an alternative histone chaperone complex consisting of a heterodimer of daxx and atrx, which loads h . onto repressed heterochromatic regions such as pericentromeric regions and telomeres, suggesting that different chaperones may load histone h . with alternative histone tail marks onto either actively transcribed or repressed chromatin , , . h the addition of a small chemical group called an acetyl group (ch co), found on rna and proteins including histones. a family of highly basic proteins used to organize genomic dna (see box ). epigenetic markers that mark a segment of chromatin for transcriptional repression, which usually coincide with 'closed chromatin' that is less accessible to transcription factors. microscopy assays in which subjects of interest are visualized though specific binding of antibodies labelled with fluorescent dyes. www.nature.com/nrmicro pml-nbs as subnuclear hubs of the epigenetic repression of viral dna. invading viral dna can also be detected by the innate immune dna sensor ifi ( fig. b ). ifi contains an oligomerizing pyrin domain (pyd) and two double-stranded dna (dsdna)-binding hin domains, and it oligomerizes on histone-free segments of dsdna longer than bp . ifi was initially characterized as a dna sensor that induces interferon-β through the signalling factors sting, tbk and irf (ref. ), and it was shown to repress gene expression from transfected plasmids as well as from viruses including herpesviruses, papillomaviruses and hbv , [ ] [ ] [ ] [ ] . interestingly, ifi suppression of hsv- and kaposi sarcoma-associated herpesvirus (kshv) transcription involves the deposition of suppressive h k me marks in an interferon-independent manner, with ifi directly recruiting h k methyltransferases to kshv dna , , . although ifi has a key role in the epigenetic repression of viral dna, it should be noted that ifi function is likely closely connected to the inhibition by pml-nbs, as ifi is known to interact with pml-nb components . herpesviruses. epigenetic repression of viruses is best understood for herpesviruses ( fig. ). herpesvirus dna is packaged into virions 'naked' (that is, free of histones) [ ] [ ] [ ] . upon release into the nucleus, viral dna is detected and then rapidly loaded with histones harbouring suppressive marks, as a form of innate immune response. upon infection of fibroblasts by the alphaherpes virus hsv- , within h viral dna is loaded with histones, mainly consisting of daxx-atrx and hira-loaded h . with repressive h k me marks [ ] [ ] [ ] [ ] . hsv- disarms this repression in two stages. first, the viral protein vp , which is packaged into the tegument layer of incoming virions, recruits host proteins, including host-cell factor (hcf- ) and octamer-binding factor (oct- ), in order to form a complex that recruits the histone demethylases lysine-specific demethylase (lsd ) and jumonji domain (jmjd ) family members as a means to remove repressive h k marks from viral immediate early promoters upon entry into the cell nucleus, the dna of many viruses initiates the replication process adjacent to subnuclear structures called pro-myelocytic leukaemia nuclear bodies (pml-nbs). however, pml-nbs are an aggregation site for many heterochromatic repression proteins, which load repressive heterochromatin onto viral dna that shuts down viral transcription. in the absence of viral de-repression factors, viral episomal dna lacks active histone marks (shown as green histones with the active marks h ac, h ac and h k me ). a | several pml-nb components -including pml itself, sp , smc , smc , daxx and atrx -are involved in the epigenetic repression of viruses, with the daxx-atrx complex having specifically been found to load the histone variant h . bearing repressive marks onto viral dna, leading to the accumulation of heterochromatic marks and blocked transcription (dna is shown associated with red histones with the repressive marks h k me and h k me ). b | the innate immune dna sensor ifi drives an alternative mechanism of antiviral epigenetic repression, promoting repressive methylations on histone tail h k . c | specifically for the retrovirus murine leukaemia virus, np and the human silencing hub (hush) complex have also been shown to deposit heterochromatic marks on unintegrated viral dna. a family of proteins that are expressed by eukaryotic cells upon invasion by viruses, used to signal neighbouring cells to mount an antiviral response through the expression of a variety of interferon-stimulated genes. the space in viral particles between the outer membrane and the inner protein capsid shell; the term is most commonly used in the herpesvirus family, where proteins packaged in this space are termed tegument proteins. a small peptide that can be ligated onto other proteins, best known to mark proteins for degradation through the proteasome. h k me from the entire viral dna genome, ensuring a productive infection . in parallel, icp has also been implicated in the degradation of ifi ( fig. c ). ifi suppresses gene expression from icp -deficient mutants of hsv- , with ifi directly binding viral dna, promoting the deposition of repressive h k me marks and the removal of active h k me marks , . however, the role of icp in counteracting ifi is somewhat controversial, with one study reporting that inhibition of hsv- gene expression by ifi occurs only in the absence of icp , whereas another study reported that ifi is also active against wild-type hsv- (refs , ). this discrepancy may be partially explained by the finding that icp -mediated degradation of ifi is efficient in primary cells yet less so in transformed cell lines such as hela . in this context, the structural maintenance of chromosome and (smc / ) proteins, which colocalize with pml-nbs, act as restriction factors for hbv transcription. in a striking parallel to herpesviruses, the hbx protein recruits the cellular e ubiquitin ligase ddb in order to induce the ubiquitylation and degradation of smc / (refs - ) ( fig. b ). importantly, artificial depletion of the pml-nb components pml and sp using rna interference not only results in the nuclear redistribution of smc but also allows the transcription of hbv cccdna in the absence of hbx. thus, hbv, like herpesviruses, disrupts pml-nbs as a means to circumvent the epigenetic silencing of viral dna. retroviruses. as we argued above, host cells can recognize incoming viral dna as targets for epigenetic silencing. whereas herpesviruses and hbv use viral proteins to disperse or degrade pml-nb components and so prevent silencing, retroviruses appear to avoid proviral dna silencing through the alternative strategy of chromosomal integration ( fig. d ). in the absence of integrase function, the potency of epigenetic repression becomes apparent, as unintegrated retroviral dna is poorly transcribed , . retroviral particles contain rna genomes, which are reverse transcribed in the cytoplasm. like herpesviral dna, retroviral dna enters the nucleus 'naked' and is rapidly loaded with histones , . specifically, unintegrated hiv- dna is loaded with the histone variant h . bearing the repressive h k me mark and only low levels of the active mark h ac. in the case of murine leukaemia virus (mlv), epigenetic repression requires the dna-binding protein np , which recruits epigenetic repressors including the h k me methyltransferase setdb , along with the human silencing hub (hush) complex , which has previously been identified as a repressor of endogenous retrotransposons and has been proposed to have a role in the epigenetic silencing of latent hiv- proviruses ( fig. c) . however, the previous study also demonstrated that the hush complex has no role in silencing unintegrated hiv- proviruses; how these are targeted for epigenetic repression remains to be determined. it is also unclear how the repressive marks deposited on unintegrated proviral dna are removed after integration; however, hiv- proviruses are known to preferentially integrate into actively transcribed chromatin, which may allow active chromatin marks of the surrounding region to spread to the proviral dna , . regardless, the ability of cells to effectively silence unintegrated retroviral dna means that integrase inhibitors are currently among the most clinically effective antiretroviral drugs. if unintegrated retroviral dna is indeed epigenetically silenced, this implies that a factor that induces the transcription of unintegrated retroviral dna should be able to rescue the replication of integrase-deficient (Δin) retroviruses. indeed, this has recently been demonstrated. specifically, the tax protein encoded by human t cell leukaemia virus , which is a potent activator of the cellular nf-κb transcription factors rela and relb, was shown to rescue the robust replication of Δin hiv- in t cells . this rescue correlated with the effective recruitment of rela and relb to the two nf-κb binding sites located in the hiv- enhancer element as well as with the acquisition of active, and the loss of repressive, epigenetic marks on unintegrated hiv- dna. thus, in the presence of tax, Δin hiv- replicates as transcriptionally active dna circles that are analogous to hbv cccdnas. upon infection of certain cell types, viruses may be epigenetically repressed, resulting in a latent infection. the successful repression of viral dna, while it prevents the killing of individual cells by lytic viral replication, does not necessarily reflect a clear victory for the host, as the establishment of latency can allow viruses to maintain long-term infections that avoid the host adaptive immune response yet revert to a lytic replication cycle at a later time. this is the case with latent infection of neurons by hsv- , of b lymphocytes by ebv and even of resting t cells by hiv- . each of these examples of latent infection is associated with the addition of repressive epigenetic marks to viral dna, including h k me and h k me , and with low levels of active markers . interestingly, latent infection by hsv- in neuronal cells may be partly explained by vp and its host cofactor hcf- being sequestered in the cytoplasm and thus unable to de-repress nuclear viral dna , . the fact that hcf- is uniquely localized to the cytosol in neurons but not in other cell types suggests that hsv- may have evolved to utilize the cytosolic localization of hcf- to identify cell types in which latency is the preferred strategy. latency establishment is an intricately regulated process that allows viruses to temporarily adopt an alternative viral state. taking ebv as an example, upon infection of b lymphocytes, the virus first activates all eight latency genes to aid in the activation of naive b cells (type iii latency), and then certain genes are switched off (type i/ii latency), to minimize the number of viral proteins that are detectable by the immune system, with different latency types utilizing distinct viral promoters that are associated with active chromatin marks only when needed [ ] [ ] [ ] . meanwhile, lytic viral genes remain transcriptionally silent and are associated with heterochromatic markers during latency , . thus, herpesviruses appear to have subverted what may have originally evolved as an innate antiviral defence mechanism (the epigenetic silencing of viral dna) as a means to maintain viral latency by selectively silencing viral genes. in this way, herpesviruses can establish a long-lived viral reservoir that avoids detection by the host adaptive immune system. in conclusion, viral episomal dna can be epigenetically silenced by the host using mechanisms analogous to host heterochromatic gene repression, while dna viruses including herpesviruses and hbv package and/or encode viral proteins that can overcome the hbv genomic dna is packaged in the viral particle as a gapped, partially double-stranded dna with open dna ends. the ends are closed up upon infection of a host cell, resulting in a fully double-stranded circular dna genome, termed the covalently closed circular dna, which acts as a functional template for gene expression and replication. an essential retrovirus enzyme that catalyses the integration of reverse-transcribed viral dna into the host genomic dna. a viral life-cycle stage in which viruses undergo active replication in the host cell, in most cases resulting in the lysis of the host cell. nature reviews | microbiology this suppression. interestingly, the identified targets of viral de-repressor proteins have highlighted pml-nbs as a key structure in the epigenetic suppression of viral dna. although it remains enigmatic how pml-nbs differentiate between host and episomal viral dna, one hypothesis suggests that ifi may act as the dna sensor that recruits pml-nb components to viral dna, because ifi associates with pml-nb components and because both ifi and pml-nbs are recruited to incoming hsv- dna. by contrast, some reports suggest that ifi and pml-nbs represent distinct mechanisms , [ ] [ ] [ ] . retroviruses appear to avoid epigenetic repression through integration of their dna into euchromatic regions of the host-cell genome, as the epigenetic repression of unintegrated retroviral dna closely mirrors that seen with mutant dna viruses that are unable to mount the necessary countermeasures. alternatively, when viruses invade host cells that are at least transiently non-permissive, viruses may utilize epigenetic suppression to enter a controlled, dormant state, thereby forming viral reservoirs that have so far proven impossible to eradicate. epitranscriptomic regulation rna transcripts are subject to a range of different covalent modifications at the single-nucleotide level, and over such modifications have been identified, particularly on trnas and other non-coding rnas (ncrnas); several of these modifications have been shown to regulate ncrna function . the repertoire of epitranscriptomic modifications (primarily but not exclusively methylations) found on eukaryotic mrnas is more limited than that found on ncrnas, and functional data exist for only a fraction of these mrna modifications (table ) . for the purposes of this review, we only discuss the four epitranscriptomic modifications that so far have been reported to affect viral gene expression -n -methyladenosine (m a), -methylcytidine (m c), n -acetylcytidine (ac c) and ′o-methylation of the ribose moiety of all four ribonucleosides (collectively nm) ( fig. ). quantification of the levels of several different epitranscriptomic modifications on highly purified samples of the genomic rna of both hiv- and the model animal retrovirus mlv has revealed that these viral rnas contain far higher levels of m a, m c and nm residues than do cellular mrnas expressed in infected cells , . specifically, m c was detected at a level - times higher on these viral rnas than on cellular poly(a) + rna; nm levels were also - times higher, and m a modifications were - times more prevalent. the level of ac c was not examined in these two studies, but it was reported to be at a level comparable to that of m a on hiv- transcripts in a third study that, despite having used virion rna samples of unknown purity, obtained m a and m c levels similar to those in the previous reports . regardless, the very high prevalence of m a, m c, nm and likely ac c modifications on these viral rnas indicates that viruses have evolved to maximize their addition to viral transcripts, which strongly suggests that these epitranscriptomic modifications are facilitating one or more steps in the viral replication cycle (fig. ). n -methyladenosine. the most common epitranscriptomic modification found on eukaryotic cellular mrnas is m a, representing . - . % of all adenosine residues , , . as a result, m a has been a major focus of epitranscriptomic research, resulting in the identification of methyltransferase-like (mettl ) as the enzyme that adds m a to mrnas (table ) . mettl , which is referred to as the m a 'writer' , functions as part of a complex with several cofactors, including mettl and wtap, and uses s-adenosylmethionine (sam) as the methyl donor , ( fig. ). this complex is predominantly nuclear, and the addition of m a to mrnas has been reported to occur co-transcriptionally . once deposited, m a residues can be detected by five m a 'readers' , including the predominantly nuclear protein ythdc and the cytoplasmic factors ythdc , ythdf , ythdf and ythdf . all five of these proteins contain a yth domain, which directly binds m a residues, and all five m a readers appear to bind to all m a sites equivalently. two m a demethylases (also known as 'erasers'), called alkbh and fto, have also been reported, and it has been proposed that mrna modification by m a may be dynamic and reversible, though this claim remains controversial , . although the presence of m a on viral transcripts was reported as long ago as the s , , analysis of the effect of m a residues on viral gene expression and replication only recently became technically feasible, with the development of techniques that allow epitranscriptomic modifications, including m a, to be mapped to specific sites on rnas (box ). initially, we and others reported that m a promotes hiv- gene expression and replication; knockdown of either mettl or the most highly expressed m a reader, ythdf , inhibited hiv- gene expression, whereas knockdown of the alkbh eraser or ythdf overexpression promoted hiv- gene expression , . subsequent studies from our laboratory and others -using a combination of gene knockdown, knockout and overexpression strategies -demonstrated that m a residues also promote the replication of a range of other viruses, including influenza a virus (iav), the picornavirus enterovirus , respiratory syncytial virus (rsv), human metapneumovirus (hmpv) and the polyomavirus sv (refs - ). in the case of iav, hmpv and rsv, viral variants carrying silent mutations that eliminated a subset cellular rnas that do not encode proteins. www.nature.com/nrmicro of the mapped m a sites but that left the underlying open reading frames unaffected were found to be not only attenuated in culture but also substantially reduced in their pathogenic potential in rodents, suggesting that the elimination of m a residues by mutagenesis might represent a novel strategy for the development of attenuated viruses that could potentially serve as vaccines . although the addition of m a residues to viral rnas clearly increases their expression , , it has recently been reported that m a addition to transcripts encoded by human hmpv also enables these viral rnas to escape recognition by the host innate immune rna sensor rig-i, thus avoiding the host antiviral response and promoting virus replication . although the majority of studies have concluded that m a promotes viral gene expression, some exceptions have been reported. for example, in the case of kshv, one group has reported that the addition of m a to viral transcripts promotes lytic replication , and another group has presented data arguing for the opposite conclusion: m a can suppress lytic replication . importantly, a third group has reported that m a can both promote and inhibit kshv lytic replication, depending on the cell type being studied . thus it appears that, in the context of the complex, temporally regulated replication cycles characteristic of large dna viruses such as kshv, m a may exert different phenotypic effects depending on the cellular context. in addition, several flaviviruses associated with acute human infections -including zika virus, dengue virus, yellow fever virus and west nile virus -have been reported to contain conserved clusters of m a residues, yet it was also reported that knockdown of the m a writer mettl increased zika virus replication, while knockdown of the m a eraser alkbh or fto exerted an inhibitory effect on viral replication , . this result, which is opposite to what was seen with the other rna viruses discussed above, is difficult to understand, as it is not apparent why m a sites would be evolutionarily conserved across several different flavivirus rna genomes if they act in cis to inhibit viral gene expression. recently, it was reported that the human m a reader ythdf can inhibit hiv- replication, though the reported effect -a less than twofold inhibition in wild-type a r t cells, when compared to ythdf knockout cells over an ~ -day infection period -was very modest . ythdf was reported to be packaged into hiv- virions and to then reduce reverse transcription by ~ % . the authors also reported that virion-associated ythdf was efficiently degraded by the hiv- protease, which they propose serves as a viral countermeasure. by contrast, we previously reported that ythdf overexpression in t cells increased hiv- replication, whereas ythdf knockout reduced hiv- replication . one possibility that was not considered is that ythdf may act by competing with ythdf for binding to m a sites on hiv- rna, thus reducing the positive effect on viral gene expression exerted by ythdf . another relatively common epitranscriptomic mrna modification is m c, which represents ~ . - . % of all cytidine residues found in cellular mrnas, but up to ~ . % of the cytidine residues in retroviral transcripts , , . although cells encode at least eight cytidine methyltransferases that act on rna, all but one of which belong to the nsun family of proteins, current data indicate that nsun is the nuclear writer responsible for the large majority of, but not all, m c residues added to mrnas, including viral mrnas , (table , fig. ). at present, no m c readers are known. analysis of the effect of loss of nsun expression, and hence loss of m c addition to mrnas, in hiv- -infected t cells revealed a specific loss of translation efficiency for hiv- and for cellular transcripts that are normally highly m c-modified . by contrast, cellular mrnas that normally lack m c, including several housekeeping genes, were unaffected by the loss of nsun expression. therefore, m c seems to increase viral gene expression and replication by acting predominantly at the level of mrna translation. n -acetylcytidine. recently, ac c residues were detected on cellular mrnas at a level of ~ ac c residue per mrna, and the ac c writer was identified as nuclear n-acetyltransferase (nat ) , (fig. ) . however, no ac c readers are currently known (table ). in the case of cellular mrnas, ac c was reported to enhance both the stability and the translation of cellular mrnas, in the latter case by acting in open reading frames to improve ribosomal decoding, especially when ac c was present in the wobble position of codons . this may relate to the fact that ac c can form more stable base pairs with guanosine residues . recently, we reported the mapping of ac c residues in hiv- transcripts (box ) and reported that the loss of nat expression results in a decline in hiv- gene expression in infected t cells . however, unlike m c, ac c was found to increase hiv- gene expression by enhancing rna stability, while viral mrna translation was unaffected. in confirmation of this result, we also observed that the mutagenesis of ac c clusters in the env gene of although high-performance liquid chromatography linked to tandem mass spectrometry (hplc-ms/ms) can identify and precisely quantify rna modifications, these methods do not provide location information. methods to map the location of modifications usually involve rna deep sequencing, which can be roughly separated into antibodydependent methods, modification-interacting protein pulldowns and chemical methods. the figure depicts the core mechanisms of modification identification used in various mapping techniques, with immunoprecipitationbased techniques on the left and chemical methods on the right. the simplest method used to map n -methyladenosine (m a) sites on mrna is m a-seq (also known as methylated rna immunoprecipitation sequencing (merip-seq)), in which rna is typically fragmented by alkaline hydrolysis and then incubated with an m a-specific antibody. the resulting rna-antibody complexes are then recovered and sequenced , . however, the resolution of this mapping technique is limited by the average rna fragment size, which is generally around nucleotides, resulting in fairly broad peaks (that is, it identifies broad regions that contain one or more m a residues). one variation of this method, called m a individual-nucleotide-resolution crosslinking and immunoprecipitation (miclip), uses ultraviolet (uv) light crosslinking (wavelength of nm) of the antibody to the motif characteristic of m a modification sites (minimally ʹ-rac- ʹ, with r representing g or a), resulting in a cytidineto-thymidine conversion of the cytidine immediately ʹ to the m a modification in the reverse-transcribed cdna library, allowing single-nucleotide resolution mapping . another variation is photo-crosslinking-assisted m a-seq (pa-m a-seq), in which cells are first pulsed with the highly photoactive uridine analogue -thiouridine ( su). the su-containing rna is then isolated and uv-light-crosslinked (wavelength of nm) in solution to an m a antibody . because this method utilizes rnase to degrade any rna not protected by the bound antibody, recovered bound rna fragments reflect the sequence protected by the antibody, which is ~ nucleotides. in addition, uv-light crosslinking of proteins to su results in a thymidine-to-cytidine conversion during reverse transcription, which allows the removal of all background reads during final data analysis . antibody-based methods can easily be adapted to mapping different modifications, as we have previously demonstrated by performing pa-m a-seq with m c and ac c antibodies , , whereas m a-seq has also been adapted to ac c , . adapting miclip to other antibodies would require additional testing to discover the preferred mutation induced by uv-light crosslinking of the specific antibody. antibody-mapping results can be corroborated using the photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (par-clip) technique to map the binding sites for modification-specific readers in living cells, if known. for example, the m a reader ythdf can be utilized in this way . in a method for mapping -methylcytidine (m c), confusingly also named miclip, mutation of the m c writer nsun at a key cysteine residue (c a) causes nsun to covalently crosslink to the target cytidine residue on the substrate rna; thus, m c modifications could be identified by immunoprecipitation of nsun -c a-bound rnas followed by rnase treatment, rna fragment recovery and deep sequencing . for sites of ′o-methylation (nm), no antibody is currently available, and mapping thus depends on chemical methods. ribomethseq exploits the fact that a methylation at the ′o position can block alkaline hydrolysis at that position , . in ′ome-seq, reverse transcription stops at an nm site under low-dntp conditions . nm-seq and ribooxiseq utilize the resistance of ′-end nm to periodate cleavage (io -) , . all three of these chemical nm-mapping methods provide single-nucleotide resolution. however, because ribomethseq and ′ome-seq depend on the absence of sequencing reads ending at the modification site, they require very high sequencing read depths. we note that the oxidation and elimination processes of nm-seq and ribooxiseq lead to the loss of most of the rna sample. thus, all current nm methods require large amounts of input rna. bisulfite sequencing has been used to map m c residues on rnas, exploiting the fact that m c residues are resistant to the cytidine-to-uridine conversion induced by bisulfite treatment . however, we have observed that cytidine residues located in rna stems are also resistant to bisulfite treatment, potentially resulting in their incorrect identification as m c residues. hiv- , which forms part of the ʹ untranslated region of the viral gag mrna, nevertheless reduced gag mrna and protein levels equivalently. thus, in addition to rna methylation, acetylations in the form of ac c can also be utilized to enhance viral gene expression, through the stabilization of viral rna transcripts. the fourth and final internal epitranscriptomic modification that has, so far, been reported to affect viral replication is ʹo-methylation of the ribose moiety of all four ribonucleosides (am, cm, gm and um, collectively known as nm). each of the four nm residues represents ~ . % of the level of the relevant nucleoside found in cellular mrnas, yet this level was found to be up to times higher when hiv- or mlv genomic rnas were analysed , . the nm writer that acts on retroviral transcripts has been identified as the nucleolar protein ftsj (ref. ), which was previously shown to function in pre-rrna processing ( fig. ). we note that ftsj was reported to be incapable of adding ′o-methyl groups to cytidine residues , which appears inconsistent with the high levels of cm detected on hiv- ( . %) and mlv ( . %) genomic rnas , . moreover, preliminary data suggest that the yeast ftsj homologue (spb ) is able to methylate cytidine residues . only one report has so far examined the phenotypic effect of nm residues on hiv- replication, and these researchers did not report any effect of nm residues on hiv- gene expression. instead, they found that hiv- virions produced in cells in which ftsj was knocked down by rna interference were potent activators of the cytoplasmic viral rna sensor mda , a key component of the host antiviral immune response, when the virions were used to infect dendritic cells . others have also reported that specific epitranscriptomic rna modifications, including not only nm but also pseudouridine, can attenuate cellular innate immune responses to transfected mrna molecules [ ] [ ] [ ] [ ] , whereas m a was recently reported to prevent the activation of a second cytoplasmic rna sensor, rig-i . clearly, it will be important to examine whether other viruses also use epitranscriptomic rna modifications, including but not limited to m a and nm, to escape viral rna detection by host innate immunity factors. in conclusion, the current evidence suggests that several different epitranscriptomic rna modifications are able to promote viral replication, either directly, by increasing viral mrna stability or translation, or indirectly, by allowing viruses to elude the recognition of their transcripts as foreign by cytoplasmic viral rna sensors. it is therefore not surprising that viruses appear to have evolved so as to maximize the level of several epitranscriptomic modifications that are added to their mrnas. by contrast, mammalian cells have evolved the capacity to recognize viral dna as foreign and seek to silence that dna epigenetically as one form of innate immune response. viruses, in turn, have evolved mechanisms that allow them to avoid epigenetic silencing, either by the targeted destruction of relevant cellular factors, such as the components of pml-nbs, or by hiding in host-cell chromosomal dna, as is seen for retroviruses. in this review, we have argued that cells use epigenetic gene regulation as a potential mechanism to silence incoming viral dna molecules, whereas viruses have evolved to recruit the cellular epitranscriptomic modification factors in order to heavily modify viral transcripts, as a means to boost viral gene expression and/or replication. although the field of epigenetic gene regulation is fairly mature, the question of how cells distinguish between viral dna and host-cell dna remains unresolved. in particular, how unintegrated hiv- proviral dna is recognized and silenced remains unknown. by contrast, the field of epitranscriptomic gene regulation is still in its infancy. for example, how writers select specific bases for modification remains largely unknown, and even in the case of m a -for which the consensus editing sequence ʹ-rac- ʹ has been defined, where r is either g or a , -only a minority of sites with that sequence are, in fact, modified. moreover, although five m a readers have been identified, the readers for all other rna modifications remain unknown, and even for m a, how the readers exert their phenotypic effects remains largely undefined. it will be important to understand why m a clearly promotes viral replication in most published studies, yet has also been suggested in other reports to inhibit viral replication, as discussed above. both epigenetic and epitranscriptomic regulatory pathways could in principle be targeted for antiviral drug development. in the case of epigenetic viral gene regulation, two strategies have emerged. in an approach termed 'shock-and-kill' , drugs that promote the formation of active chromatin, such as histone deacetylase (hdac) inhibitors, have been proposed as tools to activate latent viruses, including ebv [ ] [ ] [ ] and hiv- (ref. ). in the case of hiv- , this strategy envisions using these drugs to reactivate latent proviruses, in individuals who are also on antiretrovirals, as a means of selectively eliminating latently infected cells. however, this strategy has yet to prove clinically useful. an alternative approach envisions the use of drugs that instead keep viral dna epigenetically suppressed, including drugs that inhibit the h k demethylases lsd and jmjd , which are recruited by hcf- to hsv- viral dna in order to remove repressive marks. inhibitors of lsd and jmjd have indeed been shown to suppress hsv- gene expression, replication and reactivation from latency, both in vitro and in vivo , , . these drugs have also proved effective against other hcf- -dependent herpesviruses, including hcmv and varicella zoster virus (vzv), and they might prove useful in the treatment of pathologies that result from the reactivation of latent herpesviruses, such as shingles. although in principle lsd and jmjd inhibitors could be used to entirely repress the reactivation of latent dna viruses, these drugs would then need to be taken long-term, and it seems unlikely that inhibition of host h k demethylases for months or even years would be well-tolerated. inhibitors that block the epitranscriptomic modification of viral rnas clearly would be potentially even more interesting, as this should inhibit viral gene expression and/or promote antiviral immune responses. these drugs would presumably be targeted to the cellular a protein that normally localizes to the subnuclear structure called the nucleolus, which is where the rna components of the ribosome are produced. nature reviews | microbiology writers that add epitranscriptomic marks to mrnas (table ) , and the emergence of drug-resistant viral mutants would therefore seem to be unlikely. conversely, as such drugs would also prevent the epitranscriptomic modification of cellular mrnas, toxicity might be an issue, and we therefore envision that such drugs would be used only briefly, during the - -day acute phase of infection by viruses such as iav, rsv and possibly severe acute respiratory syndrome coronavirus , in order to reduce the peak viral load and limit viral pathogenicity until the adaptive immune system becomes effective. because the addition of m a to mrnas has been implicated as a driver in some forms of cancer [ ] [ ] [ ] , several biotech companies are already attempting to identify effective inhibitors of mettl function, and it would clearly be of interest to test these, once they have been identified, in animal models that support pathogenic infections by human viruses. in the interim, some data in fact already suggest that drugs that inhibit mrna methylation could prove to be effective pan-viral inhibitors. specifically, several drugs, including -deaza-adenosine (daa), are known to act as inhibitors of s-adenosylhomocysteine (sah) hydrolase and, as a result, to deplete cells of sam, the methyl donor used not only by mettl but also by nsun , and likely also by the enzymes that add nm residues to mrnas. importantly, daa was shown to inhibit m a addition to mrnas, while the formation of -methylguanosine, which forms part of the mrna ʹ cap, was largely unaffected . daa acts as a potent pan-viral inhibitor in culture and is able to effectively inhibit a wide range of dna and rna viruses at doses that are > -fold lower than the level found to exert a toxic effect on cultured primary cells . in vivo, daa is also a remarkably effective broad-spectrum antiviral. for example, a single dose of daa given one or two days after infection reduced peak viraemia in ebolavirus-infected mice by > , fold and resulted in the survival of almost all the treated animals, whereas untreated animals showed a % survival rate . similarly, daa drastically reduced viral titers in cotton rats infected with rsv at doses of daa that were not detectably toxic . although daa may not itself be a potentially useful drug, these studies do make the point that the epitranscriptomic modification of viral mrnas may represent a potential viral achilles heel, and that the identification of inhibitors of this process could therefore lead to the development of a novel class of potent, broad-spectrum antivirals. published online xx xx xxxx perceiving the epigenetic landscape through histone readers editing the epigenome: reshaping the genomic landscape the pivotal regulatory landscape of rna modifications dynamic rna modifications in gene expression regulation regulation of gene expression by n -methyladenosine in cancer rna modifications regulating cell fate in cancer cancer epigenetics: moving forward nuclear sensing of viral dna, epigenetic regulation of herpes simplex virus infection, and innate immunity viral gene products actively promote latent infection by epigenetic silencing mechanisms viral dna sensors ifi and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection nuclear interferon-inducible protein promotes silencing of herpesviral and transfected dna regulation of alphaherpesvirus infections by the icp family of proteins pml and pml nuclear bodies: implications in antiviral defence structure, dynamics and functions of promyelocytic leukaemia nuclear bodies pml nuclear bodies are highly organised dna-protein structures with a function in heterochromatin remodelling at the g phase nuclear domain , the site of dna virus transcription and replication ifn enhance expression of sp , an autoantigen in primary biliary cirrhosis the acute promyelocytic leukaemiaassociated pml gene is induced by interferon transcriptional induction of the pml growth suppressor gene by interferons is mediated through an isre and a gas element disruption of host antiviral resistances by gammaherpesvirus tegument proteins with homology to the fgarat purine biosynthesis enzyme sp b, a repressor of gene expression preferentially binds to dna with unmethylated cpgs interaction of sp with hp proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment rnai reveals anti-apoptotic and transcriptionally repressive activities of daxx daxx and histone deacetylase ii associate with chromatin through an interaction with core histones and the chromatin-associated protein dek daxx is an h . -specific histone chaperone and cooperates with atrx in replicationindependent chromatin assembly at telomeres the atrx syndrome protein forms a chromatin-remodeling complex with daxx and localizes in promyelocytic leukemia nuclear bodies the innate immune sensor ifi recognizes foreign dna in the nucleus by scanning along the duplex ifi is an innate immune sensor for intracellular dna this study reports the discovery of ifi as an antiviral dna sensor ifi restricts hsv- replication by accumulating on the hsv- genome, repressing hsv- gene expression, and directly or indirectly modulating histone modifications the intracellular dna sensor ifi gene acts as restriction factor for human cytomegalovirus replication the nuclear dna sensor ifi acts as a restriction factor for human papillomavirus replication through epigenetic modifications of the viral promoters nuclear sensor interferon-inducible protein inhibits the function of hepatitis b virus covalently closed circular dna by integrating innate immune activation and epigenetic suppression ifi , a nuclear innate immune dna sensor, mediates epigenetic silencing of herpesvirus genomes by its association with h k methyltransferases suv h and glp interactions of the antiviral factor interferon gamma-inducible protein (ifi ) mediate immune signaling and herpes simplex virus- immunosuppression host and viral proteins in the virion of kaposi's sarcoma-associated herpesvirus proteins of purified epstein-barr virus identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome herpes simplex virus icp promotes both histone removal and acetylation on viral dna during lytic infection promyelocytic leukemia (pml) nuclear bodies (nbs) induce latent/quiescent hsv- genomes chromatinization through a pml nb/histone h . /h . chaperone axis temporal association of the herpes simplex virus genome with histone proteins during a lytic infection the histone variant h . regulates gene expression during lytic infection with herpes simplex virus type inhibition of the histone demethylase lsd blocks alpha-herpesvirus lytic replication and reactivation from latency targeting the jmjd histone demethylases to epigenetically control herpesvirus infection and reactivation from latency the coactivator host cell factor- mediates set and mll h k trimethylation at herpesvirus immediate early promoters for initiation of infection herpesviral icp protein promotes two waves of heterochromatin removal on an early viral promoter during lytic infection relative contributions of herpes simplex virus icp and vhs to loss of cellular ifi vary in different human cell types the emerging role of nuclear viral dna sensors human cytomegalovirus (hcmv) ul gene product (pp ) relieves hdaxx-mediated repression of hcmv replication human cytomegalovirus protein pp displaces the chromatin-associated factor atrx from nuclear domain at early stages of infection inactivating a cellular intrinsic immune defense mediated by daxx is the mechanism through which the human cytomegalovirus pp protein stimulates viral immediate-early gene expression ebv tegument protein bnrf disrupts daxx-atrx to activate viral early gene transcription structural basis underlying viral hijacking of a histone chaperone complex viral reprogramming of the daxx histone h . chaperone during early epstein-barr virus infection human cytomegalovirus pul stimulates activity of the viral immediate-early promoter through its interaction with the cellular ifi protein human cytomegalovirus tegument protein pul inhibits ifi -mediated dna sensing for immune evasion innate nuclear sensor ifi translocates into the cytoplasm during the early stage of in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage epigenetic regulation of hepatitis b virus covalently closed circular dna: implications for epigenetic therapy against chronic hepatitis b hepatitis b virus x protein promotes degradation of smc / to enhance hbv replication the smc / complex restricts hbv when localized to nd without inducing an innate immune response and is counteracted by the hbv x protein shortly after infection this study reports the identification of smc / as novel viral restriction factors active against hbv integration is essential for efficient gene expression of human immunodeficiency virus type construction and analysis of deletion mutations in the pol gene of moloney murine leukemia virus: a new viral function required for productive infection unintegrated hiv- dnas are loaded with core and linker histones and transcriptionally silenced histones are rapidly loaded onto unintegrated retroviral dnas soon after nuclear entry np mediates silencing of unintegrated retroviral dna this study identifies the cellular factors that silence unintegrated murine leukaemia virus dna hush, a link between intrinsic immunity and hiv latency hiv- integration in the human genome favors active genes and local hotspots this study is the first to report the preference of the hiv- provirus for integration into open chromatin nuclear landscape of hiv- infection and integration reversal of epigenetic silencing allows robust hiv- replication in the absence of integrase function this article provides a useful review of the epigenetic regulation of viral latency nuclear localization of the c factor (host cell factor) in sensory neurons correlates with reactivation of herpes simplex virus from latency ctcf prevents the epigenetic drift of ebv latency promoter qp an atlas of the epstein-barr virus transcriptome and epigenome reveals host-virus regulatory interactions first days in the life of naive human b lymphocytes infected with epstein-barr virus epigenetic regulation of ebv persistence and oncogenesis dynamic response of ifi and promyelocytic leukemia nuclear body components to herpes simplex virus infection mechanisms of host ifi , pml, and daxx protein restriction of herpes simplex virus replication the viral ubiquitin ligase icp is neither sufficient nor necessary for degradation of the cellular dna sensor ifi during herpes simplex virus infection extensive epitranscriptomic methylation of a and c residues on murine leukemia virus transcripts enhances viral gene expression this study reports the comprehensive quantitative analysis of rna modifications on hiv- genomic rna, along with the first characterization of the role of the m c rna positive-sense rna viruses reveal the complexity and dynamics of the cellular and viral epitranscriptomes during infection the dynamic epitranscriptome: n -methyladenosine and gene expression control epitranscriptome sequencing technologies: decoding rna modifications m a mrna modifications are deposited in nascent pre-mrna and are not required for splicing but do specify cytoplasmic turnover our views of dynamic n -methyladenosine rna methylation jr pre-mrna processing includes n methylation of adenosine residues that are retained in mrna exons and the fallacy of influenza viral mrna contains internal n -methyladenosine and ′-terminal -methylguanosine in cap structures methylated simian virus -specific rna from nuclei and cytoplasm of infected bsc- cells this study represents the first report of the methylation of internal (non-cap) residues on viral transcripts posttranscriptional m a editing of hiv- mrnas enhances viral gene expression dynamics of the human and viral m a rna methylomes during hiv- infection of t cells epitranscriptomic enhancement of influenza a virus gene expression and replication this study is the first to report that epitranscriptomic modification of viral rna enhances virus replication and pathogenesis in vivo addition of m a to sv late mrnas enhances viral structural gene expression and replication viral n -methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus n -methyladenosine modification and mettl modulate enterovirus replication this study reports that the m a rna modification can prevent the detection of viral transcripts by the host innate immunity factor rig-i kaposi's sarcomaassociated herpesvirus utilizes and manipulates rna n -adenosine methylation to promote lytic replication viral and cellular n -methyladenosine and n , ′-o-dimethyladenosine epitranscriptomes in the kshv life cycle n -methyladenosine modification and the ythdf reader protein play cell type specific roles in lytic viral gene expression during kaposi's sarcoma-associated herpesvirus infection dynamics of human and viral rna methylation during zika virus infection n -methyladenosine in flaviviridae viral rna genomes regulates infection hiv protease cleaves the antiviral m a reader protein ythdf in the viral particle -methylcytosine promotes mrna export-nsun as the methyltransferase and alyref as an m c reader insights into rna biology from an atlas of mammalian mrna-binding proteins acetylation of cytidine in mrna promotes translation efficiency conformational preferences of modified nucleoside -taurinomethyluridine, taum u occur at 'wobble' th position in the anticodon loop of trna acetylation of cytidine residues boosts hiv- gene expression by increasing viral rna stability ftsj is an rna ′-o-methyltransferase recruited by hiv to avoid innate immune sensing the human nucleolar protein ftsj associates with nip and functions in pre-rrna processing conserved methyltransferase spb targets mrnas for regulated modification with ′-o-methyl ribose ′-o-methyl-modified rnas act as tlr antagonists suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna incorporation of pseudouridine into mrna enhances translation by diminishing pkr activation rnas containing modified nucleotides fail to trigger rig-i conformational changes for innate immune signaling virally targeted therapies for ebv-associated malignancies epstein-barr virus post-transplant lymphoproliferative disease and virus-specific therapy: pharmacological re-activation of viral target genes with arginine butyrate administration of vorinostat disrupts hiv- latency in patients on antiretroviral therapy emerging strategies to deplete the hiv reservoir a novel selective lsd /kdm a inhibitor epigenetically blocks herpes simplex virus lytic replication and reactivation from latency inhibition of lsd reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes n -methyladenosine (m a): a promising new molecular target in acute myeloid leukemia the critical role of rna m a methylation in cancer emerging links between m a and misregulated mrna methylation in cancer effects of the s-adenosylhomocysteine hydrolase inhibitors -deazaadenosine and -deazaaristeromycin on rna methylation and synthesis broad-spectrum antiviral activity of the carbocyclic analog of -deazaadenosine treatment of lethal ebola virus infection in mice with a single dose of an s-adenosyl-l-homocysteine hydrolase inhibitor evaluation of the toxicity and antiviral activity of carbocyclic -deazaadenosine against respiratory syncytial and parainfluenza type viruses in tissue culture and in cotton rats transgenerational epigenetic inheritance: myths and mechanisms epigenetic modifications: basic mechanisms and role in cardiovascular disease the double face of the histone variant h . the death-associated protein daxx is a novel histone chaperone involved in the replication-independent deposition of h . distinct factors control histone variant h . localization at specific genomic regions this early study uses merip-seq to characterize how the m a modification regulates human mrna function comprehensive analysis of mrna methylation reveals enrichment in ′ utrs and near stop codons single-nucleotide-resolution mapping of m a and m am throughout the transcriptome high-resolution n -methyladenosine (m a) map using photo-crosslinking-assisted m a sequencing transcriptome-wide identification of rna-binding protein and microrna target sites by par-clip nsun -mediated cytosine- methylation of vault noncoding rna determines its processing into regulatory small rnas illumina-based ribomethseq approach for mapping of ′-o-me residues in rna profiling of ribose methylations in rna by high-throughput sequencing high-throughput single-base resolution mapping of rna -o-methylated residues nm-seq maps ′-o-methylation sites in human mrna with base precision high-throughput and site-specific identification of ′-o-methylation sites using ribose oxidation sequencing (riboxi-seq) this research was funded in part by nih grants r -da and u -ai to b.r.c., and by a duke university center for aids research (cfar, p -ai ) pilot award to k.t. the authors contributed equally to all aspects of the article. the authors declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -khrljehj authors: principi, nicola; piralla, antonio; zampiero, alberto; bianchini, sonia; umbrello, giulia; scala, alessia; bosis, samantha; fossali, emilio; baldanti, fausto; esposito, susanna title: bocavirus infection in otherwise healthy children with respiratory disease date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: khrljehj to evaluate the role of human bocavirus (hbov) as a causative agent of respiratory disease, the importance of the viral load in respiratory disease type and severity and the pathogenicity of the different hbov species, we studied all hbov-positive nasopharyngeal samples collected from children who attended an emergency room for a respiratory tract infection during three winters ( – , – , and – ). human bocavirus was detected using the respiratory virus panel fast assay and real-time pcr. of the , nasopharyngeal samples, ( . %) were positive for hbov; a similar prevalence was observed in all three periods studied. among hbov-infected children, . % were between – years old, and hbov was detected alone in / ( . %) cases. all of the detected hbov strains belonged to genotype . the median hbov load was significantly higher in samples containing strains with both the n h and t s mutations compared to other samples (p< . ). children with a single hbov- infection more frequently had upper respiratory tract infections (urtis) than those who were co-infected ( . % vs . %, respectively, p = . ). the duration of hospitalization was longer among children with high viral loads than that observed among children with low viral loads ( . ± . days vs . ± . days, respectively, p = . ), and the use of aerosol therapy was more frequent among children with high viral loads than among those with low viral loads ( . % vs . %, respectively, p = . ). this study shows that hbov is a relatively uncommon but stable infectious agent in children and that hbov seems to be the only strain detected in italy in respiratory samples. from a clinical point of view, hbov seems to have in the majority of healthy children relatively low clinical relevance. moreover, the viral load influences only the duration of hospitalization and the use of aerosol therapy without any association with the site of the respiratory disease. human bocavirus (hbov) is a recently identified viral agent that belongs to the family parvoviridae and contains a single linear positive-sense or negative-sense single-stranded deoxyribonucleic acid genome [ ] . this virus has been detected mainly in younger children, in nasopharyngeal secretions, in sera and blood samples of patients with upper (urti) and lower (lrti) respiratory tract infections and in faecal specimens of subjects with gastroenteritis [ ] . currently, hbovs are classified into species through ; hbov is predominantly found in the respiratory tract, and hbov , hbov , and hbov are found mainly in stool [ ] . despite there are studies suggesting that hbov is able to infect the lower airways causing severe infections in both children and adults, the role of hbov as a causative agent of respiratory disease is frequently questioned due to its common detection with other potential pathogens [ ] and the evidence that in some studies co-infections can have a significantly greater clinical and socioeconomic impact on infected children and their households than hbov infection alone [ ] . moreover, the importance of the viral load in determining the type and severity of respiratory disease as well as the pathogenicity of the different hbov species [ ] are not precisely defined. the main aim of this study was to contribute to resolving these problems. the circulation of hbov during several winter seasons in italy was investigated, and a phylogenetic analysis of detected strains was performed. in addition, correlations between different hbov strains and the severity of disease in cases with infections due to hbov alone or due to co-infections were studied. finally, the role of the viral load was analysed. to evaluate the circulation of the different hbov types and the possible relationships between viral load, virus genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children attending the emergency room of the fondazione irccs ca' granda ospedale maggiore policlinico, university of milan, italy, due to a respiratory tract infection arising between november and march during winters ( - , - , and - ) . the study was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy. written informed consent of a parent or legal guardian was required, and children years of age were asked to give their written assent. patients' demographic characteristics and medical histories were retrieved from hospital charts and were systematically recorded before and after the first visit to the emergency room using standardized written questionnaires [ ] . the study patients were classified into disease groups (i.e., acute otitis media, rhinosinusitis, pharyngitis, croup, infectious wheezing, acute bronchitis, pneumonia) on the basis of signs and/or symptoms using well-established criteria and were finally subdivided into two subgroups: upper (urtis) and lower respiratory tract infections (lrtis) [ ] . nasopharyngeal secretions were collected from all of the children immediately after admission to the emergency room using a paranasal flocked swab ( swab per child), which was stored in a tube containing ml of universal transport medium (kit cat. no. c, copan italia, brescia, italy). viral nucleic acids were extracted from each swab by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extract was tested for respiratory viruses using the respiratory virus panel xtag rvp fast v (luminex molecular diagnostics, inc., toronto, canada), which simultaneously detects influenza a virus (subtypes h or h ); influenza b virus; respiratory syncytial virus (rsv) types a and b; human parainfluenza virus types - (hpiv - ); adenovirus (adv); human metapneumovirus (hmpv); coronaviruses (hcov) e, nl , oc and hku , enterovirus/rhinovirus (ev/hrv); and hbov, in accordance with the manufacturer's instructions [ , ] . samples that were positive for hbov were stored at - °c. hbov real-time polymerase chain reaction (pcr) viral nucleic acid extracts previously testing positive for hbov were re-tested for confirmation by two different singleplex real-time pcrs using taqman universal master mix ii (applied biosystems, california, usa). amplification and detection of viral dna were performed with a ht real-time pcr system machine (applied biosystems, california, usa). conserved regions for rt-pcr primers and probes were identified in the hbov ns- and np- genes from the nucleotide sequence alignments available from genbank (for ns , dq - , dq - , and dq , and for np- , dq - , ab - , dq - , dq - , dq , dq - , dq , dq , and am - ; http:// www.ncbi.nlm.nih.gov/genbank/). each μl singleplex reaction mixture consisted of . μl of forward primer '-tgcagacaacgcytagttgttt- ' and reverse primer '-ctgtcccg cccaagataca- ' for the base pair ns- target or forward primer '-agcatcgctcctacaaaagaaaag- ' and reverse primer '-tcttcatcacttggtctga ggtct- ' for the np- target, . μl of probe '-ccaggattgggtggaacctgcaaa- ' or '-aggctcgggctcatatcatcaggaaca- ', and . μl of sample viral dna. pcrs were conducted at °c for min and then at °c for min, followed by cycles at °c for sec and at °c for min. taqman probes were labelled at the ' ends with the reporter molecule -carboxyfluorescein and at the ' ends with black hole quencher (biosearch technologies, inc., novato, ca). each run included one synthetic template control and one no-template control for each target. specimen extracts were also tested by real-time pcr for the human rnase-p gene to monitor specimen quality and the presence of pcr inhibitors. a positive test for both the ns and np- targets or for a single target confirmed from a second extraction from a new sample aliquot was considered definitive evidence of hbov infection. the viral load was obtained using real-time pcr with the ns primers and probe previously described and a dna plasmid used as the standard calibrator. the amplified target fragment of the plasmid was verified by sequencing. plasmid dna concentrations were detected with an nd- spectrophotometer (nanodrop products, wilmington, de, usa). each run included plasmid and negative controls. standard precautions were taken throughout the pcr process to avoid cross-contamination. negative controls and serial dilutions of the positive controls were included in every pcr assay. finally, quantitative results were reported as dna copies/ ml of respiratory samples. the viral load was defined as low for values log (copies/ml) and as high for values > log (copies/ml). for genotyping, the viral vp- / gene was amplified using a conventional pcr assay. briefly, sets of forward and reverse primers ( '-cacagacagaagcagacgagat- ' and '-ggtg agaagtgacagctgtattg- '; '-ttcagaatggtcacctctaca- ' and '-ctgtgc ttccgttttgtctta- '; '-aactttgactgtgaatgggtta- ' and '-aaatagtgcc tggaggatgat- '; '-ctatcaccagagaaaatccaatc- ' and '-gagacggtaaca ccacta- ') were used in pcr amplification and the quantitect probe master mix (qia-gen, venlo, netherlands) was used as the basis for the reaction mix. viral products were analysed by electrophoresis on a . % agarose gel and purified with the qiaquick gel extraction kit (qiagen, venlo, netherlands). sequencing reactions were set up with purified dna, one of the specific primers used in the pcr and bigdye terminator v . cycle sequencing kit (applied biosystems, california, usa) according to the protocol recommended by the manufacturer. sequencing and sequence analysis were performed on a genetic analyser (applied biosystems, california, usa). all alignments were performed using clustalx . and bioedit (version . . . ) software (ibis biosciences, carlsbad, ca). phylogenetic trees of the vp- / protein gene were generated using the neighbour-joining method and p-distance model of the molecular evolutionary genetics analyses (mega) software, version . [ ] . bootstrap probabilities for , iterations were calculated to evaluate confidence estimates. the graphs were made using graphpad prism version . for windows (graphpad software, san diego, ca). all genotyped sequences of the hbov vp- / gene were submitted to genbank (accession numbers kr -kr ). tests for positive selection were conducted on the datamonkey server [ ] using the singlelikelihood ancestor (slac), and the fixed-effects likelihood (fel) [ ] , the internal branch fixed-effects likelihood (ifel) [ ] , the mixed effects model of evolution (meme) [ ] , and fast unconstrained bayesian approximation methods (fubar). the dn/ds ratios were calculated using the slac and fel codon-based maximum likelihood approaches. the slac approach counts the number of non-synonymous changes per non-synonymous site (dn) and tests whether it is significantly different from the number of synonymous changes per synonymous site (ds). the fel approach estimates the ratios of non-synonymous to synonymous changes for each site in an alignment. the ifel method is similar to the fel, but tests site-bysite selection along only the internal branches of the phylogeny. in order to avoid an excessive false-positive rate, sites with slac, fel, ifel and meme p-values of < . and a fubar posterior probability of > . were accepted as candidates for selection. descriptive statistics of the responses were generated. continuous variables were presented as mean values and standard deviations (sds) and categorical variables as numbers and percentages. for categorical data, comparisons between groups were performed using a contingency table analysis with the Χ or fisher's exact test when appropriate. for ordered categorical data, a cochran-armitage test for trend was used to compare the groups. continuous data were analysed using a two-sided student's t-test after ensuring the data were normally distributed (based on the shapiro-wilk statistic) or using a two-sided wilcoxon's rank-sum test if the data were non-normal. all analyses were two tailed, and p-values of . or less were considered to be statistically significant. all analyses were conducted using sas version . (cary, nc, usa). during the three study periods, , nasopharyngeal samples were collected in the emergency room. of these, ( . %) tested positive for hbov (table ) . among hbov infected children, . % were between - years old, whereas . % and . % were aged < and years, respectively. the prevalence of hbov detection was quite similar in the three studied periods; hbov was the only virus detected in / ( . %) cases and was detected in association with one ( . %) or more ( . %) viruses in ( . %) cases. ev/hrv and rsv were the most common co-infecting viral agents and were found, respectively, in and samples. subjects with co-infection were younger than those without (p = . ). considering dna copies/ml as a cut-off, the viral load was classified as low in ( . %) cases and as high in ( . %) cases. the phylogenetic tree constructed using the vp /vp sequences showed that all of the italian hbov strains detected during the three study periods belonged to hbov genotype (fig ) . no unusual clustering was observed among the identified strains; hbovs circulating in were closely related to strains circulating in . the sequence identity matrix of the vp /vp gene showed minimum to maximum identity ranges of . - % between the italian strains and . - . % with respect to hbov st reference strains (dq ). in comparison to the reference strain, / ( . %) strains had only one amino acid difference, / ( . %) strains had two amino acid differences, and the remaining strains ( / ; . %) had at least three amino acid changes. a total of / ( . %) amino acid positions were observed to have at least one change in the vp /vp sequence alignments (fig ) . of these, / ( . %) changes occurred within the vp unique (vp u) region corresponding to the first amino acids at the n-terminus of the vp gene. specifically, the following changes were observed: r k, g d, e k, l s, h q, d n, and g e (fig ) . the vp u region includes the conserved phospholipase a (pla ) motif (nt - ). the vp u sequences of all hbov isolates identified in this study revealed conserved yxgxg (nt [ ] [ ] [ ] [ ] [ ] and hdxxy (nt - ) motifs in the catalytic site of secreted pla . in addition, the amino acid residues at positions , , and have been hypothesized to form the catalytic network for enzymatic activity. in our hbov strains, all the sequences had amino acids associated with efficient enzymatic activity (p , h , d , and d ). of note, two hbov strains (mi- -jan and mi- -jan ) had a peculiar amino acid sequence in the -amino acid segment starting at amino acid (kvptrrvqpyirqtnwkhr), which has not been previously reported in hbov strains included in the genbank database (in red, fig ) . overall, these two strains had and amino acid changes compared to the hbov st strain, and almost all these changes were included in the region described below. the origin of this highly divergent region, which occurred in spite of the conservation displayed in the rest of the hbov dna genome, remains to be defined. a global analysis of selective pressure made using the slac model indicated an estimated overall dn/ds ratio of . . overall, the site-specific analyses identified three sites ( , , and ) as under positive selection by at least two methods used (slac, fel, fubar, and meme). the ifel model was used to determine the selection pressure acting on the vp /vp codons along the internal branches of the tree. two positively selected codons ( and ) were identified. the selected sites, highlighted with arrows in fig , were presumably located in of these strains, / ( . %) were also characterized by the n k, q p, and q d mutations. several negatively selected sites were identified by different methods (table ). regarding the viral load, a wide range of hbov dna levels from . x to . x copies/ml were found in the clinical samples. in fig (right side) , the viral load of each italian hbov strain is reported near the aligned mutations. in the group of strains (n = ; . %) harbouring at least mutations in addition to a t, the values of the hbov load greater than x dna copies/ml are reported in grey. a total of strains had a very high viral load, and / ( . %) harboured the t s mutation. this percentage is nearly significantly different than the overall frequency ( / ; . %) of t s in the group of strains described below (p = . ). seven of the strains with the t s mutation had an additional mutation in one of the sites under positive selection (reported in fig with an asterisk) . in detail, / ( . %) had the n h change, / ( . %) had the a d change, and / ( . %) had the a t change. based on the observed data, we hypothesize that the double mutation of n h with t s may positively affect viral replication and specific immune response. as shown in table , the median hbov load was significantly higher in samples of strains with the n h and t s changes than that in samples of wild type t strains, strains with only the t s mutation, and strains with only the n h mutation (p-values of . , . and . , respectively). finally, the two divergent strains (mi- -jan and mi -jan ) with unusual amino acid changes were observed with viral loads lower than dna copies/ml. this could suggest that these mutations do not confer a replicative advantage in these virus strains. in table , data regarding demographic, clinical and laboratory characteristics of children infected by hbov- alone or co-infected with hbov- and one or more other respiratory viruses are reported. because a preliminary evaluation did not find any differences among subjects co-infected with ev/hrv, rsv or other viruses, all co-infections were considered together. as shown, children infected with only hbov- had urtis more frequently than those with a co-infection ( . % vs . %, respectively, p = . ). moreover, a similar illness within the family in the days since patient enrolment was significantly more common among co-infected children than among those with a single infection ( . % vs . %, respectively, p = . ). no other significant differences between the groups were observed. table shows the demographic, clinical, and laboratory characteristics of the enrolled subjects according to the hbov load. subjects with low and high viral loads were quite similar. the only significant differences were found in the duration of hospitalization, which was longer among children with a high viral load than among those with a low viral load ( . ± . days vs . ± . days, respectively, p = . ), and in the use of aerosol therapy, which was more frequent among children with a high viral load than among those with a low viral load ( . % vs . %, respectively, p = . ). moreover, mutations leading to a high or low viral load were not associated with atypical clinical characteristics. this study shows that in italy during the winter periods - , - , and - , the incidence of hbov infection among children with respiratory disease was relatively low, limited to approximately % of cases, and did not significantly vary from year to year. the phylogenetic analysis showed that all of the strains detected in this study belonged to hbov genotype and were closely related to the prototype strain identified by allander et al. [ ] . this was expected because this genotype is the most common among hbovs associated with respiratory infections [ ] . most of the patients in whom hbov was identified were younger than years of age, further highlighting that younger children are the individuals most frequently infected by this viral agent [ ] . serological studies have shown evidence that the number of subjects positive for anti-hbov antibodies continuously increases with increasing age group from the ages of months to years, and by the age of years approximately % of children have been infected with hbov [ , ] . more than % of the children infected by hbov in this study were coinfected with at least one other respiratory virus. moreover, co-infected patients had lrti more frequently than those infected by hbov alone. these findings are not surprising because simultaneous detection of hbov and other viruses in children with respiratory disease and greater severity in co-infected cases have been already reported in studies in which it was also demonstrated that hbov can frequently be identified in the respiratory secretions of asymptomatic subjects [ ] [ ] [ ] [ ] [ ] [ ] . recently, it has been reported that hbov can be shed for several days or months after a previous infection [ ] , which could explain the simultaneous identification of hbov and other respiratory viruses, the frequency of asymptomatic infections and the generally greater severity of infections in co-infected individuals compared to those with hbov alone. in most of the co-infected cases, detection of hbov in the respiratory secretions with the new sensitive molecular methods able to identify very low viral loads might be a consequence of a previous clinically resolved disease, and a virus other than hbov was therefore the real cause of the disease. however, reports of severe clinical manifestations in patients infected with only hbov have been published [ ] , highlighting that the assessment of the real importance of hbov infection in a single patient remains very difficult. studies on children with severe pneumonia, acute wheezing, asthma and/or bronchiolitis suggested that hbov is able to infect the lower airways down to the bronchioles [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, hbov has been found as the only infectious agent in adult lung transplant recipients with severe lrti, whereas it was not detected in respiratory secretions of asymptomatic transplanted subjects [ ] . this would indicate that hbov is not always a bystander or the cause of mild respiratory problems but rather a real, although relatively rare, causative agent of severe disease in both children and adults, particularly when they are immunocompromised. on the other hand, hbov can cause serious neurological infections [ ] and contribute to chronic disease in adult patients mainly because it can persist after childhood infection and reactivate [ ] . evaluation of the viral load has been considered a possible method to define when this virus is the real cause of a respiratory disease and when it is only a secondary infection. unfortunately, this approach has had no success because although some studies have shown evidence for a strict correlation between high viral load and severe lrti in children with a single hbov infection [ ] [ ] [ ] , others, including the present study, did not show a clear relationship between these two variables [ ] . however, the evolution of virulence appears to involve a variety of mechanisms in different viral systems, including mutations in regulatory regions and viral adaptation for utilization of alternative or expanded repertoires of cellular receptors. an alternative hypothesis to evaluate the importance of hbov concerns the correlation between viral load levels and the presence of specific mutations. however, mutations associated with increased or reduced replication are rarely reported for hbov. recently, hao et al. have reported that few nucleotide changes were correlated with a lower viral load [ ] . in the present study, a double mutant (n h and t s) was observed in samples with a significantly higher viral load. however, further phenotypic validation studies are required to draw major conclusions regarding the impact of these mutations on viral replication. furthermore, as reported by qu et al. [ ] , it seems that nucleotide changes in the vp u region could affect the replication efficiency of hbov. likewise, in our strains all the amino acids of the catalytic site were conserved, and no mutations that affect spla activity were identified. in agreement with others [ ] , the phylogenetic analysis of this study confirmed the very low degree of variability in the hbov genomic region encoding proteins that are exposed to the virus surface and are therefore under immunologic pressure. only % ( codons) of amino acids were found to have at least one change in the vp /vp gene, a finding not substantially different from that reported by hao et al. in a different geographic area [ ] . in our study, several amino acid changes were observed in strains circulating in almost all the respiratory seasons. this finding provides evidence that the selection of those variants best adapted to each particular environment might select for variants with an evolutionary advantage. seven of these mutations were located in a genomic region (i.e., vp u) previously reported to be involved in the mechanisms of virus replication. for instance, the vp u amino acid variations r k and l s have been previously reported [ ] , whereas the remaining variations have not yet been described. interestingly, two hbov strains identified in respiratory samples collected in january had unusual amino acid sequences in a somewhat conserved genomic region. the reason for this genetic diversity is still undefined. however, as described for hbov, other parvoviruses [ ] , and enteroviruses, a series of α-helices and β-barrels in the vp protein were intercalated by an external loop, in which the majority of the genetic variability accumulated. nevertheless, these two divergent strains were found in samples with low viral loads, and we might hypothesize a loss of replication advantage for these strains. in the present study, the dn/ds ratios for all pairwise comparisons were < , which is in line with previous results showing that positive selection was extremely limited in parvoviruses [ ] . in fact, selective pressure analyses have identified codons under positive selection. in a previous paper, a different codon (l ) was identified as being under positive selection [ ] . nevertheless, the great majority of codons were under negative or neutral selection, which has also been confirmed by others [ ] . this finding suggests that only a few amino acids of the vp /vp proteins present on the surface of the virion are potentially subjected to a strong selective pressure by the host immune response. in conclusion, this study confirms that hbov is less common than other respiratory viruses but that the frequency of its detection in children with respiratory disease is in time stable. it was detected with a prevalence of about % in several consecutive seasons and no unusual clustering was observed among identified strains, with strains circulating in being closely related to those circulating in . moreover, only a minority of virus sites were found to be under positive selective pressure, and all the strains detected in respiratory tract infections of this italian study belonged to genotype . from a clinical point of view, this study highlights that in otherwise healthy children, hbov seems to have relatively low clinical relevance, because patients infected with hbov alone mainly suffered from an urti. the viral load was not associated with clinical characteristics of the infection, and viral mutations, despite affecting viral replication, did not affect the conditions or severity of the clinical presentation. further studies are needed to clarify the clinical relevance of hbov in children, particularly in those at risk for severe chronic underlying disease, and to evaluate the role of viral modification in conditioning the degree of viral virulence and the specific immune response. conceived and designed the experiments: np se. performed the experiments: ap az as. analyzed the data: fb. contributed reagents/materials/analysis tools: sbi gu as sbo ef. wrote the paper: np ap se. cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infections human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections impact of viral infections in children with community-acquired pneumonia: results of a study of respiratory viruses impact of human bocavirus on children and their families the human bocavirus role in acute respiratory tract infections of pediatric patients as defined by viral load quantification clinical and socioeconomic impact of different types and subtypes of seasonal influenza viruses in children during influenza seasons textbook of pediatric infectious diseases comparison of the luminex respiratory virus panel fast assay with in-house realtime pcr for respiratory viral infection diagnosis comparison of the luminex xtag respiratory viral panel with 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amongst an allages population hospitalised with acute lower respiratory infections in cambodia complete coding sequences and phylogenetic analysis of human bocavirus (hbov) human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection rapid molecular evolution of human bocavirus revealed by bayesian coalescent inference key: cord- -jizitnfl authors: meyer, r.f.; morse, s.a. title: viruses and bioterrorism date: - - journal: encyclopedia of virology doi: . /b - - . - sha: doc_id: cord_uid: jizitnfl the use of viral agents for biological warfare has a long history, which predates their recognition and isolation by culture. advances in viral culture and virus stabilization made during the second half of the twentieth century raised the level of concern by facilitating the large-scale production of viral agents for aerosol dissemination. furthermore, the nucleic acid of many viruses, including some that are currently not threats, can be manipulated in the laboratory. thus, the potential for genetic engineering and misuse of biotechnology is a serious threat. an effective defense against viral agents requires a comprehensive approach including restricting access to viral stocks, detecting deliberately induced disease outbreaks, rapid laboratory identification of viral agents in clinical specimens, preventing person-to-person transmission, using reliable decontamination procedures, and developing effective vaccines and antiviral drugs. between virus names and species names would be to change the current names of virus species into nonlatinized binomial names. such a system, which has been advocated by plant virologists for many years, consists in replacing the word 'virus' appearing at the end of the existing species name by the genus name which also ends in '-virus'. measles virus then becomes measles morbillivirus, hepatitis a virus becomes hepatitis a hepatovirus, and tobacco mosaic virus becomes tobacco mosaic tobamovirus. the advantage of such a system, which could be implemented without problems for about % of all virus species names, is that inclusion of the genus name in the species name provides additional information about the properties of the virus. a changeover to binomial species names would not affect the common names of viruses in english or other languages since names such as measles virus or 'virus de la rougeole' would remain the same. the ictv is currently debating the possibility of introducing binomial species names and some decision is likely to be made in the near future. given that the common names of viruses are used repeatedly in scientific texts there is a need for abbreviating them and the ictv has published several lists of recommended acronyms for virus names. since the names of virus species are used only very seldom in publications, there is no need to abbreviate them. if binomial names of virus species were introduced in the future, the abbreviations of common names of viruses will of course not be affected. man has known that biological organisms and toxins were useful as weapons of war long before the germ theory of disease was understood. however, as the twentieth century came to a close, the perceived difficulties in production, weaponization, and deployment of these biological weapons as well as a belief that moral restraints would preclude the use of these weapons gave many a false sense of security. recently, a number of events have served to focus attention on the threat of terrorism and the potential for the use of biological, chemical, or nuclear weapons against the military, civilian populations, or agriculture for the purpose of causing illness, death, or economic loss. this possibility became a reality in october when someone sent spores of bacillus anthracis to media companies in new york city and boca raton, florida, resulting in five deaths, considerable panic throughout the united states and other countries, and raised the awareness of our vulnerability. there are more than species of infectious organisms that are known to be pathogenic for humans; many additional organisms are capable of causing disease in animals or plants. realistically, only a few of these infectious agents pose serious problems or are capable of affecting human, animal, or plant health on a large scale. even fewer of these agents are viruses. viruses that could be used as weapons against humans, animals, or plants generally possess traits including ease of production and dissemination, transmissibility, environmental stability, and high morbidity and mortality rates. the use of biological agents is often characterized by the manner in which they are used. for the purposes of this article 'biological warfare' is defined as a special type of warfare conducted by a government against a target; 'bioterrorism' is defined as the threat or use of a biological agent (or toxin) against humans, animals, or plants by individuals or groups motivated by political, religious, ecological, or other ideological objectives. furthermore, terrorists can be distinguished from other types of criminals by their motivation and objective; however, criminals may also be driven by psychological pathologies and may use biological agents. when criminals use biological agents for murder, extortion, or revenge, it is called a 'biocrime'. the use of viral agents for biological warfare has a long history, which predates their recognition and isolation by culture. their early use is consistent with what, at the time, was known about infectious diseases, particularly smallpox. in the sixteenth century, the spanish explorer, francisco pizarro, presented the indigenous peoples of south america with variola-contaminated clothing, which resulted in widespread epidemics of smallpox. during the french and indian war ( - ), sir jeffrey amherst, commander of the british forces in north america, suggested the deliberate use of smallpox to 'reduce' native american tribes hostile to the british. captain ecuyer (one of amherst's subordinates), fearing an attack on ft. pitt from native americans, acquired two variola-contaminated blankets and a handkerchief from a smallpox hospital and, in a gesture of good will, distributed them to the native americans. as a result, several outbreaks of smallpox occurred in various tribes in the ohio river valley. in , during the revolutionary war, the british attempted to spread smallpox among the continental forces by inoculating (variolation) civilians fleeing boston. in the south, there is evidence that the british were going to distribute slaves who had escaped during hostilities, and were sick with smallpox, back to the rebel plantations in order to spread the disease. the use of viruses other than variola major is a more recent phenomenon and reflects an increased knowledge of how to grow and stabilize viruses for delivery purposes. allegations have been made by the government of cuba that the cia was responsible for the massive outbreaks of swine fever in and dengue fever in that ravaged the country. however, subsequent investigations have failed to find substantive proof of cia involvement in these outbreaks. the aum shinrikyo, a religious cult responsible for the release of sarin gas in the tokyo subway system, was also involved in biological warfare activity and sent a team of people to zaire to acquire ebola virus. fortunately, they were unsuccessful in this endeavor. in , unknown farmers in new zealand deliberately and illegally introduced rabbit hemorrhagic disease virus (a calicivirus) onto the south island as an animal control tool to kill feral rabbits. over the past two decades, the human immunodeficiency virus (hiv) has been involved in a number of biocrimes. this most likely reflects the availability of hivcontaminated blood as a source of this virus. for example, in , graham farlow, an asymptomatic hiv-positive inmate at a prison in new south wales, australia, injected a guard with hiv-contaminated blood. the guard became infected with hiv; farlow subsequently died of aids. in , brian t. stewart, a phlebotomist at a st. louis, mo hospital, injected his -month-old son with hivcontaminated blood during a fight over payment of child support. in , iwan e. injected his former girlfriend with . ml of hiv-contaminated blood after she broke up with him. in , dr. richard j. schmidt, a married louisiana gastroenterologist, injected a former lover with hivcontaminated blood. molecular typing of the hiv strains demonstrated that she contracted the same strain of hiv as found in one of dr. schmidt's patients. in perhaps the most famous case, dr. david acer, a florida dentist infected with hiv, transmitted the disease to six of his patients between and . the intentional infection of these patients is a possibility although there is no direct evidence. in spite of these incidents, hiv has not been included on lists of threat agents for public health bioterrorism preparedness. however, some contend that hiv has great weapon potential if the goal is to destabilize a society. viruses have also been involved in suspected incidents or hoaxes. in , an article appeared suggesting that the cia was investigating whether iraq was responsible for causing the outbreak of west nile fever in the new york city area. the story relied heavily on a previous story written by an iraqi defector, claiming that saddam hussein planned to use west nile virus strain sv to mount an attack. the investigation indicated that there was no known evidence of bioterrorism involved in the spread of west nile virus. a fictional 'virus' was also involved in one of the largest bioterrorism hoaxes in . according to e-mail messages widely circulated on the internet, an organization known as the klingerman foundation was mailing blue envelopes containing sponges contaminated with a fictional pathogen called the 'klingerman virus'. according to the e-mail alert, people had been infected with the virus, including who died. advances in viral culture and virus stabilization made during the second half of the twentieth century facilitated large-scale production of viral agents for aerosol dissemination. a report for the united nations on chemical and biological weapons and the effects of their possible use gave estimates on the numbers of casualties produced by a hypothetical biological attack (table ) . three viruses (rift valley fever virus, tick-borne encephalitis virus, and venezuelan equine encephalomyelitis (vee) virus) were evaluated in a scenario in which kg of the agent was released by aircraft along a km line upwind of a population center of . the viral agents produced fewer casualties and impacted a smaller area when compared with the bacterial agents used in this hypothetical model. of note, smallpox was apparently not evaluated because it had not yet been eradicated and level of vaccine-induced immunity in the population was high. viral agents were part of the biological weapons arsenal of both the soviet union and the united states ( table ) . vee virus was stockpiled by both countries as an incapacitating agent; variola major and marburg viruses were stockpiled as lethal agents by the soviet union. the soviet union reportedly conducted a live field test of variola major virus on vozrozhdeniye island in the aral sea in the s, in which g of the virus was released into the atmosphere by explosion. unfortunately, a laboratory technician who was collecting plankton samples from an oceanographic research vessel km from the island became infected. it was reported that after returning home to aralsk, she transmitted the infection to several people including children. all those infected died. a number of other viruses that infect humans (e.g., ebola virus, lassa fever virus, enterovirus ) or livestock (e.g., foot and mouth disease virus, rinderpest, newcastle disease virus) have also been studied for their offensive capabilities or for the development of medical and veterinary countermeasures. today, with the increased level of concern, a number of viruses have been cited as possible weapons for use against humans or animals ( table ). the requirements for an ideal biological warfare agent include availability, ease of production, stability after production, a susceptible population, absence of specific treatment, ability to incapacitate or kill the host, appropriate particle size in aerosol so that the virus can be carried long distances by prevailing winds and inhaled deeply into the lungs of unsuspecting victims, ability to be disseminated via food or water, and the availability of a vaccine to protect certain groups. other factors such as the economic and psychological impact of an attack on animal agriculture with a viral agent must also be considered. variola major is considered to be the major viral threat agent for humans. thus, considerable effort has been expended toward preparing the public health and medical communities for the possibility that this agent will be employed by a terrorist. variola major is considered to be an ideal terrorist weapon because it is highly transmissible by the aerosol route from infected to susceptible persons; the civilian populations of most countries contain a high proportion of susceptible persons; the disease is associated with a high morbidity and about % mortality; initially, the diagnosis of a disease that has not been seen for almost years would be difficult; and, other than the vaccine, which may be effective in the first few days post infection, there is no proven treatment available. alphaviruses ( table ) are also of concern because they can be produced in large amounts in inexpensive and unsophisticated systems; they are relatively stable and highly infectious for humans as aerosols, and strains are available that produce incapacitating (e.g., vee) or lethal infections (eee case fatality rates range from - %). furthermore, the existence of multiple serotypes of vee and eee viruses, as well as the inherent difficulties of inducing efficient mucosal immunity, make defensive vaccine development difficult. the filoviruses and arenaviruses that cause hemorrhagic fever have also been considered as agents that might be used by terrorists because of their high virulence and capacity for causing fear and anxiety. the filoviruses, ebola and marburg, can also be highly infectious by the airborne route. humans are generally susceptible to infection with these viruses with fatality rates greater than %, and infection can be transmitted between humans through direct contact with virus-containing body fluids. there are five species of arenaviruses (lassa fever, junin, machupo, guanarito, and sabia) that can cause viral hemorrhagic fevers with a case fatality rate of about %. large quantities of these viruses can be produced by propagation in cell culture. infection occurs via the respiratory pathway suggesting that dissemination via aerosol might be used by a terrorist. human to human transmission has also been reported with aerosol transmission the most likely route for at least some of the secondary cases. the filoviruses and arenaviruses discussed above are bsl- bacillus anthracis > note. these estimates are based on the following scenario: release of kg of agent by aircraft along a km line upwind of a population center of . agents and diagnostic capacities for infections caused by these viruses are limited. because the nucleic acid of many viruses, including some that are currently not threats, can be manipulated in the laboratory, the potential for genetic engineering remains a serious threat. biotechnology, which has had a tremendous impact on the development of medicines, vaccines, and in the technologies needed to counter the threat of naturally occurring disease, can also be used to modify viruses with unintended consequences or even for the development of novel biological agents. several examples involving viruses are presented below. an australian research group was investigating virally vectored immunocontraceptive vaccines based on ectromelia virus, the causative agent of the disease termed mousepox. they created a recombinant virus, which expressed the mouse cytokine il- in order to enhance the antibodymediated response to other recombinant antigens carried on the virus vector. instead, the ectromelia virus vector expressing il- altered the host's immune response to this virus resulting in lethal infections in normally genetically classification of viral agents that are considered to be of concern for bioterrorism and biowarfare and those that have been weaponized or studied for offensive or defensive purposes as part of former or current national biological weapons programs resistant mice (e.g., c bl/ ). additionally, this virus also caused lethal infections in mice previously immunized against infection with ectromelia virus. the creation of this 'supermousepox' virus led to speculation that similar genetic engineering could be performed on variola major leading to a biological weapon that would be effective against an immunized population. the influenza pandemic of - , which followed world war i, was uniquely severe, causing an estimated - million deaths globally. this pandemic happened before the advent of viral culture and very little was known about the virus until the discovery of the polymerase chain reaction (pcr). recently, the complete coding sequences of all eight viral rna segments has been determined by using reverse transcription-pcr (rt-pcr) to amplify the viral rna sequences from formalin-fixed and frozen tissue samples from individuals who died during this pandemic in an effort to shed light on both the reasons for its extraordinary virulence and evolutionary origin. more recently, researchers reconstructed the spanish influenza pandemic virus using reverse genetics and observed that this reconstructed virus exhibited exceptional virulence in the model systems examined and that the hemaglutinin and polymerase genes were essential for optimal virulence. a full-length poliovirus complementary dna (cdna) (c. bp) has been synthesized in the laboratory by assembling oligonucleotides of plus-and minus-strand polarity. the synthetic poliovirus cdna was transcribed by rna polymerase into viral rna, which translated and replicated in a cytoplasmic extract of uninfected hela s cells, resulting in the de novo synthesis of infectious poliovirus. the publication of this research raised concerns that more complicated viruses (e.g., variola major or ebola) could be synthesized from scratch based on publicly available sequences, or that viruses could be created that do not exist in the wild. an effective defense requires a comprehensive approach that includes: prevention of access to viral stocks; improved means of detecting deliberately induced disease outbreaks; rapid medical recognition of specific syndromes (e.g., hemorrhagic fever syndrome); rapid laboratory identification of viruses in patient specimens; prevention of person-person transmission; reliable decontamination procedures; development of effective vaccines; and development of effective antiviral therapy. rapid and accurate detection of biological threat agents is the basis of an effective public health response to bioterrorism. in order to address this issue, cdc in collaboration with other partners established a national network of laboratories called the laboratory response network (lrn), which was provided with the tools to accomplish this mission. rapid assays utilizing advanced molecular and immunological technologies for detection of agents such as variola virus, as well as emerging public health threats such as sars coronavirus and h n influenza virus, were distributed to member laboratories. equipment, training, and proficiency testing are elements of the lrn and contribute to a uniform operational plan. the importance of high-quality standardized testing for detection of these agents is exemplified by the rapid need for medical countermeasures to protect or treat civilian populations. accurate laboratory analysis is a major element in the decision process for deployment of the federal government's strategic national stockpile (sns) of medical countermeasures. as part of the effort to deter biological terrorism and strengthen the law enforcement response to such an act, the us recently established a microbial forensic laboratory known as the national bioforensics analysis center that operates in partnership with the federal bureau of investigation. scientists are already developing methods for the forensic investigation of incidents involving viruses. for the terrorist, the use of a viral agent would pose a challenge due to problems associated with acquisition, cultivation, and dissemination. the target for an attack with a viral agent can range from humans to animals and plants. therefore, agricultural targets are also a major concern. nature has provided many challenges to combating viral diseases. viral agents are much more prone to genetic variation and mutation, and can be manipulated or created in the laboratory to take on desired characteristics. differentiating between natural and intentional viral disease outbreaks can be challenging. unlike bacterial diseases, many of which are treatable, there are fewer medical countermeasures to employ when dealing with viral infections. laboratory diagnostic methods and reagents must continuously be refined to account for genetic changes and variants. thus, the challenge of developing bioterrorism countermeasures is significant. fortunately, this effort contributes to combating natural disease events more effectively, which has global benefits. see also: aids: disease manifestation; aids: global epidemiology; aids: vaccine development. defense against filoviruses used as biological weapons microbial forensics bioterrorism and biocrimes. the illicit use of biological agents since chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template arena viruses other than lassa virus did bioweapons test cause a deadly smallpox outbreak? smallpox as a biological weapon princes and peasants. smallpox in history expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states molecular evidence of hiv- transmission in a criminal case public health assessment of potential biological terrorism agents viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control characterization of the reconstructed spanish influenza pandemic virus world health organization( ) health aspects of chemical and biological weapons alkaliphilic having a requirement for an environment with a high ph. burst size the number of infectious virus particles released per cell. carrier state persistent infection of a host cell by a virus, with the surviving host persistently carrying and continually producing virus without entering a lysogenic state. circular permutation a change in the sequence of the linear dna termini that does not alter the relative sequence (e.g., circular permutation of abcdefgh could generate bcdefgha, cdefghab, etc). concatamer two or more dna molecules that are linked together to form a long, linear dna molecule. cured a host cell that was once a lysogen, but no longer carries viral dna in any form. halophilic having a requirement for an environment with a high salt concentration. headful packaging the mechanism of packaging viral dna based on the size of the virus head, rather than the length of the viral genome. hyperthermophile having a requirement for an environment with a high temperature (> c).insertion sequences repetitive sequences of dna that can move from one site to another within the viral dna. integrase/recombinase an enzyme which can integrate viral dna into the genome of its host cell. lysogen a host cell that has been infected by a virus that remains dormant, despite the presence of viral dna. lytic virus a virus that is able to infect a host, replicate, and subsequently leave the host cell by rupturing (lysing) the host cell. methanogenic having the ability to produce methane. monovalent a virus that has a host range limited to one species. prophage a virus that is dormant within the host cell. protein-primed replication replication of dna via the interaction of the dna polymerase with specific proteins, rather than dna or rna primers. temperate virus a virus that is able to infect a host, but remain dormant within the host cell. terminal redundancy linear dna with the same sequence at each end. transduction the transfer of host dna from one host cell to another by a virus. transfection the introduction of pure viral genomic dna into a host cell, producing viable virus. key: cord- -leicos j authors: ketzinel‐gilad, mali; shaul, yosef; galun, eithan title: rna interference for antiviral therapy date: - - journal: j gene med doi: . /jgm. sha: doc_id: cord_uid: leicos j silencing gene expression through a process known as rna interference (rnai) has been known in the plant world for many years. in recent years, knowledge of the prevalence of rnai and the mechanism of gene silencing through rnai has started to unfold. it is now believed that rnai serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing rnai have been found in various viral pathogens. during the past few years, it has been demonstrated that rnai, induced by specifically designed double‐stranded rna (dsrna) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. however, many key questions and obstacles in the translation of rnai into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsrna that induces the system. it is expected that for the specific examples in which the delivery issue could be circumvented or resolved, rnai may hold promise for the development of gene‐specific therapeutics. copyright © john wiley & sons, ltd. the battle against viral infections is ferocious. since viruses are developing resistance to therapy, novel antiviral therapeutic modalities are in great demand. the currently approved antiviral therapies are based on the use of small molecular weight drugs, utilization of proteins simulating the innate immune response, and the adaptive immune system for both passive and active vaccination [ ] . recently, an antisense drug against viral infection has also been approved, suggesting that newly developed approaches are acceptable. the first drug using antisense technology, fomivirsen (vitravene), developed by isis pharmaceuticals, was approved for the treatment of cytomegalovirus (cmv) retinitis. in general, small molecular weight drugs are mainly used today for chronic viral disease, although there are exceptions such as the anti-influenza compound, oseltamivir/tamiflu, which is used for acute infection. passive and active vaccination approaches are being developed for the prevention of severe acute diseases (in this case, there are also exceptions, such as life administration of anti-hepatitis b virus (hbv) antibodies to liver transplant patients at risk for hbv infection recurrence). the development and use of specific antiviral drugs and vaccines have been slower than expected and face major challenges. one particular example is the sluggish progress in generating effective drugs against the hepatitis c virus (hcv), even though the genomic sequence of the virus was unfolded over years ago. hiv drug development also faces major drawbacks. although hiv replication is efficiently inhibited by the combination of highly active antiretroviral therapy (haart), long-term complications of haart include significant morbidity on the one hand, and the generation of multi-drug-resistant hiv strains on the other in a large proportion of patients. in spite of the comprehensive advancement in understanding the biology of the immune response, translating these findings into rational therapeutic platforms remains slower than expected. the need for preventive and effective vaccines remains as much a requisite today as it was in previous times in the history of mankind. indeed, at the beginning of the previous century, the world suffered devastating viral infection pandemics such as the one that occurred in where a quarter of the world population fell ill and the death toll reached over million people from acute influenza infection. today, the achievement of peak vaccine efficacy to treat influenza stands at % among those under years old and just % among the elderly. even today, the annual death toll from acute influenza infection in the usa tops , in spite of national vaccination programs. thus, different strategies are currently being suggested in an effort to be prepared for future pandemics [ ] , and these include the development of escape mutants (antigenic drift) and reassortment of genetic segments of different quasispecies of the same virus or of different viruses (antigenic shift). presently, human viral pathogens are spreading worldwide, such as the much publicized menacing spread of the avian flu which is reported to have expanded to remote sites of russia and kazakhstan from the south-east, posing a major health threat to the entire world. in an effort to identify a novel class of efficient antiviral therapeutics, numerous technologies are currently being assessed for their antiviral potential. these include antisense oligonucleotides, antisense phosphorodiamidate morpholino oligonucleotides, ribozymes and, in recent times, rna interference (rnai). incidentally, we have been encouraged to learn that sirna (short interfering rna) against vascular endothelial growth factor (vegf) has recently been administered to patients in a clinical study without major side effects (asgt meeting, ) . in this review, we will summarize the recent developments in the use of rnai as an antiviral agent. rna interference (rnai) is a sequence-specific silencing of genes, induced by small molecules of double-stranded rna (dsrna). this phenomenon was first observed in plants in the late s, but its molecular mechanism remained unclear until it was discovered in by fire et al., in the nematode c. elegans [ ] . they showed that the presence of a very small quantity of dsrna led to almost a complete shut-off of the expression of the gene that was homologous to the dsrna. the interference process starts with cleavage of the dsrna that induced it into small rna duplexes, - nucleotides (nt) long, called sirnas (short interfering rnas) [ , ] . these small dsrna duplexes have nt overhang at their end, a -monophosphate and a -hydroxyl group [ ] . the enzyme responsible for that first step is dicer, a dsrna-specific nuclease that belongs to the rnaseiii family, and acts in an atp-dependent manner [ ] . in the next step, the sirnas generated by the dicer are incorporated into the rna-induced silencing complex (risc), a multi-component enzymatic complex [ ] . the risc unwinds the sirna in an atp-dependent manner, and uses its single-strand form to target the homologous transcript by base-pairing interactions. it then cleaves the mrna by its endonuclease component in a homology-dependent manner, only in the region corresponding to the sequence of the sirna. this process leads to degradation of the mrna [ , ] . sirna-mediated gene silencing has also been found in lower organisms, such as plants, fungi, worms and flies [ ] . it is a conserved mechanism of intracellular antiviral immunity that also protects the host genome from foreign genetic elements such as retroviruses, transposons and retrotransposons. these elements may have deleterious effects on the genomic dna of the host, and thus their mrna elimination may represent an earliest form of innate immunity. rnai was first suggested to evolve as a natural antiviral defense in plants, especially against rna viruses [ , ] . in mammalians, rnai has also been reported to have gene-silencing properties. the rnai machinery was triggered experimentally by the introduction into the cells of artificially designed dsrna molecules, nt in length, and the target gene was inhibited in a sequencespecific manner [ ] . this effect has become very effective in silencing and knocking-down expression of specific genes in the cells. sirnas have become the method of choice for mammalian cell genetics as well as for potential sequence-specific therapeutic approaches. the inhibitory action of sirnas has been documented for numerous viruses. it works against rna viruses with negative-or positive-strand genome polarity, as well as against dna viruses. the sirna, as a therapeutic tool, can be targeted against the various phases of the viral life cycle of dna and rna viruses including replication, transcription, assembly of new virions, and budding out of the target cells ( figure ). proteins for transcription. many rna viruses encode their own transcription factors. for example, the gag, pol and env genes of retroviruses are needed for an efficient transcription. indeed, sirnas directed against the gag and env genes of hiv- [ , ] or the avian sarcoma leucosis virus [ ] significantly reduce their overall transcription. another example is the dna human papilloma virus; sirna directed against the viral transcription factor e inhibits viral gene expression and growth in tissue culture [ , ] . baculoviruses infect many different insect species. over many years, the autographa californica nucleopolyhedrosis virus has caused severe economic losses in the silk industry. inhibition of that virus was achieved by a pair of sirnas that target specifically the viral coded early transcription activator, and the major nucleocapsid [ ] . one of the main steps in the process of viral infection is dna and rna genome replication of the virus. the genome of rna viruses, especially with plus polarity, serves both as mrna and as a replication template. many research groups applied the sirna method to inhibit replication of viruses in vitro and in vivo. in the absence of an efficient cell culture system for growing hcv, the sensitivity of hcv to rnai was shown in the replicon-based system. an hcv replicon is derived from an hcv consensus genome that was cloned from a viral rna isolated from an infected human and used to construct subgenomic selectable replicons. upon transfection of these subgenomic selectable constructs into a cell line, these rnas were found to replicate to high levels. in several studies, sirnas were directed against different targets in the virus genome [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, sirna specific for the untranslated region (utr) of the hcv genome, introduced into huh cells carrying the replicon system, inhibits hcv replication by up to % [ ] , as measured by the expression level of the replicon luciferase reporter gene. sirnas targeting the viral polymerase ns b region reduced expression of ns b-luc chimera in mice [ ] or in the replicon system in vitro. other studies that target other regions of the hcv genome reported a significant decline in the level of hcv proteins and the level of both the sense and antisense rna strands [ ] . the sirna effect shown for hcv is ifn-and cell-cycleindependent [ ] . in the hepatitis a virus (hav) replicon-based system, it has been reported that sirnas targeting the regions coding for the non-structural proteins of the virus give rise to partial inhibition of hav replication [ ] . in that study, two sirnas specific for hav sequences increased rather than inhibited hav replication. this could be due to complex secondary structures of the target region that can limit and reduce the efficiency of the rnai process [ ] . in another study, sirna targeted to various domains of the hav internal ribosomal entry site (ires) induced efficient and sustained suppression of viral genome translation and replication [ ] . poliovirus is a highly cytopathic rna virus. sirnas specific to the poliovirus genome inhibited viral replication, as was demonstrated in a poliovirus replicon system. the sirna effect led to viral genome clearance from the infected cells, without destruction of the cells harboring the virus [ ] . additional examples of inhibition of viral replication by sirna originated from the study of positive rna viruses such as dengue (denv), west nile (wnv), and severe acute respiratory syndrome (sars) [ ] [ ] [ ] [ ] [ ] . sirnas targeting the -utr sequence of denv, in a region that is conserved in all the dengue serotypes, reduced viral replication and infection in dendritic cells [ ] . sirnas targeting the sars-cov rna polymerase gene inhibited viral rna replication, protein synthesis and reduced the viral cytopathic effects on vero cells [ ] . likewise, expression vectors of sirnas specific for two different regions of the wnv genome protected t cells from wnv infection, and significantly reduced viral rna replication and virus production [ ] . coxackievirus b (cvb ), a member of the picornaviridae family, is a major cause of many human diseases, such as meningioencephalitis and myocarditis. synthetic sirna targeted to the vp or to the viral polymerase showed antiviral effects in infected hela cells by inducing a significant reduction of viral replication [ ] . the footand-mouth disease virus (fmdv) replication was inhibited in bhk- cells by sirnas targeting various conserved regions of the fmdv genome [ ] . multiple sirnas have been used to target multiple conserved viral genes that are essential for virus replication, including a long non-coding region, a short -non-coding region, the viral protein vpg, the viral polymerase, and the viral capsid protein vp . the combination of those sirnas gave rise to a - , -fold inhibition in virus yield by specific inhibition of viral replication [ ] . the antiviral properties of rnai have not been assessed in comparison for their effectiveness upon targeting the different intracellular stages of the viral life cycle. however, from current reports, we could surmise that targeting viral replication, similar to what has been described in several other types of antiviral methods, would probably be the suggested approach to suppress viral infection. replication of dna viruses can be inhibited by targeting their viral mrna, whereas replication of rna viruses can be inhibited by targeting either their mrnas or their viral rna, as was elegantly demonstrated for hiv [ ] . in the later stages of the virus life cycle, the structural proteins are produced to assemble and form mature virions before egress. rotavirus causes severe diarrhea in infants and children worldwide. to combat this virus, dector et al., utilized sirna directed to the vp , a viral structural protein that is essential for the attachment of the infecting virus to the cell surface. they showed a significant reduction in the number of viral particles produced in ma , an infected monkey kidney cell line. moreover, most of the viral particles that were produced were poorly infectious [ ] . however, there are only few reports assessing specifically the potential of the rnai effect on viral assembly [ ] . the antiviral properties of rnai against viral assembly, a late stage in the intracellular viral life cycle, is expected to be less effective than rnai in the early steps of the viral life cycle. in many rna viruses, there is emergence of quasispecies that contain point mutations in the sirna's target sequences leading to evasion from inhibition by sirna. using a pool of sirnas to simultaneously target multiple sites in the viral genome can prevent the emergence of these resistant viruses [ , ] . another approach that may partially solve this problem is targeting cellular factors rather than viral genes. during their life cycle, viruses apply cell membrane receptors for penetration, and cellular transcription factors for viral replication, harnessing very efficiently the cellular transcription and translation machinery for their life cycle. targeting those cellular genes may be another strategy for inhibition of viruses. and indeed, zhang et al., for example, succeeded in suppressing the replication of hcv in the replicon system by the expression of sirnas against cellular cofactors that are needed for viral replication, the polypyrimidine tract-binding protein (ptb) or eif bg [ ] . inhibition of the ptb alone by sirnas resulted in an efficient decrease in the levels of hcv proteins as well as hcv rna replication in huh cells harboring the hcv replicon [ ] . in another study, sirna against cellular rna helicase p reduced hcv negative strand replication [ ] . the e protein of human papillomaviruses (hpvs) contributes to oncogenesis. e was found to specifically activate the transcription of the cellular transcription factor e f in an in vitro system of differentiating human keratinocytes. while suppression of e f levels through the use of sirna decreased hpv replication, this loss did not affect cell proliferation. thus, e f is a potential target for antiviral therapies [ ] . the human cyclin t (hcyct ) is a cellular factor essential for transcription of messenger and genomic rnas from the long terminal repeat (ltr) promoter of the hiv- provirus. sirna directed against hcyct could effectively suppress hiv- replication without any induction of apoptotic cell death [ ] . in previous studies, downregulation of other cellular factors, such as cd , cxcr , ccr , nf-kb, p-tefb, cyclophilin a, dc-sign, spt- and parp- , successfully inhibited hiv- replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, since many of these molecules are essential for cellular processes (cd , e.g., is a cell-surface molecule important for adaptive immune response), not all of them can serve as a practical target for hiv gene therapy. to conclude, sirnas can be used for inhibition of both rna and dna viral infections from the early stage of viral attachment to the cell to the late stage of viral assembly. sirnas can be targeted directly to the viral genes involved in the viral life cycle, or against cellular genes which are used by the viruses. in each case, the best strategy for viral inhibition needs to be assessed according to the virus type and its unique life cycle. interestingly, a combination therapy of two sirnas, targeting different viral sequences, each with inhibitory function, did not have an enhanced effect. this was also found by other groups, assessing the rnai effect in other human and non-human pathogens. these repeated observations could be related to the early saturation of the rnai cellular machinery. however, this issue will need further investigation, which could lead to improvement of the efficacy of rnai against viral infections. in some specific cases of acute viral infections, in particular those cases which could pose a major threat to the health care system, several hurdles remain to be overcome for the development of vaccines and specific small drugs. however, in most cases, the viral agent causing the acute severe endemic, possibly pandemic disease, could be rapidly identified and sequenced, as was the case during the recent outbreak of sars. in these cases, the development of sirnas targeted against various regions of the viral genome could lead to a quick development of a therapy against the acute viral infection. the production, delivery, dose, and modes of administration of sirnas could be tailor-designed for any group of targets. suffice to say, it is imperative that the timetable for the generation of a new sirna-based rna silencing drug could be shorter, so that a therapeutic platform against many specific infectious agents with pandemic potential could be forthcoming. obviously, it must be remembered that additional important factors need to be implemented for the development of an antiviral drug, such as in vitro and in vivo models, although these requirements are essential for any other type of therapeutic modality to be produced and tested. one recent report nicely exemplified the fact that synthetic sirna could be generated against a single (respiratory syncytial virus, rsv) or a double infection (with parainfluenza virus, piv), and rapidly tested in vitro, as a sufficiently predictive tool for an in vivo effect [ ] . in this report, the sirnas against both rsv and piv were administered nasally with profound antiviral preventive and therapeutic effects without inducing interferon production. as mentioned in previous sections, sirna is very effective against other life-threatening pandemic threats such as influenza infections [ ] . however, it must be remembered that we are probably just at the beginning of experimental assessments to determine the potential antiviral effects of sirna. the antiviral effects of short hairpin rnas (shrna) or sirnas were also assessed in other viral infections where there is practically no available therapy (shrnas are short hairpin rnas expressed by plasmid and viral vector systems and are subsequently processed to sirnas by the cellular machinery). sirna targeting the protease a region of the most common viral agent causing myocarditis, coxsackievirus b , was found to be effective in inhibiting viral replication in vitro [ ] . in addition, this same group also showed that the antisense sirna strand is critical for the rnai effect and that single nucleotide mutations at the central or regions are detrimental for the antiviral effect. sirna was also effective against sars caused by the newly discovered coronavirus in a preventive model in vitro [ ] . currently, since there is no available effective specific therapy against sars, rnai could be developed for this serious infection. in other cases of severe human infections by viral pathogens, there are fewer promising results than for the antiviral potential against viral replication by inducing rnai. sirna was also assessed against wnv infection. in one report, while the investigators failed to show an antiviral effect in active replicating cells [ ] , they showed prevention of infection in a previous report. another group assessed the potential effect of sirna against wnv infection in vivo. again, only a preventive mode of therapy was found partially effective against viral replication and disease outcome in mice [ ] . from the reports on the use of sirna against human viral pathogens causing acute disease, we could learn that for each specific pathogen infecting a specific cell lineage or tissue, we would probably need to perform an indepth assessment, with proper in vitro and in vivo models, and develop specific delivery systems. although the road towards rnai development could be visible for some of its destinations and the traveling speed could be changing, the target time remains unknown and unpredictable. an interesting new approach of preventing viral infection was reported by the group of judy lieberman [ ] . in an effort to suggest a method of prevention of hsv sexual transmission, intravaginal installation of sirnas targeting two hsv- genes protected mice from a lethal hsv- challenge without inducing interferon-responsive genes. this encouraging result once again proves that combining a realistic method of gene delivery with a specific genetic drug payload for a specific disease could result in a beneficial gene therapy outcome. in most acute viral infections, the host overcomes the invading pathogen through a robust innate and adaptive immune response. however, in those cases where the virus causes severe disease as a result of a significant cytopathic effect, which could be related to a high multiplicity of infection (moi), low immunogenicity, high replication capacity or direct toxic effects, or a combination of all, could result in organ failure or even death. in these cases, rnai could significantly support, or even enhance, the antiviral effects for a short period of time and this could be achieved by the administration of sirnas. characterizing the specific viral pathogen, even in a pandemic situation, could enhance the rational design of a sequence-specific sirna that can be used as therapy. this situation is very different from cases of chronic viral infections of rna viruses, in that there could be quasispecies; in addition, the effect of the rnai should be prolonged and generated through an expression system rather than through a synthetic sirna administered once or only a few times. human chronic viral infections such as hbv, hcv and hiv are a worldwide threat. for hcv and hiv infections, there are no available vaccines, and, in addition, in both the prospect of vaccine development is not encouraging. furthermore, current therapeutics for both of these infections are suboptimal. for these viral infections, numerous gene-based approaches have been developed. although there are effective vaccines against hbv infection, chronic infection is still a major therapeutic challenge. the rnai was used to inhibit replication of dna viruses. hbv replication was inhibited in vitro and in vivo by rnai by us and by others [ ] [ ] [ ] [ ] [ ] . sirnas targeted to different regions of the hbv surface antigen gene robustly inhibited viral gene expression and replication both in vitro and in vivo [ ] . due to the overlapping gene structure of the hbv genome, targeting a region in the open reading frame (orf) of the x gene which is shared by all the viral transcripts resulted in a significant reduction of up to % in all viral transcripts and proteins and in a dramatic reduction of ∼ % in viral replication [ ] . the x gene of hbv was also recently assessed as a target for rnai in vivo [ ] . using two hbv mice models as a naked dna approach with the hydrodynamic method or expressed from an adenovector, the pol iii u promoter encoded short shrnas targeting conserved sequences of the hbx orf. the anti-hbv effect was significant without stimulating the interferon system. it is also known that hbx plays an important role in hepatocarcinogenesis. sirna against hbx was also used to test its effect against hepatocellular carcinoma cell lines which express hbx sequences [ ] . this group demonstrated a significant reduction in cell proliferation, cell growth, anchorageindependent growth in soft agar, and tumor development in nude mice following the expression of sirna against hbx. a recent report suggested that the inhibitory effect of rnai on hbv expression is stronger than that of lamivudine in vitro [ ] . we could further speculate that a combination of rnai and nucleoside analogs might encounter a synergistic effect, although this is yet to be determined. the most challenging part of rnai approaches for chronic viral infections is to design the best delivery method that would facilitate the targeting of the specific organ/cells with the appropriate expression system, for durable intracellular levels of gene-silencing effect. this also applies when designing an rnai approach for hcv infection, as well as for other chronic viral infections. the studies assessing the effect of rnai against hcv were mostly restricted to in vitro replicon systems, as discussed above. alternative in vivo systems were also adopted by some investigators, with reporting proteins used to assess the antiviral effect [ ] . in early studies using the in vitro hcv replicon system, it was shown that a synthetic sirna targeting the core region of hcv inhibited viral proteins and significantly suppressed viral replication for at least days [ ] . at about the same time, a different group showed, also in the replicon system, that sirna against the ns b region (viral polymerase) is most effective in suppressing hcv replication [ ] . this group had also transfected the huh replicon cells with a vector expressing complementary strands of sirna, again targeting the ns b region, under the control of two separate h promoters (pcep , invitrogen). in this case, the suppression effect on hcv replication lasted over weeks. another group targeted similar hcv regions, ns and ns b [ ] ; they introduced shrnas targeted against these two genes into huh -replicon cells. the delivery systems that were used in their study were plasmids or lentiviral vectors harboring shrnas against ns or ns b, expressed from the u promoter. in both cases, they observed similar effects, i.e., suppression of hcv proteins and viral replication. however, shrna against the -utr of hcv resulted in very low levels of inhibition of hcv replication. as mentioned earlier, viral replication is dependent on numerous cellular factors. targeting these viral replication/gene expression cofactors is a potential target for inhibition of viral replication. however, this approach should always be balanced against the potential of generating side effects which could overshadow the beneficial antiviral outcome. a recent study assessed this approach in vitro, targeting two hcv replication cofactors: proteasome α-subunit and hu antigen r. shrna targeted against these two genes that were expressed from an expression vector transfected into huh hcv replicon cells showed a modest reduction of hcv transcription [ ] . however, this modest inhibition on the one hand, and the potential role of both proteins in normal cellular function on the other, might suggest that it is advisable to abstain from such approaches, if possible. an interesting study in vivo targeting the hcv ires, translating a luciferase reporter protein, revealed that the in vitro synthesized shrna, administered systemically by the hydrodynamic method, encountered a sustained antiviral effect lasting over days compared to synthetic sirna [ ] . the use of rnai against hiv infection was reported by a number of groups (table ) . although the results of these studies suggest that hiv could be targeted by rnai, there are major obstacles in translating this therapeutic approach into the clinical setting. most reports have used sequences from laboratory hiv strains. viruses with mutations at the rnai recognition site produced an escape mutant after a long-term rnai pressure. targeting relatively conserved hiv sequences could improve the efficacy of the rnai effect. a recent study looked at the protective effect of shrnas targeting the rev, gag, and vif sequences of a panel of hiv clades [ ] . this study showed that targeting the vif hiv region had a significant inhibition effect on hiv replication. however, the long-term use of any specific sirna or shrna against hiv could probably induce the generation of escape mutants containing nucleotide substitutions at or near the target sites. furthermore, the escape from rnaimediated inhibition could also signify the emergence of mutations that change the hiv rna secondary structures [ ] . all of these data emphasize the significance of hiv evolution during rnai pressure and its potential impact on the use of rnai against hiv. the virus also harbors a specific mechanism that evades the nucleicacid-based innate immunity of human cells against hiv. the genome of hiv contains a plethora of dsrna regions capable of being processed into sirnas targeting the viral genome to suppression [ ] . however, the virus has evolved by a counter process, rendering itself resistant to rnai through the tat protein, altering the dicer effect on viral sequences, and abrogating the host cell innate immunity against hiv infection. interestingly, on the other hand, it is possible that hiv does apply the cellular rnai machinery for regulation of its own gene expression. the hiv nef region encodes a microrna, mir-n , which can block nef expression [ ] . later, it was shown by the same group that mir-n targets the hiv ltr promoter region, downregulating viral transcription; this might be a mechanism by which the virus regulates its own transcription [ ] . single anti-hiv therapy is ineffective against viral replication and gene expression due to the high mutation rate of the virus. one option of overcoming this major obstacle is to generate therapeutics against highly conserved viral genomic regions. a recent report showed that it is possible to clone shrnas against the conserved regions of the hiv genome into hiv vectors, and to suppress hiv infection upon targeting the gag, pol, int and vpu sequences [ ] . however, although this approach could be applied for prevention of infection, cessation of an ongoing hiv replication is prone to failure due to the high mutation rate of the virus. an alternative strategy could be to target essential cellular determinants for hiv infection. the early step of hiv infection would be attaching to the viral cellular receptor. during the progressive stage of the disease, most hiv isolates use the chemokine receptor cxcr for viral attachment and penetration into the host cells. patients with mutations of the cxcr receptor are less prone to hiv infection and are healthy by any significant measure. this finding was the rationale used to develop an anti-hiv approach by targeting cxcr expression and inhibiting viral fusion with the cellular membrane [ ] . it is expected that such an approach will pose a major obstacle to viral evolution and prevent infection. other groups have also adopted this strategy to render cells resistant to hiv infection [ , , [ ] [ ] [ ] . the hiv regulatory protein, rev, is essential for viral life cycle in a number of ways including splicing, translation and trans-activation. with regard to rev trans-activation properties, it needs to interact with the hypusine-containing protein eif- a. the eif- a rev cofactor is activated following a catalytic step performed by the human deoxyhypusine synthase (dhs). a recent study has suggested that rna interference inhibiting dhs blocked hiv replication [ ] . again, as with other drugs in development against hiv infection, we are confronted with the following major questions: what are the potential side effects from such an approach, and what would be the therapeutic dose window? indeed, studies aimed at unfolding these issues are crucial before entering any clinical studies. other groups have also suggested the targeting of other cellular factors essential for the hiv life cycle, including parp- [ ] , the elongation transcription factor p-tefb [ ] , cyclophilin a, dc-sign [ , ] , the human elongation transcription factor spt [ ] , and human cyclin t [ ] . the road towards the development of an efficient therapeutic modality using the anti-hiv rna interference strategy is bumpy, due to potential side effects; moreover, significant strides will have to be made towards harnessing clearcut approaches as described, as well as designing additional rational studies through wet and dry investigations [ ] . reduction in cell-to-cell spread [ ] the cellular level of dicer could be crucial for gene therapy approaches while utilizing the rnai machinery in targeted cells. recent data suggest that, although the expression of dicer and other proteins that participate in digesting long dsrnas into - nt, e.g., eif c ∼ , decreases in differentiated cells, they retain a sufficient amount of enzymatic activity to induce rnai. however, when designing and planning any specific approach using sirna for gene therapy, it is advisable to assess the dicer activity in the targeted tissue if the expected sirna is unmet. since rna silencing acts as an antiviral defense mechanism in plants [ ] , insects [ ] and other eukaryotes, including mammalian cells [ ] , it is not surprising that viruses have developed strategies to interfere with this effect. many plant dna and rna viruses have developed proteins that function as suppressors of rna silencing [ ] [ ] [ ] [ ] [ ] . since the silencing suppression reduces the antiviral effects, the viruses can accumulate in their target cells and reach higher titers. comparing those suppressor genes did not reveal any sequence homology between them. the rna silencing suppressors can act upon the various sequences of the rnai machinery in several ways such as inhibition of sirna processing, inhibition of the incorporation step into the risc, or preventing the action of its effector molecules. although the mechanisms of inhibition of silencing are not fully understood, there are several suppressor genes that their targets have identified. the p protein from the tombusvirus was shown to bind specifically to the sirnas, and thus may inhibit the incorporation step of the sirnas into the silencing effector complexes [ ] . the hc-pro protein, expressed by polyviruses, acts by targeting the risc [ , ] . another example is the mosaic virus b protein (cmv b). this nucleus-localized rnai suppressor protein inhibits the activity of the spreading signal of the rnai, and inhibits dna methylation processes in the nucleus that control the silencing pathways [ , ] . the coat protein of the turnip crinkle virus (tcv) strongly suppresses the rna silencing process at an early initiation step, probably by interference with the function of the dicer cleavage reaction [ ] . since the rnai machinery is conserved in mammals, it appears possible that, similar to plant viruses, viruses that infect invertebrates and vertebrates, including human viruses, have developed strategies to suppress rna silencing. and indeed, there is evidence that such inhibitors are not limited to plant viruses. in insects, the flock house virus (fhv) is a target of rna silencing. it has been shown that in drosophila and mosquito cultured cells, the fhv-encoded protein, b , inhibited rna silencing, but the mechanism is still unknown. this protein also inhibited rnai in transgenic plants, suggesting that the rna silencing pathway is conserved in plants and animals [ , , ] . recent evidence also indicates that human virus genes have the ability to inhibit the rnai pathway. both the influenza virus ns protein and the vaccinia virus e l protein can inhibit rnai in drosophila s cultured cells [ ] . the adenovirus encoded inhibitor of rnai is functional in mammalian cells. the human adenovirus inhibits rnai in the later stages of infection by suppressing the activity of dicer and the risc. the virus-associated rnas, va rnai and va rnaii, bind directly to dicer and function as competitive substrates, squelching it, and the resulting sirnas are incorporated into the active risc [ , ] . the antiviral therapeutic potential of rnai would require identifying possible viral suppressors of the rnai machinery and designing strategies to inhibit their expression. one major drawback for most antiviral approaches is the development of resistance. this is most apparent in cases where the fidelity of the viral polymerase is low, especially in viruses with an rna genome. to overcome this hurdle, most antiviral therapeutic protocols harness a strategy that uses multiple drugs targeting different viral proteins or steps in its life cycle. one example where such resistance has been developed in vitro is in the case of rnai against the nef gene of hiv [ , ] . to overcome this problem, it may be advisable to utilize a multi-targeted rnai approach possibly in combination with additional antiviral modalities. although targeting multiple genomic sites has probably no advantage with regard to the direct antiviral effect, it could repress the development of resistance. the initial steps towards focusing into this avenue have already been established. the group that initially described the sirna effect in vitro in the replicon system against hcv [ ] have shown that, following several rounds of treatment with the same sirna against hcv, the replicon became resistant to that specific sirna, developing a point mutation at the target site. however, the replicon was still sensitive to a sirna targeting a different hcv region. in addition, the development of a resistant replicon was limited by the use of a combination of two sirnas targeting simultaneously different hcv genomic regions [ ] . a similar approach has also been suggested by other groups [ ] . the off-target effect of rnai is the silencing of nontargeted genes containing partial sequence identity to the sirna. in experiments conducted on specificity of sirna in cultured human cells, jackson et al., have demonstrated that sirna can cause direct downregulation of unintended targets containing as few as contiguous bp complementarities [ ] . to increase sirna specificity, there are many sirna design programs that employ various sequence alignment algorithms; however, maximum complementarity, by itself, is not enough for accurate prediction of off-targeting. a study done in dharmacon inc., identified a significant association between off-targeting and exact complementarity between the seed region of the sirna (bases - ) and their offtargeted genes. this pattern has been recognized in mirna-mediated gene silencing, thus suggesting that sirna off-targeting may operate by a mechanism similar to that of mirna targeting (a. birmingham, personal communication). until the off-target mechanism of sirna is understood, this issue can encounter deleterious side effects on the use of rnai. the role of interferon signaling in rnai has given rise to a series of conflicting reports. although most studies suggest that there is very little non-specific effect of sirna, others have shown that the jak-stat pathway is activated following sirna transfection. this effect is mediated by dsrna-dependent protein kinase (pkr) [ ] . while we expect that this issue will foster additional debates, at this point, it would be important to impart cautious interpretations upon describing rnai effects. in addition, recent studies have suggested that although sirna does not activate the intracellular interferon machinery in mammalian cells upon entrance or in situ propagation inside the cells, if they are shorter than -nt dsrna, there is a non-specific innate immune response depicted by cytokine production [ ] . furthermore, this effect is dependent on the toll-like receptor (tlr ) that senses dsrna and serves as its receptor. tlr is located intracellularly on the endosome membrane and signals through nfκb nuclear translocation for the production of inflammatory cytokines. incubation of immune cells with sirna induces the activation of cells [ ] . all of these effects could be dependent on the concentration of sirna. recent works identified putative immunostimulatory motifs within sirnas, and showed that even a slight change of these motifs did not significantly hamper the rnai process [ ] . this research provides a basis for the design of synthetic sirnas that avoids activation of the innate immune response, and helps to minimize immunotoxicity. the group of reuven agami was the first to report a new vector system, called psuper, which directs the synthesis of sirnas in mammalian cells. they used the poly iii h -rna gene promoter to express shrnas that specifically down-regulated gene expression, resulting in functional inactivation of the targeted genes [ ] . recent reports showed that the expression of shrna from the h or the u pol iii promoters in a hiv-based vector induces the expression of interferon-stimulated genes (isgs). this effect is dependent on the presence of an aa dinucleotide near the transcription starting site. preserving a c/g sequence at positions − / + prevents this effect [ ] . in some cases, the expression from the u promoter is relatively low. the enhancer of the cmv immediateearly promoter enhances the u promoter activity [ ] . others have also tested various promoters [ ] reporting some beneficial effects on expression with the modified trna(met)-derived (mtd) promoter, upon expressing shrna against hiv- compared to u or h promoters [ ] . it may happen that for each specific application, we would need to compare numerous regulatory elements to achieve the desired rnai effect. in some systems, it may be important to tightly control the expression of the shrna. although most controlled systems do not reach the desired stringency in vivo, some reports have suggested the use of specific systems. one such method is the tet-onoff expression technology [ ] . again, each investigator should specifically assess the potential of this inducible system in a specific tissue culture or animal model. the teton-off systems were developed for naked dna transfection systems or incorporated into viral vectors like the lentiviral vector [ ] . an additional long debate in the literature questions the choice of the loop structure to apply for the design of shrna. one specific strategy was to adopt the natural loop structure of microrna [ ] . this was also used for shrna against hcv [ ] . the major challenge in rnai gene therapy is to transform the in vitro robust effects of sirna into an in vivo genesilencing method. in other words, what would be the preferred delivery system to use in animals and later on in humans? as for gene therapy in general, and the specific aims of delivering rnai platforms, we need to tailor-design the tools to be used to the sought objective. this includes targeting the tissue, adjusting the desired level of expression (high level of sirna could induce nonspecific silencing [ ] ), the longevity, and the specific maladies that we wish to treat. this is a complex situation, especially since our major barrier is the lack of a simple, non-immunogenic, targeted delivery system without side effects. however, in spite of all of these hurdles, we would like to discuss the potential available methods to deliver synthetic sirna. the most straightforward method of using sirna in vivo is by administering synthetic sirna. upon coinjecting sirna and its target being expressed from a plasmid vector, we could achieve knockdown of expression in the liver following a hydrodynamic injection. in our laboratory, we could show that hbv expression out of an hbv head-to-tail plasmid that supports viral replication in the liver of mice could effectively be silenced transiently with synthetic sirna [ ] . this effect was dosedependent. however, the kinetics of this effect revealed that the silencing was transient as was also shown by other groups using a similar approach [ , [ ] [ ] [ ] . the effect subsides after to h and is probably completely lost after days. the silencing effect of sirna following systemic administration of duplex rna could have been hampered by various factors, thus imposing some logistic hurdles upon translating this approach into the clinical setting (additional examples of rnai against viral infections are depicted in table ). although duplex rna is quite stable in serum [ ] , and more stable than ssdna or ssrna, a high serum concentration could reduce stability. the introduction of phosphorothioate linkages could enhance stability in the serum [ ] . others have used chemically modified sirna with the complete absence of -oh residues on the sense and antisense strands of the dsrna, including -fluoro, -omethyl and -deoxy sugars. these chemical modifications of sirna were produced in order to enhance its stability and effect [ ] . not all modifications have resulted in beneficial silencing effects [ ] [ ] [ ] ; a short review of these modifications was reported by paroo and corey [ ] . one particular interesting report [ ] shows that a specific sirna with a combined chemical modification had a significant and a relatively sustained effect in vitro and in vivo as compared to non-modified sirna. however, although sirna is relatively stable in the serum, there are disadvantages of using synthetic sirna: ( ) the effect of synthetic sirna is transient; in order to impose a long-term effect, repeated administrations would be needed, and this might still be true for the chemically modified sirnas. ( ) the production of synthetic sirna is expensive, making repeated administrations for longterm effect very costly. ( ) it is very complicated to target synthetic sirna to a specific cell or tissue. however, in specific cases, the use of sirna directly administered into the target tissue could encounter a significant effect. in a recent report by dorn et al., they used sirna against the pain-related cation-channel p x , by intrathecal injection of phosphorothioated (ps) sirna in a rat model of neuropathic pain [ ] . although they did not compare the non-ps-modified versus the ps-modified sirna, they have clearly shown a significant effect of sirna in relieving chronic pain. furthermore, the effect was superior to the comparable p x antisense oligonucleotides. one specific case where such an approach could be translated into an applicable clinical therapeutic modality is in post-herpetic neuralgia which could follow varicella-zoster infection. however, since chronic pain is a condition that is generally expected to last for months, this type of treatment would need to be readministered several times. a recent report took advantage of the knowledge generated regarding the stabilization of sirna by chemical modifications, and protection of the modified sirna with pegylated liposomes upon delivery. they assessed the antiviral effect of sirna against hbv in vitro and in vivo. interestingly, the modified sirna had induced less non-specific cytokine secretion combined with an effective and anti-hbv effect of up to weeks after repeated weekly administrations [ ] . in an effort to enhance and prolong the effect of sirna, various approaches have been undertaken using nonviral reagents. tailoring the specific delivery tactic and method is essential when designing a specific therapeutic strategy. one example is the use of sirna targeting the influenza virus genome. this malady can cause moderate to severe illness, can affect millions of people each year, and could be life-threatening. for the gene therapist, this means that the genetic therapy effect could be designed for a short window of time. in this case, the use of synthetic sirna with an enhanced transduction is an appropriate approach. in a study by ge et al., the systemic and intratracheal delivery of polyethylenimine (pei), a cationic polymer, promoting sirna delivery in mice, is beneficial for prophylaxis and therapy of the influenza virus infection [ ] . pei was developed for in vitro and in vivo local and systemic gene delivery and pei-mediated gene delivery/transduction into the lung following systemic and intratracheal administrations. some investigators reported safety problems in specific cases upon the use of pei in animals. however, plasmid dna mixed with pei administered into the human bladder was safe (a. hochberg, personal communication). tompkins et al., also assessed the effect of sirna against the same pathogen, the influenza virus [ ] . this group used a different therapeutic regimen. they used a preventive measure by administering naked sirna systemically, i.e., intravenously, before infection, and at the time of infection, they administered the sirna/oligofectamine  (a lipid carrier from invitrogen) intranasally. in this model, the undertaken therapeutic approach prevented death of animals. however, this study was limited to asking the general question of sirna effect against influenza virus infection rather than comparing different sirna delivery systems. thus, we cannot draw any conclusions that would suggest a preference for any specific delivery method in this disease model system. future animal studies would be required to determine the preferred delivery method. in addition to the lipid carriers, traditionally developed for in vitro and in vivo delivery of dna and now for sirna, alternative approaches have also been developed. one specific interesting report by minakuchi et al. describes the use of atelocollagen (at) [ ] . at is prepared from type i collagen from calf dermis. this is a low immunogenic product that is already available in clinics for various indications like promoting wound healing. the same investigators showed that at enhanced dna delivery and supported prolonged expression. at mediated the delivery of sirna in vitro and was found in vivo to encounter a significant advantage over the sirna/liposome complex in inhibiting tumor growth in mice. recently, more sophisticated delivery methods of sirna have also been developed to treat brain tumors [ ] . the obtained results could be important for those interested in developing sirna therapeutics for viral encephalitis. the model that they used in vivo to assess their delivery and rnai effects was an immunodeficient mouse with an inoculated intracranial human u glioma tumor that was dependent on egf signaling for growth. one of the major barriers for macromolecules to travel to diseased tissue in the brain is the blood-brain barrier (bbb). passing the bbb is a major challenge faced for the development of any pharmacological compound with high molecular weight. pegylated (polyethylene glycol) immunoliposomes ( nm size liposomes designed with monoclonal antibodies over its outer surface) were able to support transvascular delivery of plasmid dna and to target and transduce specific cells in the brain. this group conjugated the pegylated liposomes with two monoclonal antibodies, one against the mouse transferrin receptor to enable bbb crossing, and the second against the insulin receptor to enhance cellular uptake. the generated pegylated immunoliposomes encapsidated a plasmid payload that was designed to express the sirna against the egf receptor in the transduced cell. the sirna complex of pegylated immunoliposomes showed an enhanced antitumor effect by prolonging survival of animals. the ability to treat brain tumors with a systemic approach rather than by stereotactic injection is a major achievement. recently, it has been reported that recombinant hbv capsids can be used as efficient vehicles for oligonucleotide delivery; they can encapsulate the oligonucleotides in vitro, and mediate their delivery into cells very efficiently. the process is not cell-type-specific. this method may be useful for in vivo systems for hbvinfected individuals or in other diseases provided that the immunogenicity of the viral capsids can be decreased; until then, it can be used in cell culture and in ex vivo systems [ ] . the potential advantage of viral-mediated sirna delivery encouraged numerous groups to clone expression cassettes in transgenes and to encapsidate these into viral particles. each type of viral vector holds specific properties. these viral vector characteristics should be those that determine which viral delivery system needs to be applied for the specific therapeutic target. the adenovector [ ] , and in particular the ad-gutless vector, hold major promise for liver-directed systemic delivery. in cases where short-term silencing effect is warranted, the non-gutless vector could then be applied; however, for prolonged silencing effects, the gutless vector might be more beneficial. in the gutless vector, there are practically no restrictions as to the size of the sequences to be incorporated and these could include marker genes, regulatory controlled cassettes or matrix-controlled regions for prolonged expression. controlling the expression of an sirna, specifically in tumor cells, could also be designed in a conditionally replicating adenovirus (crad). crads are designed to replicate and specifically kill tumor cells without harming normal cells. carette et al. [ ] applied crad, which is dependent on rb deficiency for replication, to test its potential in silencing expression by shrnas, a marker gene in vitro in tumor cells. they showed that the silencing effect of a marker gene is dependent on crad replication. the combination of the crad antitumor effect with a sirna against a tumor-dependent growth gene should be assessed in the future; this possibility was not considered by this group, whether in vitro or in vivo. these issues would need further studies in an effort to assess and enhance their therapeutic potential. the major advantage of the retroviral delivery method is the potential to incorporate the payload transgene they 'carry' into the host cell genome. the integration site could not be specifically targeted and this could cause side effects. integration that occurs near cellular protooncogenes can lead to their aberrant expression from the viral ltr, or alternatively this could cause the disruption of a tumor suppresser gene expression. in the past years, numerous groups have reported on various retroviral deliveries [ ] , including lentiviral systems to express sirnas against viral pathogens and tumor cells. ralf bartenschlager and associates reported the use of the moloney murine leukemia virus (mo-mulv)-based vector (pbabe) as a delivery system for sirna targeting hcv [ ] . in their publication, they also assessed a unique rnai approach against hcv infection. this was done in an effort to overcome the low fidelity of the viral polymerase, establishing a state of quasispecies by generating endoribonuclease-prepared sirna to simultaneously target multiple sites of the viral genome in order to prevent escape. as for the retroviral delivery approach, this group designed their sirna mainly against the viral ires sequences. their readout system to assess the silencing effect involved tissue culture cells transfected with the subgenomic hcv replicon. this replicon harbors the hcv ires upstream of the luciferase gene and the neomycin resistance region, and an additional non-hcv ires upstream of the viral non-structural sequences. the presence of the luciferase gene enables the determination of hcv ires activity in vitro. this system enables the assessment of the effect of sirna against the hcv ires as well as against the viral non-structural genome. the sirna in the retrovirus was expressed from the h promoter. they designed numerous sirnas in the vector and found that a specific region in the hcv ires, near the beginning of the viral coding nucleotides, was the most sensitive to rnai effect. although this is an interesting approach with clearcut results, significant developmental steps and modifications are required in order to translate this modality into the clinical setting. however, the results described in this report again suggest that the rnai encounters a therapeutic potential for chronic viral infection. similar observations were reported by other groups ( [ , ] , see table ). presently, most groups who use retroviral vectors to express sirna apply lentiviral vectors. however, it must be kept in mind that the clinical experience with this vector is very limited. numerous papers were recently published describing differently designed lentiviral vectors to meet copyright [ ] or conditioning [ , ] of sirna expression. veerle baekelandt and associates have designed a study to assess the potential use of lentiviruses in delivering sirna into brain tissue. in their recent report [ ] , they constructed a lentiviral vector with sirna against the marker gene egfp. upon simultaneous administration into the brain tissue by stereotactic injection of the lentivirus expressing the egfp and the lentiviral vector expressing the sirna against the same gene, they were able to show almost complete knockdown of egfp expression. in two additional experiments in which the sirna lentivirus was administered before or after the marker gene, they were also able to show significant silencing of expression. interestingly, they claimed, albeit without showing the data, that this effect persisted for months. however, the issue of delivery is again a major barrier. stereotactic administrations are possible, but alternative approaches of systemic delivery or intra-organ spreading of the vector would be beneficial. lentiviruses hold major promise in gene therapy. once the issue of integration and production is overcome, we expect that the lentiviralbased vector will be integrated into the clinical setting. the aav vector is currently perceived as a relatively safe vector since it supports long-term expression in most tissues from an episome. beverly davidson and associates reported an interesting study on the suppression of polyglutamine-induced neurodegeneration in a mouse model of spinocerebellar ataxia (sca ), a disease of the polyglutamine-expansion group which also includes huntington chorea [ ] . they expressed the sirna under a modified cmv promoter due to its enhanced expression/silencing effect compared to the pol iii promoter, in their hands. in addition, they revealed that incorporating the mir loop ( -nt loop sequence) into the sirna expression cassette enhanced the silencing effect, resulting in improved suppression of the ataxin protein levels [ ] . however, this effect was only apparent in the pol iii expression vector. how to select the best loop in any specific case is still an open question, and, presently, this is a matter of empiric assessment. in their mouse model, they injected the aav vector expressing the shrna against the mutant sca message directly into the brain tissue. this treatment showed longterm therapeutic effect on motor coordination as well as a histological improvement by reducing intranuclear inclusions. although this represents a step forward from previous studies with similar models that used antisense and ribozymes as therapeutic agents, we are still far from the clinic. the direct administration of a viral vector into brain tissue is a significant drawback for the current delivery systems. the potential side effects of aav administration into the brain may soon be revealed once results of clinical studies using the aav for direct brain administration in parkinson and alzheimer disease studies are available [ ] . one specific point of importance should be mentioned on the issue of designing loop sequences stated previously. a number of investigators have assessed this matter as related to the effectiveness of silencing. we must bear in mind that for each specific case, there is a need to develop a specific structure to improve the silencing effect [ ] . chemically modified oligonucleotides (oligos) vs. si/shrna as antiviral drugs the jury is still out as to when an antisense approach should be adopted against viral infections or when an si/shrna strategy should be applied. although antisense oligos were discovered more than years ago [ ] their role as antiviral tools in the clinic is still in progress. early studies with antisense oligos have shown promising antiviral potency. however, later reports determined that such approaches encounter significant problems. although comprehensive stringent comparison studies in vitro and in vivo between antisense oligos and sirna were not performed, we would like to stress a few practical points which characterize each group of compounds, in particular those which are important for those investigators interested in designing an antiviral strategy. antisense oligos with a high phosphorothioate content, which are early-generation antisense, interact directly and non-specifically with proteins, potentially interfering with their function [ ] . however, recently developed nd and rd generation rnas -like oligos -have improved significantly the binding affinities of antisense oligos, as well as their nuclease and non-specific proteinbinding characteristics. the new generations include -o-methyl ( om) and the additional version -omethoxyethyl ( ome), both encountering improved nuclease resistance, binding affinity and reduced nonspecific binding affinity. recently, additional novel chemically modified versions of antisense oligos were developed including the locked nucleic acids (lna) [ ] , anhydrohexitol nucleic acids (hnas), peptide nucleic acids (pnas), morpholino nucleic acids (mnas), and other uncharged oligos. all these novel chemicals have been tested and proven to hold significant antisense properties, with antiviral effects in models in vitro and in vivo. their effects are due to the high binding affinity and nuclease degradation resistance, without significant rnaseh activity. to overcome this hurdle, a chimeric version of oligos was generated, the gapmers, containing a core region of a phosphodiester/phosphorothioate flanked on both sides with modified oligo backbones. these gapmers also hold the rnaseh activity of traditional antisenses. when designing any specific experiment aimed to assess an antisense effect, it is important to select the best fitted oligo by testing to different sequences, and compare the selected oligo with appropriate controls containing scrambled, mismatched and irrelevant antisense oligos. although a significant amount of investigations would be needed to confirm which specific antiviral nucleic acid approaches would be best fitted for a specific disease therapy, by comparing antisense or ribozymes and sirna, some general guidelines can be phrased: ( ) for acute viral infections, the preferred type of rna therapy could be a synthetic designed antisense or sirna. ( ) for chronic viral disease targets, a preferred treatment would be such that continuously generates an antiviral drug. this could include an expression vector for any type of nucleicacid-based drug, including antisense, ribozyme or shrna. ( ) for diseases of simple accessible organs, such as ocular cavity, oral cavity, vagina and epidermis, synthetic rnabased drugs might be beneficial. the need for repeated administration might be simple even in chronic disease states in cases of accessible organs. ( ) for chronic multiorgan, or internal organ, involved in viral infections, e.g., chronic viral hepatitis b or c, intracellular continuous expression of high-level non-toxic, effective therapy is desirable. in this case, an expression system delivered into infected cells is warranted. designed shrna based on up-to-date criteria of shrna [ ] [ ] [ ] might be the preferred gene therapy based treatment. ( ) special attention must be given as to the specific type of virus to be targeted; whether a dna or a rna agent. furthermore, it is important to know where, inside the cellular compartments, should the rna-based drug be concentrated, e.g., cytoplasmatic vs. nuclear. for rna viruses, such as hcv, which replicate in the cytoplasm, an antisense approach could be suggested, although it should be kept in mind that the rnaseh effect is preferentially restricted to the nuclear compartment of the cell. however, for chronic hepatitis b virus infection, in which there is a nuclear reservoir of the virus in the form of super-coiled species, a vector which will express its shrna payload in the nuclear compartment is preferred. numerous new expression systems were recently suggested to encounter improved silencing properties [ ] . most of these methods are waiting to be assessed as for their truly beneficial properties as antiviral reagents. in our modern world, viral infections still pose a major threat to mankind. since viruses are developing resistance to the current available therapies, there is an ongoing battle between the viruses and our ability to develop novel strategies to fight them. in vitro and in vivo experiments carried out so far conceivably demonstrate the effectiveness of rnai in inhibiting many viruses that cause severe health and economical problems. even though in vivo experiments in larger animals as well as developing efficient delivery methods have to be done before applying rnai in humans, this fascinating phenomenon will undoubtedly continue to provide new and exciting data regarding its mechanism of action and therapeutic applications in the years to come. strategies and mechanisms for host and pathogen survival in acute and persistent viral infections strategies for containing an emerging influenza pandemic in southeast asia potent and specific genetic interference by double-stranded rna in caenorhabditis elegans rnai: doublestranded rna directs the atp-dependent cleavage of mrna at to nucleotide intervals an rnadirected nuclease mediates post-transcriptional gene silencing in drosophila cells rna interference is mediated by -and -nucleotide rnas role for a bidentate ribonuclease in the initiation step of rna interference duplexes of -nucleotide rnas mediate rna interference in cultured mammalian cells reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants initiation and maintenance of virus-induced gene silencing rna silencing in plants -defense and counterdefense prevention of hiv- infection in human peripheral blood mononuclear cells by specific rna interference inhibition of hiv- infection by small interfering rna-mediated rna interference rna interference against retroviruses in vitro and in vivo growth suppression of human papillomavirus -positive cervical cancer cells by e sirna selective silencing of viral gene e and e expression in hpv-positive human cervical carcinoma cells using small interfering rnas silencing structural and nonstructural genes in baculovirus by rna interference inhibition of intracellular hepatitis c virus replication by synthetic and vector-derived small interfering rnas rna interference blocks gene expression and rna synthesis from hepatitis c replicons propagated in human liver cells small interfering rna-mediated inhibition of hepatitis c virus replication in the human hepatoma cell line huh- clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas alternative approaches for efficient inhibition of hepatitis c virus rna replication by small interfering rnas interference of hepatitis c virus rna replication by short interfering rnas inhibition of hepatitis c virus protein expression by rna interference interfering with hepatitis c virus rna replication suppression of hepatitis c virus replicon by rna interference directed against the ns and ns b regions of the viral genome rna interference in adult mice interference of hepatitis a virus replication by small interfering rnas local rna target structure influences sirna efficacy: systematic analysis of intentionally designed binding regions suppression of hepatitis a virus genome translation and replication by sirnas targeting the internal ribosomal entry site short interfering rna confers intracellular antiviral immunity in human cells attenuation of dengue virus infection by adeno-associated virus-mediated sirna delivery rna silencing of dengue virus type replication in transformed c / mosquito cells transcribing an inverted-repeat rna derived from the virus genome rna interference, arthropod-borne viruses, and mosquitoes the utility of sirna transcripts produced by rna polymerase in down-regulating viral gene expression and replication of negative-and positivestrand rna viruses inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells a small interfering rna targeting coxsackievirus b protects permissive hela cells from viral challenge cross-inhibition to heterologous foot-and-mouth disease virus infection induced by rna interference targeting the conserved regions of viral genome modulation of hiv- replication by rna interference rotavirus gene silencing by small interfering rnas rotavirus replication: plus-sense templates for double-stranded rna synthesis are made in viroplasms sequence homology required by human immunodeficiency virus type to escape from short interfering rnas poliovirus escape from rna interference: short interfering rna-target recognition and implications for therapeutic approaches down-regulation of viral replication by adenoviral-mediated expression of sirna against cellular cofactors for hepatitis c virus the polypyrimidine tract-binding protein (ptb) is required for efficient replication of hepatitis c virus (hcv) rna cellular rna helicase p relocalization and interaction with the hepatitis c virus (hcv) ns b protein and the potential role of p in hcv rna replication hpv e facilitates replication by activating e f transcription through its interaction with hdacs specific inhibition of hiv- replication by short hairpin rnas targeting human cyclin t without inducing apoptosis specific inhibition of hiv- gene expression by double-stranded rna sirna-directed inhibition of hiv- infection rna interference directed against viral and cellular targets inhibits human immunodeficiency virus type replication bispecific short hairpin sirna constructs targeted to cd , cxcr , and ccr confer hiv- resistance inhibition of human immunodeficiency virus type replication in primary macrophages by using tat-or ccr -specific small interfering rnas expressed from a lentivirus vector inhibition of hiv- fusion with small interfering rnas targeting the chemokine coreceptor cxcr rna interference directed against poly(adp-ribose) polymerase efficiently suppresses human immunodeficiency virus type replication in human cells inhibition of human immunodeficiency virus type replication by rna interference directed against human transcription elongation factor p-tefb (cdk /cyclint ) suppression of chemokine receptor expression by rna interference allows for inhibition of hiv- replication protection from hiv- infection of primary cd t cells by ccr silencing is effective for the full spectrum of ccr expression potent suppression of hiv type infection by a short hairpin anti-cxcr sirna suppression of chemokine receptor expression by rna interference allows for inhibition of hiv- replication cyclophilin a retrotransposition into trim explains owl monkey resistance to hiv- rna interference of hiv replication lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells modulating hiv- replication by rna interference directed against human transcription elongation factor spt inhibition of respiratory viruses by nasally administered sirna the promise of sirnas for the treatment of influenza inhibition of coxsackievirus b replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand inhibition of sars-cov replication by sirna actively replicating west nile virus is resistant to cytoplasmic delivery of sirna use of rna interference to prevent lethal murine west nile virus infection an sirna-based microbicide protects mice from lethal herpes simplex virus infection small interfering rna inhibits hepatitis b virus replication in mice short interfering rna-directed inhibition of hepatitis b virus replication inhibition of hbv replication by sirna in a stable hbv-producing cell line inhibition of hepatitis b virus expression and replication by rna interference inhibition of hepatitis b virus in mice by rna interference effective inhibition of hbv replication in vivo by anti-hbx short hairpin rnas knock-down of hepatitis b virus x protein reduces the tumorigenicity of hepatocellular carcinoma cells genomic analysis of anti-hepatitis b virus (hbv) activity by small interfering rna and lamivudine in stable hbv-producing cells small hairpin rnas efficiently inhibit hepatitis c ires-mediated gene expression in human tissue culture cells and a mouse model inhibition of hepatitis c virus translation and subgenomic replication by sirnas directed against highly conserved hcv sequence and cellular hcv cofactors lentiviral delivery of short hairpin rnas protects cd t cells from multiple clades and primary isolates of hiv hiv- can escape from rna interference by evolving an alternative structure in its rna genome evidence that hiv- encodes an sirna and a suppressor of rna silencing hiv- nef suppression by virally encoded microrna regulation of human immunodeficiency virus transcription by nef microrna lentiviral sirnas targeting multiple highly conserved rna sequences of human immunodeficiency virus type identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy computational design of antiviral rna interference strategies that resist human immunodeficiency virus escape rnai induction and activation in mammalian muscle cells where dicer and eif c translation initiation factors are barely expressed rna silencing as a plant immune system against viruses induction and suppression of rna silencing by an animal virus nucleic acid-based immune system: the antiviral potential of mammalian rna silencing adenosine kinase inhibition and suppression of rna silencing by geminivirus al and l proteins effects and side-effects of viral rna silencing suppressors on short rnas the coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step a viral protein inhibits the long range signaling activity of the gene silencing signal a viral protein suppresses rna silencing and binds silencing-generated, -to -nucleotide double-stranded rnas virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway p /hc-pro, a viral suppressor of rna silencing, interferes with arabidopsis development and mirna unction suppression of posttranscriptional gene silencing by a plant viral protein localized in the nucleus interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing a virus-encoded inhibitor that blocks rna interference in mammalian cells adenovirus va noncoding rna can inhibit small interfering rna and microrna biogenesis suppression of rna interference by adenovirus virus-associated rna human immunodeficiency virus type escapes from rna interferencemediated inhibition human immunodeficiency virus type escape from rna interference hepatitis c virus replicons escape rna interference induced by a short interfering rna directed against the ns b coding region inhibition of hepatitis b virus gene expression by single and dual small interfering rna treatment expression profiling reveals off-target gene regulation by rnai activation of the interferon system by short-interfering rnas cationic liposome-mediated delivery of sirnas in adult mice small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna a system for stable expression of short interfering rnas in mammalian cells determinants of interferon-stimulated gene induction by rnai vectors an enhanced u promoter for synthesis of short hairpin rna short hairpin type of dsrnas that are controlled by trna(val) promoter significantly induce rnaimediated gene silencing in the cytoplasm of human cells promoter choice affects the potency of hiv- specific rna interference development and application of sirna expression vector conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible rna interference short-term cytotoxic effects and longterm instability of rnai delivered using lentiviral vectors efficient delivery of sirna for inhibition of gene expression in postnatal mice rna interference in mammalian cells by chemically-modified rna rna interference targeting fas protects mice from fulminant hepatitis in vivo activity of nuclease-resistant sirnas functional anatomy of sirnas for mediating efficient rnai in drosophila melanogaster embryo lysate sirna function in rnai: a chemical modification analysis functional anatomy of a dsrna trigger: differential requirement for the two trigger strands in rna interference challenges for rnai in vivo activity of stabilized short interfering rna in a mouse model of hepatitis b virus replication sirna relieves chronic neuropathic pain potent and persistent in vivo anti-hbv activity of chemically modified sirnas inhibition of influenza virus production in virus-infected mice by rna interference protection against lethal influenza virus challenge by rna interference in vivo atelocollagenmediated synthetic small interfering rna delivery for effective gene silencing in vitro and in vivo intravenous rna interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into cells adenovirus vectormediated doxycycline-inducible rna interference conditionally replicating adenoviruses expressing short hairpin rnas silence the expression of a target gene in cancer cells inducible, reversible, and stable rna interference in mammalian cells cre-lox-regulated conditional rna interference from transgenes cre recombinase-inducible rna interference mediated by lentiviral vectors lentiviral vector-mediated delivery of short hairpin rna results in persistent knockdown of gene expression in mouse brain rnai suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia first parkinson gene therapy trial launches optimization of an sirna-expression system with an improved hairpin and its significant suppressive effects in mammalian cells inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide antisense oligonucleotides: promise and reality lna: a versatile tool for therapeutics and genomics rational sirna design for rna interference asymmetry in the assembly of the rnai enzyme complex functional sirnas and mirnas exhibit strand bias attenuation of sars coronavirus by a short hairpin rna expression plasmid targeting rnadependent rna polymerase silencing sars-cov spike protein expression in cultured cells by rna interference using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque rna interference targeting vp inhibits foot-and-mouth disease virus replication in bhk- cells and suckling mice small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism susceptibility of human hepatitis delta virus rnas to small interfering rna action rna interference against enterovirus infection inhibition of porcine endogenous retroviruses by rna interference: increasing the safety of xenotransplantation enhanced gene silencing of hiv- specific sirna using microrna designed hairpins expression of small hairpin rna by lentivirus-based vector confers efficient and stable gene-suppression of hiv- on human cells including primary non-dividing cells inhibition of hiv- by lentiviral vector-transduced sirnas in t lymphocytes differentiated in scid-hu mice and cd + progenitor cellderived macrophages efficient gene transfer of hiv- -specific short hairpin rna into human lymphocytic cells using recombinant adeno-associated virus vectors inhibition of virus production in jc virus-infected cells by postinfection rna interference inhibition of the epstein-barr virus lytic cycle by zta-targeted rna interference selective inhibition of hepatitis b virus replication by rna interference clearance of hepatitis b virus from the liver of transgenic mice by short hairpin rnas short interfering rna-mediated inhibition of herpes simplex virus type gene expression and function during infection of human keratinocytes the investigators are supported by the israeli ministry of science, through a grant to the national gene therapy knowledge center and through the ec grant lshb-ct- - . the investigators are also supported by the blum, grinspoon, horowitz & wolfson foundations and an isf grant. we would like to thank michal gropp and hilla giladi for carefully reading the review. key: cord- - w v fm authors: to, kelvin k.w.; chan, kwok‐hung; li, iris w.s.; tsang, tak‐yin; tse, herman; chan, jasper f.w.; hung, ivan f.n.; lai, sik‐to; leung, chi‐wai; kwan, yat‐wah; lau, yu‐lung; ng, tak‐keung; cheng, vincent c.c.; peiris, joseph s.m.; yuen, kwok‐yung title: viral load in patients infected with pandemic h n influenza a virus date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: w v fm viral shedding profile of infections caused by the pandemic h n influenza a virus has not been reported. the aim of this study was to determine the viral load in different body sites. viral loads of pandemic h n virus in respiratory specimens, stool, urine, and serum were determined by quantitative reverse transcriptase‐polymerase chain reaction (rt‐pcr). respiratory specimens from patients with seasonal influenza were used as historical controls. initial pre‐treatment viral load were compared between these two groups. serial respiratory specimens from patients with pandemic h n virus infection were obtained for analysis of viral dynamics. twenty‐two pandemic h n cases and seasonal influenza historical controls were included. the mean initial viral load before oseltamivir therapy was . × ( ) copies/ml for pandemic h n virus compared with . × ( ) copies/ml in seasonal influenza historical controls (p = . ). among patients with pandemic h n virus infection, peak viral load occurred on the day of onset of symptoms, and declined gradually afterwards, with no virus being detectable in respiratory specimens by rt‐pcr days and by culture days after the onset of symptoms respectively, except in one patient. pandemic h n virus was detected in stool and in urine from / and / patients, respectively. viral culture was also positive from the stool sample with the highest viral load. younger age was associated with prolonged shedding in the respiratory tract and higher viral load in the stool. data from this quantitative analysis of viral shedding may have implications for formulating infection control measures. j. med. virol. : – , . © wiley‐liss, inc. the pandemic h n influenza virus has disseminated globally after being identified in mexico and the united states in april [novel swine-origin influenza a (h n ) virus investigation team, ]. due to sustained and widespread human-to-human transmission, the world health organization raised the pandemic alert level from phase to on june . unlike seasonal influenza, people below years of age were infected preferentially worldwide [world health organization, ] , with unusually high rate of severe respiratory disease and mortality among young patients in mexico [chowell et al., ] . this phenomenon may be attributed to the lack of pre-existing cross-reactive antibody against pandemic h n virus in this age group [centers for disease control and prevention, ]. on the contrary, most if not all of these individuals have pre-existing antibody against the prevailing seasonal influenza virus. apart from cross reactive antibody level, viral load profile from different body sites is also important in predicting disease severity and transmissibility in viral infections [hayden et al., ; chu et al., ; hung et al., ; cheng et al., a; de jong et al., ; lowen et al., ] . the correlation between the virological profile and clinical characteristics of pandemic h n virus infection would provide important knowledge for epidemiological control and clinical management in terms of antiviral therapy and infection control measures. clinical specimens of respiratory secretion taken by nasopharyngeal aspirate or nasopharyngeal-throat swab, serum, urine, and stool were collected from patients with clinical suspicion of pandemic h n virus infection in hong kong. serial sampling of respiratory specimens was conducted during hospitalization or upon outpatient follow-up. archived respiratory specimens from patients with seasonal influenza a virus infection from year to were selected as historical controls. patients' records were reviewed to determine the demographic and clinical information. total nucleic acid extraction was performed by using nuclisens easymag instrument (biomerieux, boxtel, netherlands) according to the manufacturer's instruction. briefly, ml of clinical specimen was added to ml of lysis buffer and incubated for min at room temperature. the lysed sample was then transferred to the well of a plastic vessel with ml of silica. it was followed by automatic magnetic separation. nucleic acid was recovered in ml elution buffer [chan et al., b] . the diagnosis of pandemic h n virus was performed by quantitative reverse transcriptase-polymerase chain reaction (rt-pcr) using primers targeting hemagglutinin gene of pandemic h n virus, while the quantitation of both pandemic h n virus and seasonal influenza a virus was performed using quantitative rt-pcr targeting influenza a virus m gene, as described previously [chan et al., a; lau et al., ] . briefly, ml eluted rna of influenza a virus was used for cdna by invitrogen superscript ii kit with random primer as described, and then, cdna was amplified in lightcycler instrument with a faststart dna master sybr green i mix reagent kit (roche diagnostics gmbh, mannheim, germany). in a typical reaction, ml cdna was amplified in a ml of lc-pcr master mix containing  fast-start dna master sybr green i mix, . mm mgcl , . mm of each primer. to determine the specificity of the assay, all pcr products were subjected to melting curve analysis ( - c; . c per second) at the end of the assay. for quantitative assay, a reference standard was prepared using pcrii-topo vector (invitrogen, san diego, ca) containing the corresponding target viral sequences. a series of log dilution equivalent to  to  copies per reaction were prepared to generate calibration curves and run in parallel with the test samples. if the specimen result was outside the upper limit of the expected range, the extract of the sample was repeated with suitable dilution. the detection limit of this assay was copies of rna per milliliter. for viral culture, madin-darby canine kidney (mdck) cell monolayer in culture tubes were inoculated with ml of clinical samples as described previously [yuen et al., ]. they were examined daily for cytopathic effect (cpe), and direct immunofluoresence test with specific antibody against influenza a virus nucleoprotein was done on fixed cell smears when cpe appeared or at the end of the incubation period. comparison was made between patients with pandemic h n virus and seasonal influenza virus infection regarding their demographics, underlying diseases, presenting symptoms, total white blood cell counts, absolute lymphocyte counts, and initial pre-treatment viral load in respiratory specimens on the day of diagnosis. among patients with pandemic h n virus infection, the same parameters was compared between those with longer duration (! days) and shorter duration ( days) of viral shedding, as defined by the time from onset of symptoms to the last positive sample by rt-pcr. for underlying disease, chronic immunosuppressive states included autoimmune diseases, malignancies, diabetes mellitus, and solid organ transplants. chronic pulmonary diseases included asthma and chronic obstructive pulmonary disease. statistical analysis was performed by fisher's exact test for categorical variables and by mann-whitney u-test for continuous variables. univariate linear regression analysis was employed to determine correlation between age and duration of viral shedding, and between age and fecal viral load. a two-tailed p-value < . was considered significant. twenty-two patients diagnosed with pandemic h n virus infection from the period of april to june were included in this study. their respiratory specimens were positive for both influenza a virus m gene and pandemic h n virus h gene by rt-pcr. a total of respiratory specimens were collected from the day of diagnosis to days after diagnosis. three or more serial respiratory specimens were obtained from of patients. within the initial days of diagnosis, respiratory specimens were collected from out of patient days ( . %). the reason for refusal was due to either the discomfort associated with the procedure or improvement in symptoms. stool, urine, and serum specimens were available from , , and patients, respectively. all patients with pandemic h n virus infection received oseltamivir, except a -year-old girl. oseltamivir therapy was initiated on the same day ( . %), day ( . %), and days ( . %) after diagnosis. forty-four patients with seasonal influenza a virus infection at the time of admission in year - were selected as historical controls randomly. none of them received any antiviral therapy. coughing, vomiting, diarrhea, and duration of symptoms before diagnosis were significantly different between patients with pandemic h n virus and seasonal influenza virus infection (table i) . demographics, underlying diseases, and initial pre-treatment viral loads were not significantly different between the two groups. for both pandemic h n cases and seasonal influenza historical controls, respiratory specimens collected on the day of onset of symptoms (day ) had the highest mean viral load (fig. ). there was a tendency for a lower initial viral load in the pandemic h n cases. though these were not serial samples from the same patient, there was an obvious decreasing trend of the initial viral load from or log on the day of onset of symptoms to or log on the third day after onset of symptoms. for seasonal influenza virus infection, this decreasing trend reversed from day after onset of symptoms. since no pandemic h n cases were diagnosed on the th, th, th, and th day after onset of symptoms, direct comparison between the two groups were not possible for those days. viral load in respiratory specimens in pandemic h n cases correlated negatively with time after onset of symptom (r ¼ . , p ¼ . ), as shown in figure . the median duration of viral shedding after onset of symptoms was days, but two patients had viral shedding for up to days after onset of symptoms. among the patients who received oseltamivir, patients had the last positive rt-pcr respiratory specimen while still receiving oseltamivir, with a median duration of viral shedding days after onset of symptoms. the remaining two patients had the last positive rt-pcr specimen day after last dose of oseltamivir, with viral shedding up to days after onset of symptoms. among the patients with viral shedding days, patients had negative rt-pcr on day after onset of symptoms. four patients, who did not have respiratory specimen obtained on day after onset of symptoms, had a low viral load at < copies/ml on day or after onset of symptoms. younger age correlated with longer period of viral shedding after onset of symptoms (r ¼ . , p ¼ . ). apart from age, there were no significant differences between patients with shorter and longer duration of viral shedding with regard to presenting symptoms, underlying diseases, blood tests, and initial pre-treatment viral loads (table ii) . viral culture of the initial respiratory specimen was positive in out of patients with pandemic h n virus infection, ranged from to days after onset of symptoms, except in one patient. serial viral culture was performed in patients, of whom had further positive culture, from to days after onset of symptoms. pandemic h n virus was detected by rt-pcr in the stool from four out of nine patients aged , , , and years old. the positive stool specimens were obtained - days after onset of symptoms. the mean viral load in stool was .  copies/ml (range .  to .  copies/ml), with a significant correlation between higher viral load and younger age (r ¼ . , p ¼ . ). viral culture was positive in the stool specimen with the highest viral load. one out of patients had detectable pandemic h n virus in urine by rt-pcr days after onset of symptoms, but was negative by viral culture. none of the available sera, which were collected from days to after onset of symptoms, had detectable viruses by rt-pcr. a -year-old boy with pandemic h n virus infection was unique in several aspects. firstly, he was the only patient with detectable viral shedding in respiratory specimen, stool, and urine. secondly, the viral load was highest among all patients. the initial pre-treatment viral load in his respiratory specimen, stool, and urine was .  , .  , and .  copies/ml, respectively. his specimens with high viral load were all collected days after onset of symptoms, whereas for j. med. virol. doi . /jmv most of the other patients, pandemic h n virus was not detectable. viral load data provide important information regarding virus-host interaction. peak mean viral load occurred early during illness in both pandemic h n virus and seasonal influenza virus infection, similar to studies in human volunteers [hayden et al., ]. for patients who presented to hospital between days and after onset of symptoms, the initial pre-treatment viral load in pandemic h n cases was lower than the seasonal influenza historical controls. this might be explained by differences in the severity of illness. less upper respiratory tract and gastrointestinal symptoms were found in the pandemic h n cases, since they were hospitalized despite mild symptoms during the containment phase of pandemic control in hong kong. many cases were transferred directly from the airport to hospitals either because of their onset of symptoms during the flight or were found to have fever by body temperature screening upon arrival at the airport. moreover, respiratory specimens were taken from every patient with suspected pandemic h n virus infection independent of severity of symptoms or underlying diseases, whereas in non-pandemic period, only a minority of patients with seasonal influenza virus infection who attended hospital service had respiratory specimen collected for virological diagnosis. as a result, these patients selected randomly as historical controls were likely to have more severe symptoms or with chronic underlying illnesses, which were associated with a higher initial pre-treatment viral load at diagnosis. for both cases and controls, initial pre-treatment viral load was lower for patients who had longer duration of symptoms before hospitalization. however, this trend was not extended to patients who attended hospital days after onset of symptoms. in the -yearold boy with pandemic h n virus infection, his viral load was exceptionally high days after onset of symptoms. the more prolonged symptomatic phase might be a reflection of poor host immunity, leading to a higher viral load. this observation would require verification by more patient data in the future. as expected, viral load in pandemic h n patients decreased gradually after onset of symptoms, as patient's immune system responded to infection [hayden et al., ; cheng et al., b] . however, significant viral shedding, up to log copies/ml, was still present days after onset of symptoms. in addition, younger age correlated with longer periods of viral shedding. this finding was similar to the previous studies in seasonal influenza virus infection, in which viral load was generally low or undetectable by day of illness, but could persist for up to days in children [world health organization writing group, ]. viral load was found to be especially high in young children [hall et al., ] . a more prolonged shedding could also occur in immunocompromised patients. antiviral therapy was also proven to reduce viral shedding for seasonal influenza [hayden et al., ; boivin et al., ; ward et al., ] . however, since most of the patients in this series received oseltamivir, the effect of antiviral therapy for this novel strain could not be determined. further studies involving patients without oseltamivir therapy would be necessary to assess the impact of antiviral treatment on viral shedding and clinical outcomes. pandemic h n virus was detected in the stool of four patients, with positive viral culture from the specimen with the highest viral load. previous studies showed that human influenza a h n virus could be detected in stool [buchy et al., ] , and this virus was further demonstrated in the biopsy of the small and large intestine of fatal cases [uiprasertkul et al., ; zhang et al., ] . other respiratory viruses that have been found in stool include sars coronavirus , respiratory syncytial virus [von linstow et al., ] , adenovirus [wilhelmi et al., ] and bocavirus [lau et al., ] . seasonal influenza virus detection by rt-pcr in stool has only been reported in very young children between the age of weeks and months, but positive viral culture has not been reported [wootton et al., ] . the finding that fecal shedding occurred in patients with pandemic h n virus infection highlights the importance of contact precaution when handling stool. this is especially important for younger children as they have higher viral loads in stool. despite its presence in the gastrointestinal tract, none of the cases developed significant gastrointestinal symptoms. this might be related to the small sample size and the relatively mild infection. vomiting and diarrhea were common among pandemic h n urinary shedding of pandemic h n virus occurred in a previously healthy -year-old boy with an apparent symptomatic phase of days before diagnosis, accompanied by a persistently high viral load in both nasopharyngeal aspirate and feces days after onset of symptoms. although influenza virus can be cultured routinely on kidney cell lines, very few clinical manifestations were attributed to the urinary tract. in tehran, out of patients with hemorrhagic cystitis during the influenza season had either virus isolated from throat swabs or a fourfold rise in hemagglutination inhibition antibody titer to influenza a/tehran/ / virus [khakpour and nik-akhtar, ] . in that series, influenza virus was isolated from urine in three patients. other respiratory viruses that have been detected in urine include adenovirus [hatakeyama et al., ] , sars coronavirus [lau et al., ] and bocavirus [pozo et al., ] . pandemic h n virus was not detected in any serum samples, which might be related to the small sample size. viremia, which has been observed in blood donors without symptoms, is a recognized although uncommon phenomenon in seasonal influenza [zou, ; likos et al., ] . in influenza a h n virus infection, serum level had prognostic significance [de jong et al., ] . this study has several limitations. the viral load in different types of respiratory specimens might be affected by the method of collection. nasopharyngeal aspirate is considered as the gold standard in obtaining nasopharyngeal epithelial cells. however, nasopharyngeal swab, nasopharyngeal flocked swab and nosethroat swabs have been suggested as alternatives because of high sensitivity [frayha et al., ; lambert et al., ; chan et al., a] . for patients with influenza a virus infection, the quantity of virus was not significantly different between nasopharyngeal aspirate and nasopharyngeal flocked swab [chan et al., a] . the analysis of serial viral changes might also be affected by the absence of specimens on consecutive 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organization consultation on clinical aspects of human infection with avian influenza a (h n ) virus clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus systemic infection of avian influenza a virus h n subtype in humans potential impact of pandemic influenza on blood safety and availability key: cord- - w qcv c authors: ayginin, andrey a.; pimkina, ekaterina v.; matsvay, alina d.; speranskaya, anna s.; safonova, marina v.; blinova, ekaterina a.; artyushin, ilya v.; dedkov, vladimir g.; shipulin, german a.; khafizov, kamil title: the study of viral rna diversity in bird samples using de novo designed multiplex genus-specific primer panels date: - - journal: adv virol doi: . / / sha: doc_id: cord_uid: w qcv c advances in the next generation sequencing (ngs) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. in addition, this approach has also helped in establishing the associations of viromes with many diseases. however, unlike the metagenomic studies using s rrna for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. on the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. the existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the pcr-based detection of particular viral species or genera, but not for families or higher taxonomic orders. in this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. we have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. we have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an ngs-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated. an increase in globalization, climate change, and interaction with livestock animals has resulted in the emergence of novel viral pathogens or zoonoses [ ] , which pose a serious health problem for birds and animals and ultimately for humans. the natural reservoirs of pathogens, such as birds, bats, rodents, and bloodsucking arthropods play a significant role in the sustenance and transmission of zoonotic infections. migratory birds warrant special attention, as their rich diversity and migratory behavior contribute to the spread of infections to considerable distances. such migrations are strongly associated with the emergence of the epidemic and enzootic outbreaks as well as the formation and activation of natural sources of viral infections. wild birds are widely acknowledged to be reservoirs and transmitters of pathogens responsible for emerging infectious diseases such as severe acute respiratory syndrome virus (sarsv), avian influenza virus a (h n ), and west nile virus (wnv), to domestic animals and humans [ , ] . the rich biodiversity of the advances in virology wild bird population may increase the risk of spread of pathogens to domesticated poultry. understanding the viral diversity is critical for predicting future risks of transmission or possible outbreaks of viral diseases. however, identifying and monitoring the transmission of novel viruses are one of the vital requisites for responding to outbreaks. the asymptomatic carriage of viruses, which could be attributed to certain characteristics of bird metabolism and the adaptive capabilities of their immune system, provides ideal conditions for coevolution, leading to the emergence of mutant and recombinant strains of viruses. hence, the majority of widely used molecular diagnostic methods, such as those employing polymerase chain reaction (pcr), are not sufficiently suitable for the identification of a wide variety of viruses, as the techniques are usually designed for the detection of highly conserved regions in the genomes, which limits the search to a restricted group of viral agents and prevents the identification of new viruses or viral variants. in addition, the sanger sequencing technique, which has been a standard diagnostic tool for the detection and identification of various pathogens, provides limited sequence information at a higher cost per nucleotide base and can only be used to identify pathogens with a high titer. moreover, a preliminary evidence of the presence of the certain viruses is required for performing the pcr, in the absence of which the process of pathogen identification could take a significant amount of time, which can be a major obstacle in the prevention and control of the infection. dna barcoding is a method which uses a short part of organism's genome (so-called barcode) to identify whether it belongs to a particular family, genera, or even species, by using extensive parallel sequencing technologies (or more commonly ngs [next generation sequencing]). this method was initially developed for studying bacterial communities (e.g., studies on gut microbiota), but today it is widely employed for various tasks, including detection of food adulteration [ ] , the study of the diets of marine communities [ ] , and biofuel analysis [ ] . unlike other taxa, viruses lack a universally shared phylogenetic marker (such as s for bacteria, cytochrome c oxidase for birds and mammals, rbcl and matk for plants, and internal transcribed spacer (its) for fungi or plants), which makes it impossible to design a universal primer pair to amplify and differentiate diverse viral sequences. furthermore, the viral taxonomy gives a better indication of the signs of diseases caused, rather than genetic similarity. this fact complicates the barcode (a short, standardized nucleotide sequence of an organism's dna) selection, even for one genus (for example, mammarenavirus can be serologically, phylogenetically, and geographically divided into two major complexes: the old world complex prevalent in africa, europe, and asia, and the new world complex found in north and south america [ ] ), let alone higher taxa. however, metagenomics still allows the detection of different viral pathogens using shotgun sequencing [ ] . despite the constant reduction in the cost of dna sequencing, this approach is still considerably expensive and is not feasible for screening a large number of samples. also, metagenomic ngs data-sets are usually predominantly composed of host-derived sequences with only a minor fraction of pathogen sequences. often, even an approximate fraction of pathogen content is initially unknown, making it practically impossible to estimate how many sequencing reads are needed per sample, in order to detect the pathogen in the final sequencing data file. another common problem for most ngs-based tests is that complex multistep workflows may pose challenges in the reproducibility of results. recently, briese et al. [ ] had developed a virome capture sequencing platform for vertebrate viruses (vircapseq-vert), which consisted of ∼ million biotinylated dna-probes for target enrichment of viral nucleic acids to increase the sensitivity of sequence-based detection and characterization of viruses. the described method allowed the identification (and possibly even sequencing the whole genome assembly of detected viruses) of a large number of viruses including the novel ones. however, the overall cost of sequencing per sample remained considerably high. other methods of enrichment are based on the targeted amplification of cdna region using genus-specific primer pairs in pcr. this approach has been known for a long time [ ] and has been used successfully by various researchers, both in the studies of the representatives of individual viral genera and in the analyses of the diversity of viruses in different types of biological material [ ] [ ] [ ] . till recently, a large number of such primers have been described [ ] [ ] [ ] . however, most of them were designed for the detection of certain species of viruses. moreover, it was impossible to use them in multiplex reactions due to primer-specific annealing temperatures, nonspecific amplification, and potential selfcomplementarity. thus, in order to analyze one sample, it is necessary to carry out a number of pcr experiments corresponding to the number of primer pairs. the effectiveness of the study could be significantly increased by combining genus-specific pcr and ngs. in this approach, the products of different pcr assays for each sample would be pooled in one tube, purified, eluted in a minimal volume, and prepared for ngs [ ] . however, this approach does not exempt the requirement of many pcrs per sample, which is a problem when a large number of samples are studied simultaneously. in addition, there are restrictions on the use of different protocols for the library preparation, particularly when the protocol includes the emulsion pcr stage, which strictly requires pcr products of the same length. in this study, we have introduced a method for designing oligonucleotide panels for targeted enrichment of viral nucleic acids, where the main objective is to use a minimum number of primer oligonucleotides to cover the maximum number of diverse viral taxa within a single pcr reaction. we have applied this approach to design genus-specific primer pairs for targeted enrichment of cdna from zoonotic rna viruses and have evaluated it using several samples from birds. we have also demonstrated a considerable increase in the viral genome coverage. panel. this section contains the technical details of the algorithm for the design of genus-specific primer panels. to enable us to process the enrichment pcr reaction in a single tube, a number of restriction parameters were applied to the positions and structures of oligonucleotides. the availability of validated reference viral nucleic acid sequences is crucial for efficient usage of the algorithm. although several viral reference sequence databases are publicly available [ ] , the medically relevant and model organisms are largely overrepresented in most of them, and the genomic diversity of circulating strains is often underrepresented. one such source is the open-source database "the virus pathogen database and analysis resource (vipr)" [ ] , which was developed back in . the most important advantage of this database is the authenticity of nucleic acid sequences, as the data are curated and managed by experts in virology and bioinformatics. this source was used to retrieve the sequences of the polymerase genes of target viral genera (or other genes in cases where the sequences of polymerase genes were not available). the sequences were filtered by length (≥ bp), quality, and intrageneric similarity and were combined to the corresponding fasta files. in order to create consensus sequences, nucleotide sequences within each fasta file specific to a genus were aligned using clustalw [ ] . if a minimum of two consensus subsequences with lengths of bp and a maximum of four ambiguous positions with minor nucleotide frequency ≥ % were not identified, the original fasta file was iteratively clustered using cd-hit [ ] , with decreasing threshold. the clusters obtained at each step were aligned independently using clustalw to identify consensus subsequence(s) for all the subsets, which must collectively represent at least % of different species within the genus. finally, at least one aligned fasta file was obtained for every genus. for extracting the common subsequences from multiple sequence alignments, a "sliding window" was used within a specified range of lengths. this window "slides" from the 'to '-end of the alignment with a step of one nucleotide and identifies all subsequences fulfilling the following criteria: (i) proportion of ambiguous positions (p amb ) ≤ %; (ii) proportion of unique species, which share the subsequence and do not contain gaps (p sh ) ≥ %; (iii) gc content of the consensus sequence (p gc ) within - % interval; (iv) absence of self-complementary regions; (v) absence of formation of homodimers; (vi) absence of formation of heterodimers with previously selected oligonucleotides. an amplicon length between two subsequences (primers) was then adjusted between and bp to make the panel compatible with the most popular sequencing platforms (for example, illumina miseq or ion s from thermo fisher scientific). two subsequences were considered to be a pair if they together covered at least % of species related to a target genus or cluster and shared over % of the species. the pairs were then filtered according to their annealing temperature ( ∘ c ≤ t a ≤ ∘ c). the selected primer pairs were then aligned with the nr database using blast to check for their specificity. nonspecific candidates were eliminated. the possibility of formation of heterodimers between sequences in the pair, and between the sequences and previously selected primers, was calculated using the software primer [ ] . then the parameters described above were calculated and the pairs were sorted accordingly. the "best" primer pair was selected as a "genus-specific pair." the parameters described above were then calculated, and the pairs were sorted accordingly. the primer pair with the best fit was selected as a "genus-specific pair." the sequences of primer pairs for the reference viral genera, designed using the developed algorithm, are presented in tables and . the ability of the developed panel to enrich the target cdna from zoonotic rna viruses was assessed using high titer solutions of viral rna (concentration ranging from to copies per ml) from species of viruses, belonging to viral genera within viral families (table ). viral rnas were sourced from the collection stored at the central research institute for epidemiology, moscow, russia. all viral rnas were stored at − ∘ c till further use for the study. the h o sterile (amplisens, russia) was used as a negative control in all experiments. the control samples cdna was obtained by reverse transcription reaction performed on l of the extracted rna using the reverta-l rt kit (amplisens; total volume of the reaction mixture is l); after that l of the reaction mixture containing cdnas was further used for evaluation of the ability of the primer pair to amplify the targeted region of viruses, both in single and in multiplex pcr format. mode. the pcr reaction mix ( l) was prepared using l of the cdna template, l of h o (milliq, amplisens), l of pcr mix fep/frt, l of . m of each primer (in the single-plex format) or . m of each primer (total concentration of . m in the multiplex format), . l of dntps ( . mm; amplisens), and . l of taqf polymerase (amplisens). the thermal cycling parameters were initial denaturation at ∘ c for min, followed by cycles of ∘ c for s, ∘ c for s, ∘ c for s, and final extension at ∘ c for min. pcr products of appropriate lengths were resolved by electrophoresis on . % agarose gel containing ethidium bromide. the amplicons were purified using a qiaquick pcr purification kit (qiagen), following the manufacturer's instructions. the purified pcr products were then sequenced using abi prism sequencer (applied biosystems) to confirm the specificity of the reactions in a single-plex format. bird samples (cloacal swabs and/or feces) were collected from the enisei ecological station, mirnoe (russian federation). the samples were collected from birds captured using mist-net for routine ornithological examination or from droppings left on the ground by geese at their stop-over sites. to prevent contamination, separate . . rna extraction and reverse transcription. total rna was extracted from l of the resuspended sample with rneasy lipid tissue mini kit (qiagen) using robotic workstation qiacube (qiagen), following the manufacturer's protocol. the cdna was obtained by reverse transcription reaction on l of the extracted rna using a reverta-l rt kit (amplisens), according to the manufacturer's instructions. the primer panel was tested with a reference set of samples ( table ). the results are presented in figure . in all cases, the products of the specified range of lengths were obtained. the multiplex system was then tested with the same set of viruses and the pcr products were reamplified using the genus-specific primer pairs. in all cases, unspecific amplification was observed as well. however, the reamplification reactions with the genus-specific primers showed the presence of the target products. the multiplex system was first tested with three bat samples infected by several known viruses (sample n : betacoronavirus, sample n : betacoronavirus, sample n : orthoreovirus). the reamplification of the pcr products was carried out with the genus-specific primers ( figure ) . the obtained results clearly demonstrated the presence of the products with target length in all three samples. advances in virology the targeted sequences were enriched by multiplex pcr with the designed primer pool described above (table ) . pcr products were cleaned using carboxyl-coated magnetic particles, commercially available as sera-mag speed beads (ge healthcare). the concentrations of the fragments were measured using qubit dsdna hs assay kit with a qubit . fluorimeter (invitrogen). the preparation of the amplicon libraries involved phosphorylation of the '-end and incorporation of barcoded adapters, followed by amplification of the final library. for this purpose, t polynucleotide kinase and t dna ligase (both from new england biolabs, neb) were used according to the manufacturer's protocol with slight modification. amplification was performed using pcr-mix fep/frt (amplisens). the two total rna libraries for ion s high-throughput sequencing were prepared from total rna of two bird samples (b and b ). the first strand of cdna was synthesized using random primers and reverta-l rt kit (amplisens). the second strand of cdna was prepared with nebnext ultra second strand synthesis module of kit #e (neb). the double stranded dna was fragmented by ion shear plus reagent kit (thermo fisher scientific) and advances in virology amplicon libraries were separated by . % agarose gel electrophoresis stained with ethidium bromide, and the fragments of target lengths were cut out and purified using the minelute gel extraction kit (qiagen). size selection of the final total rna libraries was done using % e-gel sizeselect ii agarose gels (thermo fisher scientific) on the e-gel electrophoresis system (thermo fisher scientific). sequencing was carried out on the ion s platform using ion / kit-chef reagent sample preparation kit and employing ion chips on the ion chef instrument (thermo fisher scientific). raw sequencing reads obtained from the platform were first filtered using the prinseq-lite tool [ ] to eliminate too short (< bp) and low-quality (min mean < ) fragments. the mean, median, and th and th percentiles of read lengths distributions for selected unfiltered fastq files are shown in the supplementary table s . the bwa software [ ] was used then to align the filtered reads to the reference birds' genomes database. the ideally aligned reads (full read length and no mismatches) were marked as nonviral reads and were eliminated. the software cd-hit [ ] was used to reduce the redundancy, i.e., the number of reads per sample, by clustering (with a similarity threshold %) and selecting the representative sequences for each cluster. briefly, the cd-hit algorithm sorts the sequences from long to short and processes them sequentially. the first sequence is automatically classified as the first cluster representative sequence. then each query sequence from the remaining sequences is compared to the representative sequences identified before it and is classified as redundant or representative based on whether it is similar to one of the existing representative sequences. the described filtering and clustering steps significantly reduced (by ∼ times) the computational time of the further steps. the software blast was then employed to first compare the representative sequences (rs) against virus-only nucleotide and protein databases that were collected by selection of virus sequences from genbank nt and genbank nr databases, respectively, to identify viral nucleotide candidate rs (nrs) and protein candidate rs (prs) with e-value cutoffs of - and - , respectively. since virus-only databases are much smaller than ncbi nt and nr databases, pairwise sequence comparisons are much faster, which maximizes the speed and allows for the more efficient use of computational resources. we subsequently aligned these candidate nrs and prs to the entire genbank nt and nr databases, respectively, to eliminate potential false positives (sequences with higher similarity to nonviral reference sequences than to viral ones in the database) among the candidates and to select the true positives. the additional step was applied to select true positives from viral prs: the prs aligned to nr database were compared with nrs aligned to nt database to eliminate viral prs, which correspond to nrs aligning to nonviral nucleotide sequences with a high e-value. to get an idea of the total number of true viral reads belonging to the same virus, the numbers of reads for each rs were eventually summed up. virus hits e< e- figure : a schematic picture of the bioinformatics pipeline developed for the analysis of the ngs data in this study. specific third-party tools that were employed are shown in parentheses. a schematic picture of the bioinformatics analysis scheme is presented in figure . a panel of primers including forward and reverse primers was designed using the developed algorithm and was synthesized accordingly (tables and ) . a preliminary analysis using the available control samples (table ) demonstrated the ability of the designed primers to perform targeted cdna enrichment in both single-plex and multiplex formats. this was confirmed by the presence of specific bands of expected lengths in electrophoresis, followed by capillary sequencing (figures and ) . subsequently, we deployed the primer panel to study bird samples (as described in the methods) and the sequencing results are presented in table and figure . only those samples, for which at least viral reads (filtered by quality and length) were identified in the final fastq files, are presented in the table. the total number of sequencing reads in such samples is also given to exemplify the percentage of pathogen reads. the alignment of sequencing reads with viral reference sequences was visually inspected to eliminate most of the potential artifacts (such as primer-dimers). as evident from table , the genera of viruses (samples b , b , and b , highlighted in bold) having a specific primer pair in the panel showed significant amplification, and percentage of the corresponding viral reads ranged between . % and . %. this confirms that our primer panel can efficiently enrich the target genera. other sequencing reads belonged to bacteriophages, bacteria, and host species. however, we also observed a significant enrichment in the samples b and b , for which a significant number of reads corresponding to sanxia water strider virus and duck adenovirus were, respectively, detected. we scrutinized these results in further detail by carrying out a blast analysis of all possible versions of the primers (since they are very degenerated) against the sequencing reads and observed that these findings were very likely due to nonspecific amplification from the primers designed for other genera (gammacorona f and flavi r), though we have not checked this by a direct experiment with a panel that lacks these primers. although nonspecific amplification allows an even more exhaustive search, it also introduces potentially undesirable enrichment. this could be a problem, especially when the lengths of obtained amplicons are outside the standard intervals suitable for sequencing on most popular platforms. we also observed a considerable number of viral reads corresponding to various genera in some samples (b , b , b , b , b , and b ), despite the absence of specific primers in the panel for their enrichment. the number of reads varied from . % (tunis virus) to . % (watercress white vein virus). the latter is a plant virus and its presence is expected as the feces of the host birds mainly contained grass. these results are possibly due to a weak, nonspecific pcr amplification. we then checked whether the obtained viral reads for samples b , b , b , b , and b were due to the amplification with the designed primers in the panel. these five samples were chosen as they indicated the most significant amplification, with . - . % of total sequencing reads in the fastq files being viral. this was done by repeating a blast analysis of all possible versions of the primer sequences against the obtained viral reads for the aforementioned samples and calculating the percentage of those containing at least one primer. we observed that over % of the sequences contained the primers validating the effectiveness of the panel. finally, in order to confirm the effectiveness of the panel's enrichment, we selected two samples (b and b ) for which the presence of viral rna was shown using amplicon sequencing data (see table ). notably, the b possessed a significant number of reads that correspond to coronaviruses ( . %) and our panel had specific primers for these genera. as for the b sample, it had a small number of reads that correspond to adenoviruses ( . %) but we did not design oligonucleotides for them. we then prepared total rna-seq libraries for these samples, and subsequently sequenced them, yielding ∼ . million of reads per sample. the percentage of the coronavirus reads in the b sample was found to be significantly lower ( . %) than that in the same sample that was previously enriched by the panel ( table ). as for the b sample, we identified that . % of reads belong to the adenoviridae family, and most of them were identified as being similar to the duck and psittacine adenoviruses. this is similar to the percentage of reads found in the b figure : a graphical representation of the percentage of the detected viral reads (grey) with respect to the total number of sequencing reads obtained for the samples listed in table . fastq file ( . %) when the primer panel was applied, i.e., no noticeable enrichment was observed as expected. at the same time, we lost ∼ . % of adenoviruses in the b , but since the amount of data was very different in the two experiments, there was a significant amplification of coronaviruses with almost a half of the reads belonging to these genera; this outcome is not totally surprising. thus, our approach allows a significant reduction in the cost of sequencing. besides, for a successful identification of viruses in a preenriched sample, , - , reads per sample are generally enough. in this study, we have presented a method of oligonucleotide design for the enrichment of viral nucleic acids. we used this method to design a primer panel, which showed a high efficiency in the detection and identification of several viruses using ngs sequencing. this is especially important for the detection of viruses known to persist in natural zoonotic reservoirs including birds, bats, and rodents, those with a high genetic diversity, and those which can be potentially dangerous to humans and animals. these factors make the development of rapid test-systems essential for the effective management of potential epidemics. the described method can be recommended to the researchers investigating diverse viromes across different types of biological samples. we deployed our panel for the screening of a number of bird samples and demonstrated a great efficiency. in future studies, the panel will be expanded to target more diverse viruses and tested with bird samples from other regions. all the fastq files used to support the findings of this study are available from the corresponding author upon request (kamil khafizov; kkhafizov@gmail.com). all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. an earlier version of this work was presented as an abstract at the rd febs congress prague - july . zoonosis emergence linked to agricultural intensification and environmental change reservoirs and vectors of emerging viruses relationship between domestic and wild birds in live poultry market and a novel human h n virus in china detection of plant-based adulterants in turmeric powder using dna barcoding dna metabarcoding for diet analysis and biodiversity: a case study using the endangered australian sea lion (neophoca cinerea) comparative and joint analysis of two metagenomic datasets from a biogas fermenter obtained by -pyrosequencing lassa virus validation of metagenomic next-generation sequencing tests for universal pathogen detection virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis detection and analysis of diverse herpes-viral species by consensus primer pcr a strategy to estimate unknown viral diversity in mammals hantavirus in bat, sierra leone identification of a novel coronavirus in patients with severe acute respiratory syndrome novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in south-east australia rt-pcr detection and identification of three species of cucumoviruses with a genus-specific single pair of primers genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction retrospective diagnosis of two rabies cases in humans by high throughput sequencing the th annual nucleic acids research database issue: a look back and upcoming changes vipr: an open bioinformatics database and analysis resource for virology research clustal w and clustal x version . cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences primer -new capabilities and interfaces quality control and preprocessing of metagenomic datasets fast and accurate short read alignment with burrows-wheeler transform the authors declare that there are no conflicts of interest regarding the publication of this paper. key: cord- - uzivsk authors: zhang, zheng; zhu, zhaozhong; chen, wenjun; cai, zena; xu, beibei; tan, zhiying; wu, aiping; ge, xingyi; guo, xinhong; tan, zhongyang; xia, zanxian; zhu, haizhen; jiang, taijiao; peng, yousong title: membrane proteins with high n-glycosylation, high expression, and multiple interaction partners were preferred by mammalian viruses as receptors date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: uzivsk receptor mediated entry is the first step for viral infection. however, the relationship between viruses and receptors is still obscure. here, by manually curating a high-quality database of pairs of mammalian virus-host receptor interaction, which included unique viral species or sub-species and virus receptors, we found the viral receptors were structurally and functionally diverse, yet they had several common features when compared to other cell membrane proteins: more protein domains, higher level of n-glycosylation, higher ratio of self-interaction and more interaction partners, and higher expression in most tissues of the host. additionally, the receptors used by the same virus tended to co-evolve. further correlation analysis between viral receptors and the tissue and host specificity of the virus shows that the virus receptor similarity was a significant predictor for mammalian virus cross-species. this work could deepen our understanding towards the viral receptor selection and help evaluate the risk of viral zoonotic diseases. systematic analysis of the characteristics of the viral receptor could help understand the mechanisms under the receptor selection by viruses. the virus-receptor interaction was reported to be a principal determinant of viral host range, tissue tropism and cross-species infection [ , , ] . the existence and expression of the virus receptor in a host (or tissue) should be a prerequisite for viral infection of the host (or tissue) [ ] . usually, a virus mainly infects some particular type of hosts or tissues. for example, the influenza virus mostly infects cells of the respiratory tract [ ] . however, the virus-receptor interaction is a highly dynamic process. some viruses can recognize one or more receptors [ , , ] , which can also differ among virus variants or during the course of infections [ , , ] . in some cases, a few amino acid mutations in the viral protein or the receptor could abolish or enhance viral infection [ ] [ ] [ ] . besides, the virus-receptor interaction is under we firstly investigated the structural characteristics of mammalian virus receptor proteins. as expected, all the mammalian virus receptor protein belonged to the membrane protein which had at least one transmembrane alpha helix ( figure s a ). twenty-four of them had more than five helixes, such as -hydroxytryptamine receptor a (htr a) and npc intracellular cholesterol transporter (npc ). the receptor protein was mainly located in the cell membrane. besides, more than one third ( / ) of them were also located in the cytoplasm, and thirteen of them were located in the nucleus. then, the protein domain composition of the mammalian virus receptor protein was analyzed. the mammalian virus receptor proteins contained a total of domains based on the pfam database, with each viral receptor protein containing more than two domains on average ( figure s b ). this was significantly more than that of human proteins or human membrane proteins (p-values < . in the wilcoxon rank-sum test). some viral receptor proteins may contain more than domains, such as complement c d receptor (cr ) and low density lipoprotein receptor (ldlr). the human viral receptors were observed to have ten or more n-glycosylation sites, such as complement c b/c b receptor (cr ) and lysosomal associated membrane protein (lamp ). figure b displayed the modeled d-structure of htr a, the receptor for jc polyomavirus (jcpyv). five n-glycosylation sites were highlighted in red on the structure, which were reported to be important for viral infection [ ] . for comparison, we also characterized the n-glycosylation level for the human cell membrane protein, human membrane proteins and all human proteins (figure a ). it was found they had a significantly lower level of n-glycosylation than that of human and mammalian virus receptors (p-values < . in the wilcoxon rank-sum test), which suggests the importance of n-glycosylation for the viral receptor. enriched, such as "regulation of leukocyte activation" and "lymphocyte activation". for the go molecular function (table s ) , besides for the enrichment of terms related to the virus receptor activity, the human virus receptor was also enriched in terms of binding to integrin, glycoprotein, cytokine, and so on. consistent with the enrichment analysis of go cellular component, the kegg pathways of "cell adhesion molecules", "focal adhesion" and "ecm-receptor interaction" were also enriched. besides, the pathway of "phagosome" was enriched (table s ) , which may be associated with viral entry into the host cell. interestingly, some pathways associated with heart diseases were enriched, including "dilated cardiomyopathy", "hypertrophic cardiomyopathy", "arrhythmogenic right ventricular cardiomyopathy" and "viral myocarditis". we next analyzed the protein-protein interactions (ppis) which the mammalian virus receptor protein took part in. as the reason mentioned above, we only used the human (table s ) . when looking at the interactions between viral receptors, we found that of viral receptors interacted with themselves. this ratio ( / = %) was much higher than that of human proteins ( %), membrane proteins ( %) and human cell membrane proteins ( %). however, we found the viral receptor tended not to interact with each other ( figure s d since the virus has to compete with other proteins for binding to the receptor, proteins (table s ) . since the viral receptor determines the host specificity of the virus to a large extent, it is expected that the closer between the viral receptor and its homolog in a species, the more likely the virus which used the receptor would infect the species. to validate this hypothesis, we firstly calculated the sequence identities between the viral receptor and their homologs in mammal species ( figure and table s ). for clarity, only mammal species, which were frequently observed, were presented in figure . then, table s . what's the relationship between glycosylation and viral receptor selection? as we know, glycosylation of proteins is widely observed in eukaryote cells [ ] . it plays an important role in multiple cellular activities, such as folding and stability of glycoprotein, immune response, cell-cell adhesion, and so on. glycans are abundant on host cell surfaces. they were probably the primordial and fallback receptors for the virus [ ] . to use glycans as their receptors, a large number of viruses have stolen a host galectin and employed it as a viral lectin [ , ] . for example, the sjr fold, which was mainly responsible for glycan recognition and binding in cellular proteins, was observed in viral capsid proteins of over one fourth of viruses [ ] . thus, during the process of searching for protein receptors, the protein with high level of glycosylation could provide a basal attachment ability for the virus, and should be the preferred receptor for the virus. secondly, our analysis showed that the viral receptor protein had a tendency to interact with itself and had far more interaction partners than other membrane proteins. besides the function of viral receptor, the receptor protein functions in the host cell by interacting with other proteins of the host, such as signal molecules and ligands. therefore, the virus has to compete with these proteins for binding to the receptor [ ] . the protein with less interaction partners are expected to be preferred by the virus. why did the virus select the proteins with multiple interaction partners as receptors? one possible reason is that the receptor proteins are closely related to the "door" of the cell, so that many proteins have to interact with them for in-and-out of the cell. this could be partly validated by the observation that for the interaction partners of human viral receptors, six of top ten enriched terms in the domain of go biological process were related to protein targeting or localization (table s ) . for entry into the cell, the virus also selects these proteins as receptors. another possible reason is that viral entry into the cell needs cooperation of multiple proteins which were not identified as viral receptors yet. besides, previous studies show that the virus could structurally mimic native host ligands [ ] , which help them bind to the host receptor. (table s ). the number of transmembrane alpha helix of the mammalian virus receptor was derived from the database of uniprotkb and the web server tmpred [ ] . the location for the viral receptor was inferred from the description of "subcellular location" for the receptor protein provided by uniprotkb, or from the go annotations for them: the viral receptors annotated with go terms which included the words of "cell surface" or "plasma membrane" were considered to be located in the cell membrane; those annotated with go terms which included the words of "cytoplasm", "cytosol" or "cytoplasmic vesicle", or shown to be in the cytoplasm in uniprotkb, were considered to be located in the cytoplasm; those annotated with go terms "nucleus" (go: ) or "nucleoplasm" (go: ) were considered to be located in the [ ] in r (version . . ) [ ] . to identify the homolog of the mammalian virus receptor in other mammal species, the protein sequence of each viral receptor was searched against the database of mammalian protein sequences, which were downloaded from ncbi refseq database [ ] on october th , , with the help of blast (version . . ) [ ] . analysis showed that in the database of mammalian protein sequences, there were mammal species which were richly annotated and had far more protein sequences than other mammal species (table s ) . therefore, only these mammal species were considered in the evolutionary analysis. based on the results of blast, the homolog for the viral receptor was defined as the hit with e-value small than e- , coverage equal to or greater than % and sequence identity equal to or greater than %. only the closest homolog, i.e., the best hit, in each mammal species was used (table s ). similar methods as above were utilized to calculate the indicators of conservation level for these proteins. for analysis of co-evolution between viral receptors, firstly for each viral receptor, a phylogenetic tree was built based on the protein sequences of the receptor and its homologs in mammal species with the help of phylip (version . ) [ ] . the neighbor-joining method was used with the default parameter. then, the genetic distances between the viral receptor and their homologs were extracted from the phylogenetic tree with a perl script. finally, for a pair of viral receptors, the spearman correlation coefficient (scc) was calculated between the pairwise genetic distances of viral receptors and their homologs, which was used to measure the extent of co-evolution between this pair of viral receptors. the set of housekeeping gene in human was adapted from the work of eisenberg et al [ ] . a total of genes were identified as the housekeeping gene. (table s ) the mammalian virus-host interactions were primarily adapted from olival's work [ ] . one hundred and fifteen viruses in our database and of richly annotated mammal species could be mapped to those in olival's work (table s ). these viruses used a total of viral receptors. the sequence identities of these viral receptor proteins to their related homologs in the corresponding mammal species were presented in table s . for comparison, we also extracted genetic distances (host relatedness) between the mammal species and the viral host with reported receptors based on olival's work (table s ) . then, the genetic distance of the mammal species to the viral host with reported receptors, and the sequence identity of the receptor homolog in the mammal species to the viral receptor protein, was respectively used to predict whether a mammal species could be infected by the virus which infected the host with reported receptors. the method of receiver operating characteristic (roc) curve was used to evaluate and compare their performance with the functions of roc(), auc(), roc.test() and plot.roc() in the package of "proc" [ ] in r (version . . ). statistical analysis all the statistical analysis was conducted in r (version . . ) [ ] . the wilcoxon rank-sum test was conducted with the function of wilcox.test(). clusterprofiler: an r package for comparing biological hz, tj and yp conceived and designed the study. zz and zzz did the computational key: cord- -eqxn auk authors: garcia‐cremades, maria; solans, belen p.; hughes, emma; ernest, jacqueline p.; wallender, erika; aweeka, francesca; luetkemeyer, anne f.; savic, radojka m. title: optimizing hydroxychloroquine dosing for patients with covid‐ : an integrative modeling approach for effective drug repurposing date: - - journal: clin pharmacol ther doi: . /cpt. sha: doc_id: cord_uid: eqxn auk hydroxychloroquine (hcq) is a promising candidate for coronavirus disease of (covid‐ ) treatment. the optimal dosing of hcq is unknown. our goal was to integrate historic and emerging pharmacological and toxicity data to understand safe and efficacious hcq dosing strategies for covid‐ treatment. the data sources included were (i) longitudinal clinical, pharmacokinetic (pk), and virologic data from patients with severe acute respiratory syndrome‐ (sars‐cov‐ ) infection who received hcq with or without azithromycin (n = ), (ii) in vitro viral replication data and sars‐cov‐ viral load inhibition by hcq, (iii) a population pk model of hcq, and (iv) a model relating chloroquine pks to corrected qt (qtc) prolongation. a mechanistic pk/virologic/qtc model for hcq was developed and externally validated to predict sars‐cov‐ rate of viral decline and qtc prolongation. sars‐cov‐ viral decline was associated with hcq pks (p < . ). the extrapolated patient half‐maximal effective concentration (ec( )) was . µm, comparable to the reported in vitro ec( s). hcq doses > mg b.i.d. for ≥ days were predicted to rapidly decrease viral loads, reduce the proportion of patients with detectable sars‐cov‐ infection, and shorten treatment courses, compared with lower dose (≤ mg daily) regimens. however, hcq doses > mg b.i.d. were also predicted to prolong qtc intervals. this prolongation may have clinical implications warranting further safety assessment. due to covid‐ 's variable natural history, lower dose hcq regimens may be indistinguishable from controls. evaluation of higher hcq doses is needed to ensure adequate safety and efficacy. membrane that reduces viral entry and inhibition of later stages of replication. hcq also causes immunomodulation, and, thus, may suppress the cytokine storm associated with advanced stages of covid- illness. in a small, nonrandomized, open-label clinical trial in france, patients with covid- , who received hcq dosed as mg t.i.d. were compared with a convenience sample of patients who only received supportive care. in this study, hcq was well-tolerated, however, approximately one third of patients remained viremic by nasopharyngeal swabs after days. a small study in china reported no apparent clinical benefit of mg daily hcq administered for days compared with placebo in patients with covid- , the majority of whom had mild disease. based on in vitro data, several hcq regimens have been proposed, including mg q.i.d. and mg b.i.d. followed by mg daily for days, but none of these regimens have been evaluated clinically. the centers for disease control (cdc) also provides anecdotal dosing suggestions, but explicitly states optimal dosing and duration of hcq for covid- are unknown. with promising initial clinical and in vitro results for hcq, our goal was to integrate all available pharmacological data and mechanistic knowledge related to covid- , to date, which include novel in vitro pharmacokinetic/pharmacodynamic (pk/pd) data for sars-cov- , historical data on viral replication of similar coronaviruses (e.g., sars-cov- , first observed in in china), historical data on population pks and safety of hcq from large patient cohorts, and newly emerging clinical pk/pd data from patients with covid- . we use translational pk/pd modeling to propose optimized hcq dosing regimens, which ensure the highest likelihood of success as a covid- treatment. in vitro hcq and cq drug sensitivity data for sars-cov- , reported as percent inhibition, were obtained from -hour and -hour experiments in vero or veroe cells derived from african green monkey kidney epithelium. , , the experiments are described in detail in the original publications. estimated, apparent half-maximal effective concentration (ec ) values were reported, whereas the % effective concentration values were obtained by digitizing the graph of antiviral activity for hcq using the software webplotdigitizer version . . in vitro viral replication data were obtained from longitudinal data profiling the growth of sars-cov- in vero cells over days. rna extraction and quantitative real-time polymerase chain reaction (pcr) were performed, and viral load was reported as cycle threshold (ct). viral load was calculated from the ct reported in the original publication as / log (ct). in vivo data was obtained from a published nonrandomized single arm open label study of hcq mg t.i.d., with or without azithromycin, for treatment of sars-cov- infection in france. participants had pcr confirmed sars-cov- infection by nasopharyngeal swab and swab samples were obtained daily. controls were selected by convenience at multiple hospitals in france. viral load was calculated from ct. one sparse serum hcq concentration was reported for each patient on days , , or of treatment. sixteen patients in the control arm (samples = ), patients in the hcq arm (samples = ), and patients in the hcq with azithromycin arm (samples = ) contributed viral load samples over days. additional patient characteristics and results are reported in the original publication. an external cohort of patients receiving mg t.i.d. for days was used for external model validation. translational and clinical pk/pd-viral kinetics models a published two-compartment population pk model for hcq was used to predict plasma drug concentrations for different dosing regimens of hcq. published scaling parameters were used to simulate lung concentrations and the fraction unbound was assumed to be the same in the lung as in the plasma ( . ). in vitro viral growth, death, and saturable growth parameters were estimated using data from sars-cov- in the software nonlinear mixed effects modeling version . (nonmen icon development solutions, ellicott city, md). the drug effect over time on viral replication rate was established by simulating unbound plasma concentrations or unbound lung tissue concentrations using a previously defined partition coefficient ( . ; hcq unbound fraction assumed to be ~ %) and using the established in vitro sigmoidal efficacy parameters. , , regimens of hcq included - mg b.i.d. for days. the drug effect on the viral replication rate was simulated by fixing ec to one of the reported in vitro values, ranging from . µm to . µm. , , each regimen was simulated with each ec values times. the pk/pd analysis of the in vivo viral load data was performed sequentially. the control patients' longitudinal viral load was analyzed to inform the unperturbed viral growth. a previously published population pk model was used to define the individual pk profile for each patient treated with hcq (with and without azithromycin). plasma and serum concentrations were assumed to be comparable. subsequently, longitudinal plasma pk and viral load were linked to predict the impact of time varying hcq concentrations on viral replication. viral dynamics (vl) were described using the viral kinetic model (eq. ), where k g and k d represent first-order growth and death rates, respectively. max virus stands for the saturated maximal viral load. drug (pk/pd) effect (edrug) was included as a linear function increasing k d (eq. ), where the slope (sl) of the function is estimated, and c p represents the drug concentration predicted with the pk model. a function (eq. ) independent of the drug effect was added during the model validation stage to improve predictions at later time points (beyond day ). this function might mimic the immune response (ir), which comes on board once the viral load is reduced to a certain level, described as a function of the immune effect (ie) and time. finally, participants' sex, age, baseline viral load, clinical status time between onset of symptoms and inclusion, and the use of concomitant azithromycin were tested as potential covariates. pk/pd parameters were estimated using the laplace estimation method with interaction, and the m method was applied to handle data below the limit of quantification by estimating the likelihood of the predicted viral load being less than the lower limit of assay quantification. , interindividual variability parameters were modeled exponentially and the residual variability was described using proportional error. model selection and evaluation were done by the likelihood ratio test, goodness of fit plots, and visual predictive checks. the final model was used to externally predict %pcr-negative patients from the new patient cohort who received mg t.i.d. with azithromycin for days. simulated regimens of hcq included , , , and mg b.i.d. for , , and days with and without loading dose. all simulations included virtual patients, simulated , times. large variation in the basal viral load range was assumed. the pk model was used to generate the virtual individual pk profiles. the percentage of patients with positive pcr results was computed longitudinally throughout and at the end of the treatment. regimens were evaluated based on the proportion of patients with viral loads above the lower limit of assay quantification. all model simulations were performed using nonmem and the pkpdsim package in r software version . . . to predict the risk of corrected qt (qtc) prolongation associated with hcq, we used a published pk-qtc model established for high-dose cq in children, describing a linear pk-qtc relationship where for every nm increase in cq concentration there was a . ms ( % confidence interval . - . ) increase in qt interval. a conservative upper boundary estimate ( . ms/nm) was used assuming that hcq and cq have a similar pk-qtc relationship. this relationship was validated as it successfully predicted qtc prolongation in adults, after a single dose of mg cq reported by mzayek et al. a conservative assumption was made that the pk-qtc relationship remains linear and it does not reach a maximal effect. we assumed a trial population of , patients with a baseline qtc of ± sd. we conducted simulations of each hcq dosing regimen in this population using the established population pk model (including intersubject variability) and predicted the peak hcq concentrations during treatment. we assumed all changes in qtc were attributed to pk variability. the maximum delta qtc during treatment was predicted using the linear pk-qtc relationship for cq ( . ms/nm); the maximum predicted qtc was the sum of the baseline and the delta qtc. although the baseline qtc distribution contained variability, the slope used ( . ms/nm) did not. the proportion of patients with a delta qtc > ms or predicted peak qtc on treatment > ms were reported. the natural course of disease progression is variable and might impact the benefit of hcq therapy. we explored various models of natural history for covid- based on the viral load distribution from the control arms in two recently reported studies. , three natural history scenarios were explored for several plausible control arms with low, high, and mixed baseline viral load. viral load decline of various hcq doses was compared with the control arm under different assumptions. translational pk/pd model the pk/pd viral kinetic model structure is shown in figure . the translational pk/pd model included the unperturbed growth rate of sars-cov- in vitro, the clinical population pk model, and the hcq in vitro efficacy parameters in sars-cov- . , , , , the in vitro viral kinetic model included a first-order growth ( . day - ) and death rate ( . day - ) and saturated maximal viral load ( table s ). the simulated unbound lung concentrations were above the ec for all evaluated dosing regimens, including a subtherapeutic mg dose. for unbound plasma concentration, the impact of the drug effect on viral replication increased with the dose but was highly sensitive to the in vitro ec values ( figure ). for all reported ec s , higher doses increased the rate of viral decline. clinical pk/pd model hcq concentrations from the clinical cohort fell within the range expected from historical population profiles (figure a left). , figure b shows the reconstructed individual pk profiles in the treatment group. the patients' viral load is displayed in figure a (right). pharmacokinetic/pharmacodynamic (pk/pd)-viral kinetics model diagram. a previously published two-compartment plasma pk model was used to simulate plasma concentration. pd compartments included a one-compartment model describing viral growth, death, and drug effect, and a model describing drug effect on qtc prolongation. e drug represents the pd relationship for hydroxychloroquine (hcq) plasma concentration (c p ). in the in vitro model, e drug is characterized by a maximum effect (e max ) function e drug =e max × cp cp +ec , whereas for the clinical model it is described using a linear function (e drug = sl × c p ). cl, clearance; ec , half-maximal effective concentration; qtc, corrected qt; v, volume of distribution. article viral response to treatment was significantly associated (p < . ) to hcq pk concentrations using a linear effect model ( figure and table s ). each μm increase in plasma hcq is associated with a % decrease in viral load per day. the pk/pd model was significantly better compared with a drug effect model (p < . ), confirming a significant concentration-effect relationship. in addition, a maximum effect (e max ) model to link drug concentrations to effect was also tested, however, it was not identifiable. this is most likely due to the limited clinical pk concentration range. the effect of azithromycin on viral load decline was not significant. among the covariates explored, two asymptomatic patients in the treatment group seemed to have a lower baseline viral load and cleared the virus by the second day ( figure s ). the visual predictive check (figure c) shows good agreement between the raw data and model simulations, both for the longitudinal viral load dynamics and for the proportion of censored pcr samples, associated with undetectable viral load. the estimated plasma concentrations for % viral inhibition in patients was . . this value is in closer agreement with the in vitro values reported after a -hour incubation (ec = . µm) compared with -hour incubation (ec figure ). , , pharmacokinetic simulations hcq pks, relative to in vitro and extrapolated in vivo ec , are summarized in figure and figure s (with population variability). simulated unbound lung concentration are shown in figure s . optimal dosing regimens are those that, to the extent possible, are close or above the clinical ec values and below . µm, identified as the mean concentration associated with > % of patients having an increase in > ms qtc while on treatment. regimens that give ~ mg/day either loaded upfront, or as mg b.i.d., figure translational pharmacokinetic/pharmacodynamic (pk/pd) simulation. simulated scenarios of drug effect on in vitro replication rate based on reported hydroxychloroquine (hcq) efficacy. , , viral kinetics were estimated from in vitro replication rate of severe acute respiratory syndrome-coronavirus (sars-cov)- and unbound drug concentration in plasma and lungs were simulated with hcq pk model. , solid continuous line represents the th percentile of the simulated data and shaded areas represent the % prediction intervals for median, . th, and . th percentiles obtained from simulated datasets. dotted horizontal lines represent the baseline level, whereas dashed vertical lines indicate the start of treatment. seem to have good efficacy and safety, and hcq is detectable in plasma for up to days (figure c ). for pk/pd-viral kinetics simulations, a median baseline ct of . ( . - . %; . - . ) was assumed (figure a) . first, we predicted the longitudinal viral loads of an external study of patients receiving mg t.i.d. of hcq with azithromycin ( figure a) . the model predicted the viral decline in the first week, however, it overpredicted later time points. after incorporating a time varying function to mimic a delayed immune effect, predictions aligned well with the data throughout treatment (figure a) . the simulations performed in order to obtain the predicted percentage of patients with positive pcr accounted for interindividual variability in the pk and growth rate kinetics. the data were not sufficient to identify variability in the drug effect and it was not included in our simulations. hcq mg b.i.d. for days was predicted to produce the lowest percentage of patients with detectable viral loads ( %), however, it was predicted to result in a significant probability of qtc prolongation (data not shown). both mg b.i.d. for or days, and mg b.i.d. for , , or days were predicted to have lower detectable viral loads than those previously studied (figure b,c) . dosing regimens that included loading doses mg b.i.d. and mg b.i.d. for or days followed by a maintenance dose of mg b.i.d. or mg t.i.d. are shown in figure s . by comparison, mg b.i.d. or t.i.d. regimens showed modest efficacy. reaching ec levels and viral load data. in the pk graph, raw data is shown in red, whereas black and grey lines represent the typical and population plasma pk simulation (n = ) using the pk model. in vitro half-maximal effective concentration (ec s ) indicated in the graph were calculated considering total drug using the values reported in yao et al. in the viral load graph (left), thick lines represent the mean profiles of each group, whereas the thin ones represent the individual profiles. (b) individual pk plasma profile predicted with the pk model for each patient treated with hydroxychloroquine (hcq). (c) visual predictive check of population pk/pharmacodynamic model. the solid continuous line represents the th percentile of the observations, dashed lines represent . th and . th percentiles of observations, and shaded areas represent the % prediction intervals for median, . th, and . th percentiles obtained from , simulated datasets. the lower panel shows the proportions of below the limit of quantification values observed (solid line), with % prediction variability shown by shaded area. ct, cycle threshold; lloq, lower limit of quantification. faster by using higher doses seems to offer more benefit compared with extending treatment duration. both longer durations and higher doses of hcq resulted in greater qtc prolongation (figure d ; table s ). an average patient with baseline qtc value of ms or less could receive higher doses of mg b.i.d. over or days with minimal risk ( . % and . %) of qtc prolongation (table s ) . given the number of assumptions, reported numbers should be interpreted more as an indication of risk, which remain to be determined in clinical trials. the viral load decline in the control arms was substantially different in the two reported clinical studies (figure a) . , we explored hcq efficacy under different control arm scenarios. different viral growth and death values were obtained when the model was fit to each study's control arm. this represents the intrinsic variability of disease progression, and, accordingly, resulted in different viral kinetics over time, suggesting large uncertainty and variability in the natural history of disease. two dosing regimens ( mg hcq daily and mg hcq b.i.d.) were simulated and overlaid for comparison with the different natural history scenarios (figure b) . this sensitivity analysis revealed that the low-dose regimen might be indistinguishable from placebo under different control group scenarios. however, higher hcq doses (≥ mg b.i.d.) are likely to show efficacy in viral clearance regardless of the control arm. for hcq to maximally suppress sars-cov- replication in vivo, the hcq dose may need to be optimized. to best define the effective hcq concentrations for treatment of covid- , all available data from in vitro and clinical studies using hcq for sars-cov- were pooled to quantify the relationship between hcq pk and sars-cov- viral decline in patients with covid- . we predicted that higher hcq daily doses (e.g., as high as mg b.i.d.), were associated with rapid rates of viral decline and increased the percentage of pcr-negative patients but could result in increased risk of qtc prolongation. regimens that give ~ mg/day either loaded upfront or as mg b.i.d., could be safely tolerated and would reduce the time with a detectable sars-cov- viral load, and, thus, improve treatment outcomes. higher hcq doses of up to mg b.i.d. could result in even faster rates of viral decline but there is limited safety information for these high doses. hcq pharmacology is complex; hcq distributes extensively into erythrocytes (whole blood to plasma ratio ~ . , exhibits a long half-life ( hours) and a large volume of distribution, all attributed to extensive tissue uptake, clearly important for treatment of covid- systemic illness. , hcq and cq are diprotic weak bases (with pka of . and . vs. . and . for hcq and cq, respectively). interestingly, both drugs experience ion-trapping in which the drug becomes ionized in acidic environments like the lysosome (ph ~ . ). this causes an irreversible accumulation, explains the large volume of distribution, and potentially impacts the amount of free drug available in tissues. , hcq is converted into at least three metabolites (desethylhydroxychloroquine, desethylchloroquine, and bidesethylhdroxychloroquine). desethylhydroxychloroquine hcq, the primary metabolite, is pharmacologically active for some nonviral illnesses, and formed by various cytochrome p isozymes. for our analysis, we focused on the parent hcq, as potent in vitro activity against sars-cov- has only been described for the parent compound. for derivation of our dosing rationale, we have utilized hcq levels in plasma, instead of the lungs. lung accumulation has been observed for hcq and cq in animal pk studies and reported to be substantial (a partition coefficient of ( . )). the partition coefficient ratio enables quantification of the total drug concentration in the tissue, and by assuming the same fraction of unbound drug in plasma and tissue, one can further estimate unbound concentrations in the tissue. by using this approach, a wide range of doses, including doses as low as mg, seem to be potentially therapeutic. the drug efficacy at the site of action is determined by the fraction of drug unbound in the tissue, which has not been studied for hcq, and, thus, the amount of free drug in tissue remains unknown. highly lipophilic drugs for other infectious diseases, like bedaquiline and clofazimine, accumulate in lungs as well, however, the accumulation correlates with binding to macromolecules in tissue, not necessarily to the free fraction. , based on the physicochemical parameters of hcq (log p of . and pka of . , . ), the fraction unbound in tissue is likely low. therefore, in our study, we conservatively assume that the free fraction in plasma equilibrates between plasma and tissue and consider that to be the fraction of drug that can contribute to drug effect. tissue binding studies using a rapid equilibrium dialysis assay with lung homogenate should be performed to define an accurate fraction unbound in the tissue. using a mechanistic pk/pd modeling approach, we were able to quantify a relationship between hcq concentration and sars-cov- viral decline. however, we were not able to differentiate if azithromycin offered any additional benefit. the group receiving hcq and azithromycin had the lowest baseline viral load and showed a similar rate of viral decline compared with the hcq group. therefore, it remains unclear if azithromycin offers any additional benefit. clinically significant qtc prolongation associated with hcq have been reported. [ ] [ ] [ ] only two small observational studies have reported associations between hcq doses of - mg daily and qtc prolongation , and a concentration-dependent qtc relationship is not available. as a result, we used cq as a model to predict qtc prolongation risk. hcq and cq have an identical structure with the substitution of a hydroxyl group for hcq, and both have been found in vitro to inhibit the inward rectifier k+ channels. , this has been associated with qtc prolongation, and docking studies suggest nitrogen in the alkylamine and quinoline ring found in both compounds are responsible for binding with potassium channels. although a dedicated study is needed, the hydroxyl group in hcq is unlikely to affect rectifier k+ channels binding as the pka for the alkylamine nitrogen is similar to that of chloroquine's. in vitro data from cq identified an herg ic of , nm. we leveraged a recent study of high-dose cq for malaria treatment to predict potential risk of qtc prolongation with hcq. in support of our findings, a maximum dose of , mg daily for - weeks has been well-tolerated without reported cardiac toxicity. , based on this evidence, and the pk-qtc relationship for cq presented here, we expect a hcq course of - mg b.i.d. for days or less is unlikely to be associated with clinically significant cardiac toxicity in patients without a known risk factor for qtc prolongation. as data for hcq and qtc prolongation are limited, we recommend the highest doses of hcq be reserved for study in dose escalation studies. additional toxicities associated with hcq include retinopathy and gastrointestinal adverse events. , the mechanism of irreversible retinal damage associated with hcq is unknown, but it has been associated with hcq doses > mg/kg and in patients who receive hcq for > years. retinopathy associated with use < month of hcq has not been reported, and this side effect is less likely in the acute setting. , gastrointestinal toxicity with hcq is concentration-related and could be a limiting factor to dosage of hcq but doses up to , mg have been reported to be well-tolerated without adverse events in patients with cancer and rheumatologic disease in other studies. there were a few limitations to this study. first, clinical hcq data are limited to nonrandomized studies, and a clear model for the natural rate of viral decline is not well defined. to explore this effect, we compared viral kinetic trends on treatment to the extracted baseline data from cao et al. (n = hospitalized patients who received supportive care). second, the translational viral replication was obtained from sars-cov- data. sars-cov- and sars-cov- share an estimated . % sequence homology. third, we imputed the pk profiles for hcq using population pk parameters derived from a pool of both healthy and malaria-infected patients. fourth, we were not able to predict how concomitant hcq and azithromycin may impact the risk of qtc prolongation or anticipate how underlying risk factors for qtc prolongation could impact the pk-qtc relationship due to the lack of available data. closely monitored clinical trials will be needed to confirm that high-dose hcq is safe with or without azithromycin. finally, our model used plasma hcq concentrations to predict nasopharyngeal viral loads, which may not fully correlate with clinical improvement or viral load measured at different sites, however, it has generally been accepted that viral decline is a desirable marker leading to clinical improvement. [ ] [ ] [ ] [ ] [ ] [ ] in addition, all relevant assumptions made during the analysis are summarized in supplementary table s . treatment options for covid- can most effectively be advanced by utilizing all available data and pharmacologically driven drug repurposing. suboptimal dosing can result in wasted time and resources. even more problematic is the potential to declare a drug ineffective because of misdosing. using pk-exposure modeling, we predict that higher doses of hcq will be needed to achieve cure within days for all patients. given the observed prolonged viral shedding in patients with covid- , these data support the possibility that early treatment with high-dose hcq could reduce transmissibility and potentially reduce the risk of late clinical decompensation. however, given the possibility of qtc prolongation with high-dose regimens, rigorous trials must precede widespread clinical usage. we predict that higher hcq doses, (> mg b.i.d.) are most efficacious for viral suppression and should be further examined in clinical trials to evaluate safety and efficacy. supplementary information accompanies this paper on the clinical pharmacology & therapeutics website (www.cpt-journal.com). coronavirus disease (covid- ) outbreak situation <https design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) information for clinicians on therapeutic options for covid- patients <https hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro screening of a fda approved chemical library reveals potential inhibitors of sars-cov- replication sars-associated coronavirus replication in cell lines a randomized, controlled trial of ebola virus disease therapeutics clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: an observational study pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by plasmodium vivax plasma or serum in therapeutic drug monitoring and clinical toxicology likelihood based approaches to handling data below the quantification limit using nonmem vi ways to fit a pk model with some data below the quantification limit high-dose chloroquine for uncomplicated plasmodium falciparum malaria is well tolerated and causes similar qt interval prolongation as standard-dose chloroquine in children randomized dose-ranging controlled trial of aq- , a candidate antimalarial, and chloroquine in healthy volunteers a trial of lopinavir-ritonavir in adults hospitalized with severe covid- pharmacokinetics of hydroxychloroquine in pregnancies with rheumatic diseases on the mechanism of chloroquine resistance in plasmodium falciparum hydroxychloroquine: a physiologically-based pharmacokinetic model in the context of cancer-related autophagy modulation the contribution of lysosomal trapping in the uptake of desipramine and chloroquine by different tissues prediction of drug penetration in tuberculosis lesions tuberculosis drugs' distribution and emergence of resistance in patient's lung lesions: a mechanistic model and tool for regimen and dose optimization hydroxychloroquine is much less active than chloroquine against chloroquine-resistant plasmodium falciparum, in agreement with its physicochemical properties toxicokinetics of hydroxychloroquine following a massive overdose life threatening severe qtc prolongation in patient with systemic lupus erythematosus due to hydroxychloroquine chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia long qt and hydroxychloroquine; a poorly recognised problem in rheumatology patients voltagedependent biphasic effects of chloroquine on delayed rectifier k(+)-channel currents in murine thymocytes urgent guidance for navigating and circumventing the qtc-prolonging and torsadogenic potential of possible pharmacotherapies for coronavirus disease (covid- ) the molecular basis of chloroquine block of the inward rectifier kir . channel pka values of medicinal compounds in pharmacy practice cardiotoxicity of antimalarial drugs hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis phase i trial of hydroxychloroquine with doseintense temozolomide in patients with advanced solid tumors and melanoma development and validation of a risk score to predict qt interval prolongation in hospitalized patients the risk of toxic retinopathy in patients on long-term hydroxychloroquine therapy accidental hydroxychloroquine overdose resulting in neurotoxic vestibulopathy a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov- viral load in upper respiratory specimens of infected patients viral load of sars-cov- in clinical samples virological assessment of hospitalized patients with covid- detection of sars-cov- in different types of clinical specimens single-cell rna-seq data analysis on the receptor ace expression reveals the potential risk of different human organs vulnerable to -ncov infection high expression of ace receptor of -ncov on the epithelial cells of oral mucosa the investigators thank the savic laboratory for all their help and support during this project. we would also like to thank tia tummino for her help addressing reviewer comments. no funding was received for this work. the authors declared no conflict of interest. this is an open access article under the terms of the creative commons attribution-noncommercial license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. key: cord- -mf ae authors: tan, cedric chih shen; maurer-stroh, sebastian; wan, yue; sessions, october michael; de sessions, paola florez title: a novel method for the capture-based purification of whole viral native rna genomes date: - - journal: amb express doi: . /s - - -y sha: doc_id: cord_uid: mf ae current technologies for targeted characterization and manipulation of viral rna primarily involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host rna background. the former strategy is non-compatible for characterizing properties innate to rna strands such as secondary structure, rna–rna interactions, and also for nanopore direct rna sequencing involving the sequencing of native rna strands. the latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral rna. to address this, we developed a novel application of current nucleic acid hybridization technologies for direct characterization of rna. in particular, we modified a current enrichment protocol to capture whole viral native rna genomes for downstream rna assays to circumvent the abovementioned problems. this technique involves hybridization of biotinylated baits at nucleotides (nt) intervals, stringent washes and release of free native rna strands using dnase i treatment, with a turnaround time of about h min. rt-qpcr was used as the primary proof of concept that capture-based purification indeed removes host background. subsequently, capture-based purification was applied to direct rna sequencing as proof of concept that capture-based purification can be coupled with downstream rna assays. we report that this protocol was able to successfully purify viral rna by - to -fold. we also report that application of this protocol to direct rna sequencing yielded a reduction in human host rna background by -fold, a . % recovery of viral genome with at least × coverage, and a mean coverage across the genome of ×. this report is, to the best of our knowledge, the first description of a capture-based purification method for assays that involve direct manipulation or characterisation of native rna. this report also describes a successful application of capture-based purification as a direct rna sequencing strategy that addresses certain limitations of current strategies in sequencing rna viral genomes. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. viruses with rna genomes are the cause of many infectious diseases with serious consequences to human health and mortality such as flaviviruses, hiv- , sarscoronavirus, htlv- and influenza virus (cantara et al. ) . it has been shown that rna-rna interactions (romero-lópez and berzal-herranz ) and intramolecular structure (witteveldt et al. ) of viral rna genomes play an important role in viral replication. however, extraction of viral rna directly from culture often yields viral rna with high host rna background (marston et al. ) . to deal with this issue, two main strategies are employed. firstly, extracted viral rna can be reverse transcribed to complementary dna (cdna) and amplified via polymerase chain reaction (pcr) to suit the sensitivities of the downstream assays (ozsolak and milos ) . following conversion to cdna and amplification, in vitro transcription is then performed to retrieve genetic information in its rna form, since both rna-rna interactions and rna intramolecular structure can only be observed whilst in this form (peattie ) . indeed, both romero-lópez and berzal-herranz ( ) and witteveldt et al. ( ) amplified viral rna obtained from cell culture via pcr amplification of viral cdna clones followed by in vitro transcription. it has been shown, however, that such indirect methods for probing inherent properties of rna increases experimental throughput at the expense of introducing pcr-induced artifacts and biases (acinas et al. ) , potentially clouding our understanding of these properties (ozsolak et al. ). secondly, isopycnic ultracentrifugation can be used to retrieve high purity samples of viral particles containing viral rna, which can be subsequently extracted for downstream rna assays. one example is the use of a sucrose density gradient to purify denv rna for rna tertiary structure analysis (dethoff et al. ) . while the use of isopycnic centrifugation allows for a high purity of viral rna, only rna from encapsulated viruses are obtained (liu et al. ) , making this technique unfeasible for the study of unassembled viral genomes, which have also been shown to be crucial to our understanding of viral replication machinery (manokaran et al. ; moon et al. ) . with the advent of rd generation nanopore sequencing, direct rna sequencing of native rna strands is heralded as a pcr and cdna-bias free sequencing technology (garalde et al. ) . however, the direct rna sequencing of rna viral genomes is similarly affected by high amounts of host rna background in viral cultures. the only method to date which addresses this issue is the use of custom sequencing adapters which has been demonstrated to yield high sequencing coverage for rna viruses. keller et al. ( ) utilized a custom sequencing adapter complimentary to the ′ end of influenza a viral genome to selectively sequence only the viral rna. however, this strategy is unviable for samples with diverse viral strains or genotypes where the ′ sequence is unknown or non-conserved. an appropriate example is the wide diversity in hepatitis c virus genotypes, which has caused issues in pcr primer design for target enrichment strategies due to sequence ambiguity (thomson et al. ) . similarly, cowan et al. ( ) noted that pcr-based enrichment techniques where a priori knowledge of target sequences is required for pcr primer design render the enrichment strategies ineffective in the characterization of novel viruses. it follows that the same sequence ambiguity in viral genomes would pose a problem for using customized sequencing adapters during direct rna sequencing. from the abovementioned problems, we identified a need for an alternative technique that isolates viral rna molecules from host rna background without pcr amplification or cdna synthesis. in the case of direct rna sequencing, such a technique should address the limitations presented by current strategies. in this study, we describe a capture-based purification method that leverages on current nucleic acid hybridization and enrichment techniques. this novel method is able to isolate native viral rna molecules from a high host rna background without the use of pcr amplification or cdna synthesis whilst retaining genetic information. our capture-based purification method is distinguished from conventional hybridization-based or pcr-based enrichment techniques explored in the review paper by mamanova et al. ( ) by virtue of not having any pcr amplification or cdna conversion steps. such a purification method, when merged with current technologies for probing and characterizing inherent rna properties, could potentially catalyze the study of viral rnas and elucidate previously inaccessible characteristics of viral rna genomes. reverse-transcription quantitative polymerase chain reaction (rt-qpcr) was used as the primary proof of concept that capture-based purification indeed removes host background. subsequently, capture-based purification was applied to direct rna sequencing as proof of concept that capture-based purification can be coupled with downstream rna assays. the application of capture-based purification to direct rna sequencing as an alternative to current strategies will also be discussed. viral inoculum and huh- hepatocellular carcinoma cells (japanese tissue repository) were cultured in dmem media ( % fetal bovine serum, % penicillin streptomycin) up to - % confluency in corning ® cm cell culture flasks (sigma aldrich). prior to infection, spent media was discarded and huh- cell monolayer was washed with ml × pbs twice. viral inoculum was provided by low et al. ( ) . a multiplicity of infection of was used, ml dengue virus strain d / sg/ k dk / - -i (denv ) was diluted with ml pure dmem media and mixed via pipetting up and down times. four milliliters of diluted virus was pipetted into each flask and incubated at °c for h. flasks were rocked every min. subsequently, inoculum was removed via pipette and ml of pure dmem media was pipetted to each flask was incubated at °c for h. extractions were separately performed on viral supernatant and cell lysates. media of infected flasks (viral supernatant) was transferred to a ml falcon tube. subsequently the cell monolayer of each flask was treated with ml of trizol ls reagent (invitrogen) and . ml of × pbs (thermo fisher scientific) followed by incubation at °c for min. one milliliter aliquots of this cell lysate mixture were then pipetted to . ml eppendorf tubes. µl aliquots of viral supernatant were also pipetted into individual tubes and treated with µl of trizol ls regeant. µl of chloroform was then added to individual aliquots of viral supernatant and cell lysate before incubation at room temperature for min. tubes were shaken vigorously by hand for s, and subsequently inverted by hand every min. each tube was centrifuged at , g, °c for min. upper aqueous phase in each tube was carefully removed and pipetted to a new . ml eppendorf tube (approximately µl). two microliters of glycoblue coprecipitant (invitrogen) and µl of isopropanol (sigma aldrich) was added to each new tube and stored in − °c overnight (or longer). prior to the capture step, tubes were centrifuged at , g, °c for min and supernatant was discarded. pellet was re-suspended in µl % ethanol diluted with nuclease-free water. tubes were centrifuged again at g, °c for min and supernatant was discarded. pellet was air dried. for the cell lysate rna, total rna concentration was determined via nanodrop (thermo fisher scientific) and re-precipitated in ethanol to obtain µg total rna pellets. viral supernatant rna either underwent capture or direct rna sequencing library preparation without re-precipitation. twenty-one denv baits were designed using bait-maker, https ://umasa nguma thi.githu b.io/baitm aker/ (plos neglected tropical diseases, kamaraj et al. , in press ). the bait stock in our experiments was previously used for hybridization-based target enrichment in a published study by the singapore zika study group (ho et al. ) . briefly, mer biotinylated dna baits (integrated dna technologies) specific to conserved regions across all serotypes of dengue virus were used. baits were tiled along the viral genome with intervals of nt. a total of pmol of baits were added to each capture. this protocol was modified from that for the use of xgen lockdown baits and reagents (integrated dna technologies), optimized for our purposes of rna capture. seqcap hybridization and wash kit (roche) was used in place of integrated dna technologies reagents. figure demonstrates the workflow for the capture protocol. rna pellet from the extraction step was re-eluted with reagents shown in additional file : table s and incubated at °c for h in a thermocycler. µl of dynabeads m- streptavidin beads (thermo fisher scientific) was pipetted to a . ml eppendorf tube and equilibrated at room temperature for min. the tube was put on a magnetic rack (invitrogen) until eluate was clear and supernatant was discarded. µl × bead wash buffer was added and mixture was vortexed for s before placing it on a magnetic rack and discarding the supernatant for a total of two washes. hybridization mixture was immediately added to the beads and incubated at °c for min. all wash buffers were diluted to × prior to washing. wash buffer i and stringent wash buffer was pre-heated for h to °c. µl of wash buffer i was added to the hybridization-bead mixture, agitated by hand and transferred to a new . ml eppendorf tube. the new tube is placed on a magnetic rack. once eluate is clear, supernatant was quickly discarded, taking caution not to remove any beads. µl of stringent wash buffer was added to the pellet and incubated at °c for min, before discarding the supernatant as per the method above, for a total of two washes. subsequent washes were performed fig. capture-based purification workflow as per the method above without the incubation step in the following sequence: wash buffer i, wash buffer ii, and wash buffer iii. viral rna was released from the beads via dnase i treatment. beads were re-suspended in µl of nuclease-free water, incubated at °c for min then cooled to °c. subsequently, . µl of × reaction buffer and µl of turbo dnase (thermo fisher scientific) was added to the reaction and mixed by pipetting up and down times, followed by further incubation at °c for min. subsequently, the tube was placed on a magnetic rack to allow the beads to aggregate. the supernatant was then transferred to a clean . ml eppendorf tube for a . × agencourt rnaclean xp beads clean-up (beckman coulter). briefly, . µl of beads were added to the supernatant and incubated for min at room temperature, and then min on a magnetic rack. the beads were washed twice with % ethanol and residual ethanol was air-dried. the beads were then re-eluted in µl of nuclease-free water. the proof of concept for our capture-based purification method was to demonstrate that host rna background could be removed from target viral rna. absolute quantification of host and viral rna was done using rt-qpcr. two . ml eppendorf tubes containing µg of total rna from cell lysate were prepared to represent pre and post-capture groups. µl and µl of nucleasefree water was added to the pre and post-capture group respectively. quantitect probe rt-pcr kit was used according to the manufacturer's instructions. primer targets were selected in the nonstructural protein region of denv and human gapdh (glyceraldehyde -phosphate dehydrogenase) as a reference gene. the volumes of reagents, shown in additional file : table s , were added to a . ml thin-walled pcr tube, and incubated in a viia real time-pcr system (applied biosystems) with the program shown in additional file : table s and analyzed using quantstudio real-time pcr software v . (applied biosystems). each plate included a non-template control for each pair of primers to check for pcr contamination. samples were quantified in duplicate and non-template controls in triplicate. a standard curve was constructed using a serial dilution of an oligo complementary to the primer target in accordance with miqe guidelines (taylor et al. ) . custom oligoes of primer targets were ordered from integrated dna technologies. serial dilution was performed with nuclease-free water to obtain concentrations of . × n copies/µl (where n = , , ,…, ). each dilution was quantified in duplicate. the sequences of the oligoes, primers and probes used are shown in additional file : table s . average c q values for the serial dilutions were linearly regressed on log (copies/µl) and pearson's product-moment correlation coefficient was calculated. equation ( ) was used to determine primer efficiency: where m is the gradient of the regression line of < c q > on log (copies/µl). absolute quantity (copies/µl) was calculated using the respective equations of the regression lines. the absolute quantity of denv rna, normalized to human gapdh reference gene, was then compared between pre and post-capture groups. the ratio of the absolute quantity of denv and human rna pre and post-capture was compared, and purification factor calculated using eq. ( ). in this study we defined purification factor as a measure of how well host rna background is removed from viral rna. pre and post-capture rna extracted from supernatant were sequenced on the minion. separately, we pooled captures to generate the concentrated post-capture group. six captures each consisting of µl of purified rna was added to the same rnaeasy minikit (qiagen) column and final elution volume was µl. prior to library preparation, all rna samples were polyadenylated using e. coli poly(a) polymerase (new england biolabs). the volumes of reagents shown in additional file : table s were added to a thin-walled . ml pcr tube. the reaction was then incubated in a thermocycler with the following program: °c for min then cooled to °c before proceeding to a . × agencourt rnaclean xp cleanup (beckman coulter). the rna was eluted in . µl nuclease-free water. subsequently, library preparation and sequencing was performed according to the protocol for direct rna sequencing kit (sqk-rna ) using . µl of rna. all ligation reactions were extended to min. the rna library was loaded on a r . min-ion flow cell and sequencing was ran for h using minknow v . . (gui . . ) interface. live basecalling was turned off. raw fast files were basecalled and end adapters were trimmed using poreplex v . (https ://githu b.com/hyesh ik/porep lex) which internally calls albacore v . . (oxford nanopore technologies). basecalled read metrics and plots were then generated using nanoplot v . . (https ://githu b.com/wdeco ster/nanop lot). subsequently, mapping and alignment was performed using graphmap v . . (https ://githu b.com/isovi c/graph map) with default parameters. mean coverage and mapping quality was calculated using qualimap v . . (garcía-alcalde et al. ) respectively. viral and human genome references used were eu . and grch respectively. based on the ratios of the absolute quantities of viral to host rna shown in table , we report a - purification factor, suggesting successful retrieval of viral rna and substantial removal of host rna background. capture yield, which is defined as the percentage of viral rna amount captured, was calculated to be . - . %. additional file : figure s shows the standard curves for denv and gapdh primers. the primer efficiency of denv and gapdh was . and . respectively. the denv primer set used was a pre-optimized primer set that is able to detect all dengue virus serotypes previously used and therefore contains ambiguous bases, explaining the lower primer efficiency. it was noted that relative quantification using the −ΔΔct method (schmittgen and livak ) was not appropriate as the primer efficiencies of denv and gapdh primer sets were not comparable. absolute quantification and subsequent ratio comparison of viral and host rna amounts is therefore more accurate in the determination of purification factor. all obtained values were within the dynamic linear range and limit of detection of the reaction conditions and primer sets used, suggesting reliability and accuracy of the reported rt-qpcr results. based on the mapping rates to human and denv reference genomes for pre and post-capture groups, shown in table , purification factor was calculated to be -fold. we also report a . -fold increase in percentage recovery of denv genome over × coverage from a single precapture to a single post-capture rna sample. on the other hand, comparing the concentrated post-capture group (containing a pool of captures) to the pre-capture group yielded a purification factor of -fold and a -fold increase in the percentage recovery of denv genome above with at least × coverage from . to . %. additional file : figures s , s , and s show representative coverage plots for the pre-capture, post-capture and concentrated post-capture groups respectively. in eqs. ( ) and ( ), a binomial distribution was used to determine the minimum read coverage, n, required to have a . confidence that k reads mapping to a particular base, x, are correctly called. we used the average error read rate of our runs as the probability, p, of correctly calling and mapping a single base. since the common heuristic for variant calling with illumina reads requires × coverage (illumina, https ://www.illum ina.com/docum ents/produ cts/ techn otes/techn ote_snp_calle r_seque ncing .pdf ), we assumed the criterion that at least reads were correctly called and mapped to a single base (k = ). based on our sequencing runs, the average error read rate of pre and post-capture sequencing runs was . ± . % (calculated from table ). with this error read rate, we substitute − p = . into eq. ( ) and determined that the minimum coverage, n, required for variant calling to be at least ×. we demonstrated with rt-qpcr that our novel rna capture technique can be used to retrieve and purify viral rna in its native form for downstream assays. this was corroborated by our direct rna sequencing results. indeed, unpurified viral rna represented by the pre-capture group did not produce sufficient throughput for any meaningful downstream analysis due to the indiscriminate nature of the sequencing of both host and viral rna. however, after treatment with our capture method, the amount of host rna background has decreased greatly as seen from the -fold increase in mapping rates to target viral rna. in comparison, even with the incorporation of pcr amplification for shotgun sequencing of enterovirus, jensen et al. ( ) reported only a -to -fold increase in mapping rates to enterovirus. moreover, wongsurawat et al. ( ) observed only a -fold increase in mapping rates to zika virus (zikv). this suggests that our capture-based purification method is highly successful in the removal of host rna background. the minimum coverage required for variant calling was, as described above, benchmarked to that of illumina reads so that the effectiveness of our capture-based purification method could be more accurately evaluated based on the higher error read rates of direct rna sequencing technology. even with the higher adjusted heuristic for variant calling of ×, the ( ) x ∼ b(n, p) ( ) p(x ≥ k) ≥ . ( ) n! !(n − )! ( . ) ( . ) n− ≥ . . -fold increase in percentage of the genome above × coverage suggests that application of our capturebased purification method increases the amount of target genetic information retrieved. our method fundamentally allows for the capture of unknown viral strains and genotypes due to the flexibility of adding multiple baits complementary to different regions of the viral genome, or even multiple baits specific to different viruses in a single capture. indeed, deviatkin et al. ( ) has reported the successful use of genus-specific degenerate baits for hybridization-based enrichment, which can be seamlessly incorporated into our bait design. by extension, the baits used can be appropriately modified according to the intended research focus. the robustness of this capture-based purification method therefore not only broadens the applicability of direct rna sequencing on studying unknown viruses but also the applicability of a diverse range of direct rna probing technologies. another major advantage to our capture-based purification method is that large machinery like an ultracentrifuge is not required. to perform our capture method, only a thermocycler and a heat block is required. this is especially applicable to research environments where an ultracentrifuge is not readily available. more fundamentally, our capture-based purification method allows for the capture of both unassembled and assembled viruses, which any method leveraging on the differential physiochemical properties of encapsulated viral rnas cannot. this would be important in studies of unassembled rna segments, which have been demonstrated to be of great biological importance. indeed, in the case of denv , subgenomic flaviviral particles have been shown to underlie mosquito immunity and viral transmission (pompon et al. ) . if so, a method which can remove host background and capture unassembled viruses would be useful for characterising inherent rna properties of unassembled viruses. one point of concern for our protocol is the low capture yield. however, it must be noted that the capture yield is still significantly higher than that of conventional viral purification methods. hall et al. ( ) , in a study evaluating conventional viral purification methods, reported a -fold decrease in viral copy number post-treatment, whereas our method results in, at most, a -fold decrease in viral copy number. in evaluating our capture-based purification method, a wide range in capture yield was observed, which was due to the high sensitivity of rna to potential contamination during handling. this is a problem common to any viral rna purification method. our capture-based purification method has a turnaround time of approximately h min (fig. ) , comparable to the time required for isopycnic gradient-based methods (buclez et al. ) . while there is a significant loss of viral rna post capture, we also note that our capture method is scalable. multiple captures can be performed and purified rna pooled together before re-concentrating in the final bead clean-up step. the concentrated post-capture group was a demonstration of how our capture-based purification method was appropriately scaled to suit the sensitivity of direct rna sequencing. indeed, after comparison of the postcapture and concentrated post-capture sequencing runs (table ) , the . -fold increase in the percentage of reads mapping to denv suggests that scaling our method greatly improved the signal-to-noise ratio of this particular downstream rna assay. while it was opined that this improvement would similarly be observed in other downstream rna assays, further experimental verification is required. the use of direct rna sequencing to study viral genomes is limited by its sensitivity. conventionally, the sequencing adapter used in direct rna sequencing was a poly(t) adapter which would hybridize with poly(a) tails, facilitating adapter ligation (garalde et al. ). this poly(t) adapter is also the sequencing adapter provided in the direct rna sequencing library preparation kit (sqk-rna ). currently, four strategies may be employed for direct rna sequencing of rna viruses. firstly, as described by keller et al. ( ) , a custom adapter can be used during library preparation. the second strategy is the use of the conventional poly(t) adapter supplied in the kit by polyadenylation of rna prior to library preparation and sequencing. the third strategy is to couple polyadenylation with capture-based purification. the fourth is to couple polyadenylation with host rrna depletion (wongsurawat et al. ) . a summary of the advantages and disadvantages for these strategies are presented in table . the custom adapter strategy employed by keller et al. ( ) greatly improves sequencing specificity, and was demonstrated to yield . % of reads mapping to influenza a. this strategy allows sequencing of rna viruses with or without an inherent poly(a) tail since the conventional poly(t) adapter was substituted with a custom sequencing adapter specific to the target. however, one drawback of this strategy is that foreknowledge of the ′ end of rna targets is required. this entails that sequencing of unknown, novel, or mutant viruses would be difficult. another limitation is that this strategy may be ineffective in cases where the rna sample is fragmented since the ′ end of each fragment of viral rna is distinct. degenerate adapters may be used to circumvent the first limitation, as with the degenerate adapters at the + position used by keller et al. ( ) . however, using degenerate adapters would only be able to address the problem of mutant viruses but not that of novel or unknown viruses. indeed, foreknowledge of target ′ sequences is still required. on the other hand, attempts at using multiple adapters at different locations on the target genome cannot address the problem of rna fragmentation. custom adapters must be specific to the ′ end of each fragment for ligation. in most laboratory samples, however, rna is stochastically fragmented. as such, determination of the exact ′ end of each fragment would be impossible and so such methods to circumvent the problem of rna fragmentation are unviable. in this study, we demonstrated the second and third strategies, which make use of the conventional poly(t) adapter. this was done using denv , a positive single stranded rna virus lacking a poly(a) tail. the protocol used to generate the pre-capture group sequencing results employ the second strategy while that for the post-capture and concentrated post-capture results employ the third. the polyadenylation of rna and subsequent use of the conventional poly(t) adapter has several advantages. first, the polyadenylation of rna prior to library preparation and sequencing allows for sequencing of rna viruses with or without an inherent poly(a) tail. second, usage of the conventional poly(t) adapter removes the need for any foreknowledge of the target sequence. third, all rna fragments would be ligated with adapters since the adapters are not sequence specific, circumventing the problem of fragmented rna. however, the main disadvantage strategies involving polyadenylation of rna is that sequencing specificity is greatly reduced as host mrna transcripts include a poly(a) tail and will also be sequenced. wongsurawat et al. ( ) independently replicated our pre-capture group results but with zikv, which is also a positive single stranded rna virus lacking a poly(a) tail in the family flaviviridae. the authors observed . % of reads mapping to zikv, which corroborates the low . % of reads mapping to denv in our pre-capture sequencing run. the authors also reported . % of reads mapping to zikv whilst employing the fourth strategy. the low percentage of target reads underscores this main disadvantage of sequencing specificity. the issue of low sequencing specificity is, however, less pronounced in the third strategy. comparing the second strategy (pre-capture) and third strategy (post-capture), there is a -fold increase in percentage of reads mapping to denv . furthermore, a comparison of pre-capture and concentrated post-capture results yields a -fold increase in mapping rates to denv . this suggests that while simply polyadenylating rna samples leads to low sequencing specificity, applying our capture-based purification prior allows us to reap the advantages of using the conventional poly(t) adapter whilst having a significantly higher sequence specificity. additionally, coupling capture-based purification with polyadenylation yields a higher target mapping rate of . % with regard to the . % observed when the fourth strategy was employed (wongsurawat et al. ) . this suggests that the third strategy is superior with respect to sequencing specificity when compared to the fourth strategy. it is noted, however, that the sequencing specificity of the third strategy was still lower than that of the first strategy. moreover, applying capture-based purification in the third strategy significantly increases the turnaround time as compared to the first, second and fourth strategy. therefore, it is suggested that the third strategy should only be employed where foreknowledge of the viral sequence at hand is limited, or where rna integrity is low. it is also suggested that the second and fourth strategy be used in cases where detection rather than characterisation of rna viruses is required due to significantly lower sequencing specificity. in this study, we demonstrated a novel capture-based method for the purification of viral rna from host rna. in addition, we described a successful attempt of applying our capture method to the direct rna sequencing of viral culture, using a denv model, exemplifying how our capture method could significantly remove host background in downstream assays requiring viral rna in its native form. further improvements to the capture yield are underway and could potentially expand its application to clinical samples where starting rna material is low. the fundamental advantages of using our capturebased purification method makes it a superior alternative to conventional viral purification methods and addresses limitations of current strategies for direct rna sequencing, potentially paving a new path for the characterization and direct rna sequencing of viral rna. additional file : table s . reagents and volumes for hybridization step. table s . reagents and volumes for rt-qpcr. table s . rt-qpcr program set on applied biosystems viia real time-pcr system. table s . primers, probes and standards used with the respective modifications for rt-qpcr. table s . reagents and volumes used for polyadenylation of rna samples. figure s . denv and gapdh standard curves used for calculation of primer efficiency. figure s . coverage plot against nucleotide position on denv reference for pre-capture minion sequencing run. figure s . coverage plot against nucleotide position on denv reference for post-capture minion sequencing run. figure s . coverage plot against nucleotide position on denv reference for concentrated post-capture minion sequencing run. csct was the primary contributor to this study with guidance from his mentor pfdes and helpful advice from oms, sm-s and yw. all authors read and approved the final manuscript. author details genome institute of singapore (gis), agency for science, technology and research (a*star), singapore, singapore. a*star graduate academy, singapore, singapore. university college london, london, uk. bioinfomatics institute (bii), a*star , singapore, singapore. national university of singapore, saw swee hock school of public health, singapore, singapore. pcr-induced sequence artifacts and bias: insights from comparison of two s rrna clone libraries constructed from the same sample rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system progress and outlook in structural biology of large viral rnas metagenomic gene discovery: past, present and future pervasive tertiary structure in the dengue virus rna genome enrichment of viral nucleic acids by solution hybrid selection with genus specific oligonucleotides highly parallel direct rna sequencing on an array of nanopores qualimap: evaluating next-generation sequencing alignment data evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery outbreak of zika virus infection in singapore: an epidemiological, entomological, virological, and clinical analysis target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery direct rna sequencing of the coding complete influenza a virus genome purification and characterization of enterovirus viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system early dengue infection and outcome study (eden)-study design and preliminary findings target-enrichment strategies for next-generation sequencing dengue subgenomic rna binds trim to inhibit interferon expression for epidemiological fitness next generation sequencing of viral rna genomes flavivirus sfrna suppresses antiviral rna interference in cultured cells and mosquitoes and directly interacts with the rnai machinery rna sequencing: advances, challenges and opportunities direct rna sequencing direct chemical method for sequencing rna dengue subgenomic flaviviral rna disrupts immunity in mosquito salivary glands to increase virus transmission a long-range rna-rna interaction between the ′ and ′ ends of the hcv genome analyzing real-time pcr data by the comparative ctmethod a practical approach to rt-qpcr-publishing data that conform to the miqe guidelines comparison of next-generation sequencing technologies for comprehensive assessment of full-length hepatitis c viral genomes the influence of viral rna secondary structure on interactions with innate host cell defences rapid sequencing of multiple rna viruses in their native form we would like to acknowledge ng hui qi amanda and niranjan nagarajan from computational systems biology , gis; aw poh kim pauline (denv primer design), kwan kam weng philip, balamurugan periaswamy, png eileen from germs platform for their technical advice throughout the course of this study; vithiagaran gunalan and dimitar kenanov from maurer-stroh's group for helpful conversations. we would also like to acknowledge members of wan yue's team, in particular, lim wei sheng shaun, aw jong ghut ashley. lastly, we would like to thank andreas wilm and shih chih chuan from the gis bioinformatics core. the authors declare that they have no competing interests. data will be made available through publication and ncbi. this article does not contain any studies with human participants or animals performed by the author. all authors listed on this manuscript have read and agreed to the publication of this research. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -fhvt ht authors: burrell, christopher j.; howard, colin r.; murphy, frederick a. title: virus replication date: - - journal: fenner and white's medical virology doi: . /b - - - - . - sha: doc_id: cord_uid: fhvt ht understanding the molecular events accompanying virus replication is essential for the proper understanding and control of all virus diseases. the virus replication cycle generates new viral genomes and proteins in sufficient quantities to ensure propagation of the viral genome; this requires that the extracellular viral genome is protected from enzymatic degradation and can be introduced into further target cells for further rounds of replication. the initial recognition between virus and host is more complex than originally supposed and may involve more than one cellular receptor. a critical first intracellular step is the generation of viral mrna by one of a limited number of strategies first described by david baltimore. lacking ribosomes, viruses have no means of producing protein and are reliant on the host cell for protein synthesis. viral proteins are often modified by host cell glycosylation during or after virus assembly. temporal regulation of intracellular events is critical in all but the very simplest of viruses, and some form of suppression of the host innate immune response is common to nearly all human viruses. infected cells often produce non-infectious particles with incomplete genomes, and these defective interfering particles may play a role in pathogenesis. understanding these processes will open up a range of targets for the development of novel therapies. understanding the molecular events accompanying virus replication has been a major focus of experimental virology, and is essential for the proper understanding and control of all virus diseases. the biological "purpose" of any replication cycle is the generation of new viral genomes and proteins in sufficient quantities to ensure propagation of the viral genome (a clear example of the "selfish gene"); this requires that the extracellular viral genome is protected from enzymatic degradation and can be introduced into further target cells for further rounds of replication. much has become known about the initial stages of attachment, and more detailed study shows that the initial recognition between virus and host is more complex than originally supposed. temporal regulation of intracellular events is critical in all but the very simplest of viruses, with some form of suppression of the host innate immune response being common to nearly all human viruses. there are examples where the innate immune response is even used to enhance virus spread to cells otherwise unavailable to the virus. over the next few years, the boundaries between virus-directed events and cellular processes that control specialized cell functions are likely to be even more complex; nevertheless understanding these processes will open up a range of targets for the development of novel antiviral therapies and immunotherapy. this chapter presents an overview of the subject, indicating similarities and differences in the replication strategies adopted by viruses of each family that contains human pathogens. major features and replication requirements of human viruses are shown in table . . more detailed information about the replication of individual viruses can be found in the relevant chapters of part ii of this book. before the development of in vitro culture techniques, viruses of humans could only be propagated in experimental animals. thus much of our knowledge about virus growth and replication relied in the first instance on the study of bacterial viruses. in it was shown that vaccinia virus and herpes simplex virus could be grown on the chorioallantoic membrane of embryonated chicken eggs. this system became routine for the study of many mammalian viruses as virtually all virus families contain viruses that can be cultured in this way. although cell culture has long since replaced the use of eggs for this purpose, the technology is still in use for the preparation of influenza virus stocks and the manufacture of influenza vaccines. the development of in vitro cell culture systems was a watershed development in virology: not only did it become possible to dissect the intracellular events accompanying virus replication in a manner similar to that of the study of bacteriophages in bacterial cells, it also provided a means of quantifying the amount of infectious virus in samples and virus stocks. artificial medium was developed to maintain cell viability independent of the source species: these cells could be in the form of organ cultures, explant cultures, primary cell culture monolayers, or monolayer cell cultures immortalized into cell lines. organ cultures maintain the three-dimensional structure of the tissue of origin and can be useful for short-term experiments that depend upon preserving fully differentiated cells. for example, tracheal epithelial cells attached to the cartilage matrix of the trachea during culture played a critical role in the isolation of many human respiratory viruses. the preparation of primary cell cultures uses proteases such as trypsin or collagenase to separate individual cells of a tissue such as fetal kidney or lung, and the individual cells then attach to a cell culture substrate where a limited number of divisions will occur. the limited life span of these cells requires repeated preparation of new cultures from source tissue, clearly presenting problems with reproducibility. in contrast, continuous propagation of cells is possible with two types of long-term culture: . "semicontinuous," "diploid" cell strains, for example, human lung or foreskin fibroblasts , in which cells eventually senesce after to divisions (the "hayflick number") due to progressive shortening of telomeres, and . continuous immortalized cell lines, for example, hela cells, bhk- cells. these are derived either from tumors or from primary cells that have undergone a spontaneous transformation event during cell culture, and can undergo an almost infinite number of cell divisions, thus generating consistency although often accompanied by a loss of differentiated cell functions. for example, hela cells were originally isolated in cell culture from a patient who died of cervical cancer chapter in , but the cells remain an important system for culturing viruses to this day, and in fact proliferate so successfully that hela cells can become contaminants of cell cultures from other sources. recognition of the presence of a virus was dependent initially on observing cultures at regular intervals under the light microscope for signs of morphological change or cell death, compared to uninoculated cell cultures acting as controls. the specific appearance of the cytopathic effect (cpe) is often diagnostic for a certain family of viruses, for example herpesviruses can cause a distinct cytopathology often accompanied by the fusion of dying cells. however, reliance on cytopathology can give false results especially if the sample is contaminated with bacterial toxins or other adventitious agents not related to the pathological process under study. using such systems, the basic mechanisms of transcription, translation, and nucleic acid replication have been characterized for all the major families of vertebrate viruses and the strategies of gene expression and regulation have been clarified. every family of viruses employs unique replication strategies. one important unifying and simplifying concept, as originally proposed by david baltimore in , was to assign all viruses to one of six classes based on their genome composition and the pathway used to produce mrnas for translation using host cell ribosomes ( fig. . ). this also reflects the parasitic property of all viruses in requiring a host cell for the synthesis of viral proteins. most studies of the replication of viruses of humans have used cultured mammalian cells, grown either in suspension or as a monolayer adherent to a plastic surface. classic studies of this kind defined the one-step growth curve, in which all cells in a culture are infected simultaneously by using a high multiplicity of infection, and the increase in infectious virus over time is measured by sequential sampling and titration of infectious virus ( fig. . ) . it is important to understand that, with synchronous infection of the whole cell population, the events observed in the total culture can be used to study events in a single cell. the time to achieve maximal yield of virus ranges from hours (poliovirus) to more than hours (adenovirus), compared with a generation time for e. coli in broth culture of to minutes. virus released into the medium can be titrated separately from virus that remains cell-associated. shortly after infection, the inoculated virus "disappears"-infectious particles cannot be detected, even if the cells are disrupted. this eclipse period continues until the first progeny virions are detected some hours later. as infection progresses, non-enveloped viruses mature within the cell and may be detected as infectious, intracellular virions for some hours before being released by cell lysis. many enveloped viruses, on the other hand, mature by budding from the plasma baltimore, d., . expression of animal virus genomes. bacteriol. rev. , - . then, modified from flint, s.j., et al., membrane of the host cell and are released into the medium. virus release via cell lysis is abrupt, whereas release via budding may continue over a protracted period of time. the eclipse period generally ranges from to hours for viruses of different families. early studies, relying on quantitative electron microscopy and the assay of infectious virions, provided an overview of the replication cycle (attachment, penetration, maturation, and release), but with little detail as to the processes involved. investigation of the expression and replication of the viral genome became possible after the introduction of biochemical methods for the analysis of viral nucleic acids and proteins. furthermore, imaging methods are also available nowadays that enable the tracking of viral and cellular proteins as the viral replication cycle proceeds. in order to initiate infection, virions must first bind to cells. binding occurs between ligands on the surface of the virion (viral attachment proteins) and receptors on the plasma membrane of the cell (table . ). this initial interaction between virus and host cell is sometimes complex and there is frequently a lack of correlation between attachment studies in cultured cells versus intact hosts. for example, several viral envelope glycoproteins of herpesviruses may serve as attachment proteins and several cellular receptors may be involved, in sequential order, first achieving a loose attachment via one receptor, then irreversibly binding via a second receptor and ligand. exploitation of more than one cellular ligand provides redundancy and also may assist herpesviruses in invading both epithelial and neural tissues to support latent/recrudescent infection cycles. while there is a degree of specificity about the recognition of particular cellular receptors by particular viruses, quite different viruses (e.g., orthomyxoviruses and paramyxo viruses) may utilize the same receptor and, conversely, viruses in the same family or genus may use different receptors. viruses have thus evolved by making opportunistic use of a wide variety of host cell surface proteins as receptors-in most cases viruses employ as receptors host cell proteins that are crucial for fundamental cellular functions, many of which are strongly conserved over evolutionary time. the receptor-ligand interaction between human cells and human immunodeficiency virus (hiv- ) illustrates the complexity of the interaction between virus and host proteins. attachment initially involves cd molecules on the surface of target cells, notably macrophages and t helper lymphocytes, via the viral gp envelope glycoprotein. this binding induces a conformational change exposing a highaffinity chemokine receptor-binding site on gp , which binds one of several chemokine receptors on the cell surface. this latter binding in turn exposes a fusogenic domain of the viral transmembrane protein gp . direct contact between the fusogenic domain of gp and the cell membrane brings about the fusion of the viral envelope with the plasma membrane of the cell, permitting the viral nucleocapsid to enter the cell cytoplasm (see fig. . ). the cellular receptor for most orthomyxoviruses is the terminal sialic acid on an oligosaccharide side-chain of a glycoprotein (or glycolipid) exposed at the cell surface: the viral ligand is in a cleft at the distal tip of each monomer of the trimeric viral hemagglutinin glycoprotein (see chapter : virion structure and composition). viruses can adapt to new hosts through modification of the relevant attachment ligand. for example, influenza a viruses originate in aquatic birds and recognize sialic acid residues on the surface of cells along the respiratory tract. however, influenza a viruses of birds have a greater affinity for sialic acid linked to the penultimate galactose moiety through α - bonding, whereas those of humans have a greater affinity for sialic figure . one-step growth curve of an enveloped and a non-enveloped virus. attachment and penetration are followed by an eclipse period of to hours during which cell-associated infectivity cannot be detected. this is followed by a period of several hours when viral maturation occurs. virions of non-enveloped viruses (e.g., adenoviruses-top) are often released late, when the cell lyses. release of enveloped virions (e.g., the togavirus western equine encephalitis virus) occurs concurrently with maturation by budding from the plasma membrane. adapted from flint, s.j., et al., . principles of virology, third ed. asm press, washington, dc. acid linked via an α - bond; this reflects differences in the relative abundance of the two linkages in the different host species. there is considerable subtlety in these interactions, and as with many human and animal viruses a single amino acid change within a receptor site may manifest as a significant change in host range and epidemiology. proinflammatory reactions in the host may also affect the expression of receptors and thereby indirectly modify interactions between virus and potential host cells. for example, human adenovirus recognizes both the coxsackievirus and adenovirus receptor (car) and αvβ / integrin in order to infect cells, yet neither is normally exposed on the apical surfaces of cells until macrophages respond to infection and secrete il- . this in turn moves the car and αvβ / integrin receptors away from the tight junctions of polarized cells toward the apical cell surface and thus become exposed and available for virus attachment. the majority of mammalian cells are continuously engaged in receptor-mediated endocytosis for the uptake of macromolecules via specific receptors. many enveloped and non-enveloped viruses use this essential cell function to initiate penetration and uncoating ( fig. . ). virion attachment to those receptors clustered at clathrin-coated pits is followed by endocytosis into clathrin-coated vesicles. vesicles enter the cytoplasm and, after removal of the clathrin coat, fuse with endosomes (acidic prelysosomal vacuoles). acidification within the vesicle triggers changes in virion proteins and surface structures. for example, the acidic ph within the endosome, induces the hemagglutinin molecules of influenza viruses to undergo a conformational change. this enables fusion to occur between the viral envelope and the endosomal membrane and results in the release of the viral nucleocapsid into the cytoplasm. many other non-enveloped and enveloped viruses undergo comparable changes. fusion at neutral ph is an alternative mechanism. the f (fusion) glycoprotein of paramyxoviruses causes the viral envelope to fuse directly with the plasma membrane of the cell, even at ph . this allows the nucleocapsid to be released directly into the cytoplasm. a number of other important enveloped viruses for example filoviruses, have the ability to fuse with the host cell plasma membrane, thereby allowing entry of viral nucleic acid into the cytosol. a third entry mechanism ("direct entry") is used by some non-enveloped viruses (e.g., poliovirus, adenovirus). this involves binding of virus to receptors at the endosomal membrane, which induces conformational changes in the viral capsid; these changes then expose regions that react with the endosomal membrane to induce channels for transport of the genome across the plasma membrane ( fig. . ) . the receptors induce conformational changes in the virion (the s infectious virion becomes a s particle), with the loss of vp and the externalization of the n-terminus of vp . the s particles are then internalized by a clathrin-and caveolin-independent, but actin-and tyrosine kinase-dependent, mechanism. the release of the viral genome takes place only after internalization from an endocytotic compartment localized within to nm of the plasma membrane. upon release of the rna genome, the empty capsid ( s) is transported away along microtubules. reproduced from brandenburg, b., et al., , imaging poliovirus in order for viral genes to become available for transcription it is necessary that virions are at least partially uncoated. in the case of enveloped rna viruses entering by the process of fusion, the nucleocapsid is discharged directly into the cytoplasm and transcription commences from viral nucleic acid while still associated with the nucleocapsid protein(s). with the non-enveloped icosahedral reoviruses, only certain capsid proteins are removed and the viral genome expresses all its functions without being released from the virion core. for most other viruses, however, uncoating proceeds to completion, otherwise genome duplication cannot proceed. for some viruses, uncoating takes place in the nucleus where later stages of replication occur. replication of viral dna different mechanisms of dna replication are employed by each family of dna viruses ( fig. . ). these involve either synthesis of daughter strands via a replication fork (papillomaviruses, polyomaviruses, herpesviruses), or via strand displacement (adenoviruses, parvoviruses, poxviruses). since cellular dna polymerases cannot initiate synthesis of a new dna strand but can only extend synthesis beginning from a short (e.g., rna) primer, one end of newly synthesized viral dna molecules might be expected to retain a short single-stranded region. various dna viruses have evolved different strategies for circumventing this problem. viruses of some families have a circular dna genome, others have a linear genome with complementary termini that serve as dna primers, yet others have a protein primer covalently attached to the ʹ-terminus of each dna strand. several virus-coded enzyme activities are generally required for replication of viral dna: a helicase (with atpase activity) to unwind the double helix; a helix-destabilizing protein to keep the two separated strands apart until each has been copied; a dna polymerase to copy each strand from the origin of replication in a ʹ-to ʹ-direction; an rnaase to degrade the rna primer after it has served its purpose; and a dna ligase to join the okazaki fragments together. often a single large enzyme performs two or more of these activities. the genomes of papillomaviruses and polyomaviruses structurally and functionally resemble cellular dna in binding to cellular histones. the viral genome utilizes host dna polymerase α-primase to synthesize the rna primer for genome replication. among the polyomaviruses an early viral antigen, large t, binds to the regulatory sequence-and in the case of papillomaviruses e and e -thereby initiating dna replication. replication of these circular double-stranded dna molecules commences from a unique palindromic sequence and proceeds simultaneously with replication forks in both directions. both continuous and discontinuous dna synthesis occur (of leading and lagging strands respectively) at the two growing forks as for the replication of mammalian dna. the discontinuous synthesis of the lagging strand involves repeated synthesis of short oligoribonucleotide primers, which in turn initiate short nascent strands of dna (okazaki fragments), each is then covalently joined by a dna ligase to form one of the growing strands. the replication of adenoviruses is distinct from that of other dna viruses. the adenovirus dna genome is linear, the ʹ-end of each strand being identical to the other (terminally repeated inverted sequences) and covalently linked to a protein, the precursor of which serves as the figure . two mechanisms of synthesis of dna virus genomes. a replication fork is used by papillomaviruses, polyomaviruses, and herpesviruses. this involves initiation from an rna primer; synthesis along the leading strand is continuous, but discontinuous along the lagging strand with formation of okazaki fragments which are subsequently ligated. with circular dna genomes, synthesis proceeds bidirectionally from the site of origin. alternatively, the dna genomes of adenoviruses, parvoviruses, and poxviruses replicate by strand displacement, using as primers either protein (adenoviruses) or dna hairpins (parvoviruses and poxviruses). reproduced from flint, s.j., et al., . principles of virology, third ed. asm press, washington, dc, with permission. primer for viral dna synthesis. dna replication proceeds from both ends, continuously but asynchronously, in a ʹ-to ʹ-direction, using a virus-coded dna polymerase. it does not require the synthesis of okazaki fragments. herpesviruses code for many or all of the proteins required for dna replication, including a dna polymerase, a helicase, a primase, a single-stranded dna binding protein, and a protein recognizing the origin of replication. poxviruses, which replicate entirely within the cytoplasm, are selfsufficient in dna replication machinery. hepadnaviruses, similar to retroviruses, utilize positive-sense single-stranded rna transcripts as intermediates for the production of progeny dna by a process of reverse transcription. singlestranded dna parvoviruses use ʹ-palindromic sequences to form a double-stranded hairpin structure to provide a primer for cellular dna polymerase binding. transcription of rna from an rna template is a phenomenon unique to viruses (fig. . ) , and requires an rna-dependent rna polymerase, a virus-coded enzyme not found in uninfected cells. the replication of viral rna requires first the synthesis of complementary rna: this in turn serves as a template for the synthesis of further copies of viral rna. for viruses where the viral rna is of negative-sense (orthomyxoviruses, paramyxoviruses, rhabdoviruses, filoviruses, bornavirus, arenaviruses, and bunyaviruses), the complementary rna is of positive-sense and the rna poly merase involved performs the same function as the virion-associated transcriptase used for the primary transcription of mrnas. most transcripts from such negative-sense viral rnas are subgenomic mrna molecules, but some full-length positive-sense strands must also be made, in order to serve as templates for full-length progeny viral rna synthesis (replication). for some viruses there is good evidence that the rna polymerases used for transcription and replication are distinct, in others the same enzyme performs both functions. in the case of the positive-sense rna viruses (picornaviruses, caliciviruses, togaviruses, flaviviruses, coronaviruses, and arteriviruses), the complementary rna is negative-sense, and its sole purpose is to provide a template for synthesis of more positive-sense rna. several viral rna molecules can be transcribed simultaneously from a single complementary rna template, each rna transcript being the product of a separately bound polymerase molecule. the resulting structure, known as the replicative intermediate, is therefore partially double-stranded with single-stranded tails. some rna-dependent rna polymerases can initiate rna synthesis de novo, while others require a primer with a free ʹ-oh group to which further nucleotides can be added. initiation of the replication of picornavirus and calicivirus rna, similar to that of adenovirus dna, requires a bound protein as primer, rather than an oligonucleotide. this small protein is covalently attached to the ʹ-terminus of nascent positive and negative rna strands in addition to virion rna, but not to molecules used as mrna. little is known about what determines whether a given picornavirus positive-sense rna molecule will be directed ( ) to a replication complex (a structure bound to smooth endoplasmic reticulum), where it serves as template for transcription by rna-dependent rna polymerase into negative-sense rna, or ( ) to a ribosome, where it serves as mrna for translation into protein, or ( ) to a procapsid, with which it associates to form a virion. other rna polymerases require an oligonucleotide ʹ cap structure, which may be synthesized by viral enzymes or derived from cellular mrnas by a process of "capsnatching" (e.g., chapter : orthomyxoviruses). retroviruses have a genome consisting of positive-sense single-stranded rna. unlike other rna viruses, retroviruses replicate via a dna intermediate. the virion-associated reverse transcriptase, using a transfer rna (trna) molecule as a primer, makes a single-stranded dna copy, while the same enzyme functions concurrently as a ribonuclease and removes the parental rna molecule from most of the dna:rna hybrid, except for several short rna stretches known as poly-purine tracts. the poly-purine tracts then act as primers for copying the negative-sense single-stranded dna strand to form a linear double-stranded dna that contains an additional sequence known as the long terminal repeat (ltr) at each end. this double-stranded dna then is processed by the viral integrase and integrates into cellular chromosomal dna. from this point on, transcription of viral rna occurs from the integrated (proviral) dna (see chapter : retroviruses). distinguishing the various strategies used by viruses for the production of viral proteins is fundamental to an understanding of virus replication (fig. . ) . a summary of the properties of viral proteins is given in table . . for many viruses, the production of viral proteins immediately after entry of the viral genome is a critical step. these early proteins act to subvert the molecular machinery of the host, to allow the later production of new virus particles and to inhibit the activation of the host innate immunity that would otherwise prevent virus replication and spread to adjacent cells and tissues. for most families of dna viruses, transcription and dna replication take place in the cell nucleus, using cellular rna polymerase ii and other cellular enzymes. for viruses such as poxviruses and herpesviruses it is possible to identify a significant number of genes by gene deletion (more than %) that are not essential for the replication of the virus in cultured cells. of course, in the strict economy of viral genomes it is likely that most or all of such genes are important for virus survival in nature. the situation is quite different for rna viruses as these are unique having genetic information coded as rna. thus an effort needs to be made to understand the distinct replication and expression strategies, particularly as these have a bearing on understanding virus pathogenesis. rna viruses with different types of genomes (single-stranded or double-stranded, positive-or negative-sense, monopartite or segmented) have necessarily evolved different routes to the production of mrna. since eukaryotic cells do not contain an rna-dependent rna polymerase, all rna viruses need to code for a unique rna-dependent rna polymerase. in the case of positive-sense single-stranded rna viruses, the incoming rna genome can bind directly to ribosomes and be translated in full or in part without the need for any prior transcription; all other forms of incoming viral rna must first be transcribed to produce mrna, in order to begin the process of expression of the infecting viral genome. thus, both negative-sense single-stranded rna viruses and double-stranded rna viruses need to carry an rnadependent rna polymerase in the virion, originating from the preceding round of infection. in the case of dna viruses dependent upon the nucleus for replication, cellular dna-dependent rna polymerase ii performs this function, while double-stranded dna viruses that replicate in the cytoplasm carry a virus-encoded dna-dependent rna polymerase. many, but not all, viruses express different genes at different stages of the replication cycle. early viral genes are first transcribed into rna, which may then be processed in a number of ways, including splicing. the early geneproducts translated from this mrna are of three main types: proteins that shut down cellular nucleic acid and protein synthesis, proteins that regulate the expression of the viral genome, and enzymes required for the replication of the viral nucleic acid. following viral nucleic acid replication, late viral genes are transcribed. the late proteins are principally viral structural proteins used for assembly of new virions; some of these are subject to post-translational modifications before use, and are often made in considerable excess. structural proteins for the coating of nascent viral genomes are required in multiple copies for every new nucleic acid molecule destined for encapsidation. for this reason, many viral strategies have evolved so that new copies of the viral genome can act as templates for the further specific transcription and translation of structural proteins required for new virus particles. the existence of overlapping reading frames, multiple splicing patterns of rna transcripts, post-translational cleavage of polyproteins, etc., mean that it is too simplistic to assume one particular gene codes for a particular protein. it is more appropriate to refer to a transcription unit, defined as a region of the genome beginning with the transcription initiation site and extending to the transcription termination site (including all introns and exons in between), the expression of which falls under the control of a particular promoter. some viral proteins serve as regulatory proteins, modulating the transcription or translation of cellular genes or down-regulating early viral genes. the large dna viruses also encode numerous additional proteins, called virokines, which do not regulate the viral replication cycle per se, but influence the host response to infection. included among these are viral protein homologues of cellular cytokines. in walter fiers and his colleagues presented the first complete description of the genome of an animal virus, namely that of the polyomavirus sv (see fig. . ). analysis of the circular double-stranded dna molecule and its transcription revealed some remarkable insights, many of which are now applicable to other double-stranded dna viruses. first, early and late genes are transcribed in opposite directions, from different strands of the dna. second, certain genes overlap, the protein products generated having common amino acid sequences. third, some regions of the viral dna may be read in different reading frames (orfs), so that quite distinct amino acid sequences are translated from the same nucleotide sequence. fourth, certain long stretches of the viral dna consist of transcribed introns that are not translated into protein as these are spliced out of the primary rna transcript (see fig. . ). adenoviruses exemplify some of the mechanisms that regulate the expression of viral genomes at the level of transcription. there are several adenovirus transcription units; at different stages of the viral replication cycle, "preearly," "early," "intermediate," and "late" transcription units are transcribed in a precise temporal sequence. a product of the early region e a induces transcription from the other early regions including e b, but following viral dna replication there is a -fold increase in the rate of transcription from the major late promoter relative to early promoters such as e b, and a decrease in e a mrna levels. a second control operates at the point of termination of transcription. early in the replication cycle transcripts terminate at a particular point in the genome, while later in infection these are read through the point of termination to produce a range of longer transcripts with different polyadenylation sites and proteins of different functions. this is but one of the many examples of the economy of viral genomes in coding for complex functions using minimal sequences of nucleic acid. for rna viruses, the regulation of transcription is, on the whole, not as complex as is the case for dna viruses. in particular, the temporal separation into early genes transcribed before the replication of viral nucleic acid, and late genes thereafter, is not nearly so well defined. in the simplest examples (e.g., picornaviruses), a full-length polycistronic mrna is translated and the resulting polyprotein is subsequently cleaved to yield equimolar amounts of all protein products. togaviruses synthesize excess amounts of structural proteins from a separate subgenomic rna. yet other mechanisms of regulation have evolved among viruses with non-segmented negative-sense rna genomes. once the nucleocapsid is released into the cytoplasm of an infected cell, the rna polymerase initiates transcription from the ʹ-end of the genome. with paramyxoviruses, discrete genes along the viral rna are each separated by a consensus sequence that includes termination and start signals as well as short intergenic sequences of u residues enabling the transcriptase to generate a long poly(a) tail for each mrna by a process of reiterative copying (also known as stuttering). each discrete mrna is released from the template while the enzyme continues to transcribe the next gene in sequence. as the polymerase moves from ʹ to ʹ along the template, decreasing amounts of each mrna are sometimes made due to decreasing efficiency of the transcription process-thus, gene order becomes an efficient way of modulating the relative amount of each protein synthesized. paramyxovirus transcription also involves a process known as rna editing. the p gene codes for two proteins, p and v, which share a common n-terminal amino acid sequence but differ completely in the c-terminal sequences as a result of a shift in the reading frame brought about by the insertion of two uncoded g residues into the rna transcript by transcriptase stuttering. eukaryotic cells are not equipped to translate polycistronic mrna into several individual species of protein, as mammalian cells cannot normally reinitiate translation partway along an rna molecule. dna viruses overcome this limitation by using cellular mechanisms for cleavage (and sometimes splicing) of viral polycistronic rna transcripts to yield monocistronic mrna molecules. rna viruses, most of which replicate in the cytoplasm, do not have access to the rna processing and splicing enzymes of the host cell nucleus, but have developed a remarkable diversity of solutions to the difficulty of punctuating a large genome to produce multiple individual gene products. some (e.g., orthomyxoviruses) have developed a segmented rna genome in which each gene is, in general, expressed and duplicated as a separate molecule. others (e.g., paramyxoviruses) have evolved a polycistronic genome but produce monocistronic rna transcripts by termination and reinitiation of transcription. yet other rna viruses (e.g., coronaviruses) make use of a nested set of overlapping rna transcripts, each of which is translated into a single gene product. finally, some (e.g., picornaviruses) have a polycistronic genomic rna, which is translated into a polyprotein that is later cleaved proteolytically to yield the final products. with certain viruses, polycistronic mrna can be translated directly to produce several gene-products as a result of initiation, or reinitiation, of translation at internal aug start codons. internal initiation of translation is facilitated by an upstream rna motif known as an internal ribosomal entry site (ires), first discovered in in poliovirus and encephalomyocarditis virus rnas (another picornavirus). where initiation of translation at an internal aug takes place, a frameshift can also occur. another mechanism, known as ribosomal frameshifting, occurs fortuitously when a ribosome happens to slip one nucleotide back and forth along an rna template. this phenomenon is exploited by retroviruses, where frameshift read through leads to about % of gag protein molecules being extended as a gag-pol polyprotein. thus, taken together with the phenomenon of rna splicing and rna editing described above, it can be seen that there are several mechanisms of exploiting overlapping reading frames to maximize the limited coding potential of viruses with comparatively small genomes. there is considerable interest in the untranslated regions of viral genomes, particularly the numerous conserved (consensus) sequences or motifs that represent responsive elements. many of the latter have a critical role to play in the regulation of transcription. for example, each transcription unit of a viral genome has near its ʹ-end an mrna transcription initiation site (start site), designated as nucleotide + . within the hundred or so nucleotides upstream of the start site is the promoter, which upregulates the transcription of a particular gene or series of genes. upstream or downstream from the start site there may be a long sequence with several, in some cases repeated, elements known as enhancers that amplify transcription even further. these regulatory regions are activated by the binding of either viral or cellular dna-reactive proteins. several such proteins may bind to adjacent responsive elements to form an interactive structure, or otherwise interact to facilitate attachment of the viral rna polymerase. viral regulatory genes encoding such regulatory proteins may act in trans as well as in cis, that is, these proteins may trans-activate genes residing on a physically separate molecule of nucleic acid. a description of the role of the regulatory genes of human immunodeficiency virus (hiv- ) serves to illustrate the sophistication of such regulatory mechanisms. a dna copy of the viral genome is integrated into a chromosome of a resting t cell, and remains latent until a t cell mitogen or a cytokine induces synthesis of the cellular nf-κb family of dna-binding proteins. nf-κb then binds to the enhancer present in the integrated provirus, thereby triggering transcription of the hiv- regulatory genes. one of these, tat, found in all lentiviruses, codes for a protein specific for a responsive element, tar, within the provirus, greatly augmenting (trans-activating) the transcription of all viral genes (including tat itself). a positive feedback loop is thereby established that stimulates the production of hiv rna transcripts. moreover, by interacting with tar present in all viral mrnas as well as in the proviral dna, tat also enhances translation. the hiv- regulatory protein rev plays a different role to modulate gene expression by regulating the nuclear export of viral mrnas. although the control of lentivirus transcription may be unusually complex compared to many rna viruses, these viruses contain only genes, compared with up to in the case of some dna viruses. primary rna transcripts from eukaryotic dna are subject to a series of post-transcriptional alterations in the nucleus prior to export to the cytoplasm as mrna. first, a cap, consisting of -methylguanosine (m gppp), is added to the ʹ-terminus of the primary transcript; this cap structure facilitates the formation of a stable complex with the host s ribosomal subunit, necessary for the initiation of translation. second, a sequence of to adenylate residues is added to the ʹ-terminus. this poly(a) tail acts as a recognition signal for processing and the transport of mrna from the nucleus to the cytoplasm, and assists in the stabilization of mrna against cytoplasmic degradation by ubiquitin. third, a methyl group is added at the sixth position to about % of the adenylate residues throughout the rna (methylation). fourth, introns are removed from the primary transcript and the exons are linked together in a process known as splicing, an important mechanism for regulating gene expression in nuclear dna viruses. a given rna transcript can have two or more splicing sites and, moreover, be spliced in several alternative ways to produce a variety of mrna species coding for distinct proteins; both the preferred poly(a) site and the splicing pattern may change in a regulated fashion as infection proceeds. for example, the hiv- protein rev assists the nuclear export of unspliced or singly spliced (intron-containing) viral mrna; thus, early in the replication cycle, the mrna present in the cytoplasm is largely doubly spliced, while later in the cycle after rev accumulates, a temporal switch in mrna species occurs. special mention should be made of an extraordinary phenomenon known as cap-snatching. the transcriptase of influenza virus, which also carries endonuclease activity, steals the ʹ-methylated caps from newly synthesized cellular rna transcripts in the nucleus and uses these host cell rna sequences as primers for initiating transcription from the viral genome. the rate of degradation of mrna provides another possible level of regulation. not only do different mrna species have different half-lives but the half-life of a given mrna species may change as the replication cycle progresses. capped, polyadenylated, and processed monocistronic viral mrnas bind to ribosomes and are translated into protein in the same fashion as cellular mrnas. the sequence of events has been closely studied in reovirusinfected cells. each monocistronic mrna molecule binds via its capped ʹ-terminus to the s ribosomal subunit and this then moves along the mrna molecule until reaching the initiation codon. the s ribosomal subunit also binds, together with methionyl-transfer rna and various initiation factors, after which translation proceeds. most viral proteins undergo various sorts of post-translational modification such as phosphorylation (for nucleic acid binding), fatty acid acylation (for membrane insertion), glycosylation, myristylation, or proteolytic cleavage. newly synthesized viral proteins must also be transported to the various sites in the cell where they are needed, for example, into the nucleus in the case of viruses where the nucleus is the major site of replication. the sorting signals that direct intracellular trafficking are only now beginning to be understood, as are the polypeptide chain-binding proteins (molecular chaperones) that regulate folding, translocation, and assembly of oligomers of viral as well as cellular proteins. the polycistronic viral rna is translated directly into a single polyprotein in the case of the positive-sense picornaviruses and flaviviruses. this large molecule carries protease activity that cleaves the polyprotein at defined recognition sites into smaller proteins. the first cleavage steps are carried out while the polyprotein is still associated with the ribosome. some of the larger intermediates exist only fleetingly; others are functional for a short period but are subsequently cleaved by additional virus-coded proteases to smaller proteins with alternative functions. post-translational cleavage occurs in several other rna virus families, for example, togaviruses and caliciviruses, in which polyproteins corresponding to large parts of the genome are cleaved. some viruses encode several different proteases. most are either trypsin-like (serine or cysteine proteases), pepsin-like (aspartyl proteases), or papain-like (thiol proteases). cellular proteases, present in organelles such as the golgi complex or transport vesicles, are also vital to the maturation and assembly of many viruses. for example, cleavage of the hemagglutinin glycoprotein of orthomyxoviruses or the fusion glycoprotein of paramyxoviruses is essential for virion infectivity. viruses frequently exploit those cellular pathways normally used for the synthesis of host cell secretory glycoproteins. the amino terminus of viral envelope proteins contains a sequence of to hydrophobic amino acids, known as a signal sequence, which facilitates binding of the growing polypeptide chain to a receptor site on the cytoplasmic side of the rough endoplasmic reticulum and its passage through the lipid bilayer to the lumenal side. oligosaccharides are then added in n-linkage to certain asparagine residues of the nascent polypeptide by en bloc transfer of a mannoserich core of preformed oligosaccharides, and glucose residues are removed by glycosidases (called trimming). the viral glycoprotein is then transported from the rough endoplasmic reticulum to the golgi complex. here the core carbohydrate is further modified by the removal of several mannose residues and the addition of further n-acetylglucosamine, galactose, and the terminal sugars, sialic acid, or fucose. the completed side-chains are a mixture of simple oligosaccharides (also called high mannose oligosaccharides) and complex oligosaccharides which are usually n-linked (to asparagine) or less commonly o-linked (to serine or threonine). a coated vesicle then transports the completed glycoprotein to the cellular membrane from which the particular virus buds. the precise composition of the oligosaccharides is determined, not only by the amino acid sequence and tertiary structure of the proteins concerned, but more importantly by the particular cellular glycosyl transferases active in the type of cell in which the virus happens to be growing. all non-enveloped viruses of vertebrates have an icosahedral structure. the structural proteins of simple icosahedral viruses associate spontaneously to form structural units (called capsomers when considered morphologically), which then self-assemble to form capsids into which viral nucleic acid is inserted, often accompanied by conformational changes to the nascent capsid structure. completion of the virion may also involve proteolytic cleavage of one or more species of capsid protein. the mechanism of packaging viral nucleic acid into a pre-assembled empty procapsid has been well elucidated for adenoviruses. a particular protein binds to a nucleotide sequence at one end of the viral dna known as the packaging sequence; this enables the dna to enter the procapsid bound to basic core proteins, after which some of the capsid proteins are cleaved to produce the mature virion. most non-enveloped viruses accumulate within the cytoplasm or the nucleus and are released only when the cell eventually lyses (fig. . ) . [rer] or golgi), then pass through the cytoplasm in smooth-walled vesicles and are released by exocytosis. reproduced from maclachlan, n.j., dubovi, e.j., . veterinary virology, fourth ed. academic press, london (fig. . ) , with permission. all mammalian viruses with helical nucleocapsids, as well as some with icosahedral nucleocapsids (e.g., herpesviruses, togaviruses, and retroviruses) mature by acquiring an envelope by budding through cellular membranes. enveloped viruses may bud from the plasma membrane, from internal cytoplasmic membranes, or from the nuclear membrane; viruses that acquire an envelope within the cell are then transported within vesicles to the cell surface. budding usually occurs through patches of membrane that contain viral glycoprotein(s) inserted into the lipid bilayer of the membrane. this occurs by lateral displacement of cellular proteins from that patch of membrane ( fig. . ) . the cleaved single molecules of viral glycoprotein associate into oligomers to form typical rod-shaped or clubshaped peplomers with a hydrophilic domain projecting from the external surface of the membrane, a hydrophobic trans-membrane anchor domain, and a short hydrophilic cytoplasmic domain projecting slightly into the cytoplasm. in the case of icosahedral viruses, for example togaviruses, each protein molecule of the nucleocapsid binds directly to the cytoplasmic domain of the membrane glycoprotein oligomer, thus molding the envelope around the nucleocapsid. in the more usual case of viruses with helical nucleocapsids, a matrix protein attaches to the cytoplasmic domain of the glycoprotein peplomer, and the nucleocapsid protein recognizes the matrix protein to initiate budding. release of each enveloped virion does not breach the integrity of the plasma membrane, hence many thousands of virus particles can be shed over a period of several hours or days without significant cell damage. many but not all viruses that bud from the plasma membrane are only slowly cytopathic: and non-cytopathic may be associated with persistent infections. epithelial cells display polarity, that is they have an apical surface facing the outside world and a basolateral surface facing the interior of the body, the two separated by lateral cell-cell tight junctions. these surfaces are chemically and physiologically distinct. viruses that are shed to the exterior (e.g., influenza viruses) tend to bud from the apical plasma membrane, whereas others (e.g., lentiviruses, such as human immunodeficiency virus) bud through the basolateral membrane (fig. . ) . in type i budding, as with the alphaviruses, both the envelope glycoproteins and the internal capsid are essential. if altered or chimeric envelope proteins reach the plasma membrane, these do not support budding, indicating that the quaternary structures of the envelope heterodimers and capsid are involved in driving the process. type ii budding, for example gag-dependent budding of many retroviruses, requires only the internal capsid or matrix proteins. type iii budding can be driven solely by the envelope proteins. finally type iv budding is driven by viral matrix proteins but requires additional components; for example, internal matrix proteins alone can drive the budding of rhabdoviruses or orthomyxoviruses. but the process is inefficient or results in deformed particles unless envelope glycoproteins or the internal ribonucleoprotein are also present. reproduced from flint, s.j., et al., . principles of virology, third ed. asm press, washington, dc (fig. . ) , with permission. figure . polarity of virus release from infected cells. viruses that bud from apical surfaces can be shed in respiratory or genital secretions or intestinal contents. viruses budding from basal surfaces are available for systemic spread via viremia or lymphatics. some viruses, for example, flaviviruses, bunyaviruses, and coronaviruses, take a more circuitous route in exiting the cell. viruses that do not bud are usually released by cell lysis. reproduced from maclachlan, n.j., dubovi, e.j., . veterinary virology, fourth ed. academic press, london (figure . ), with permission. flaviviruses, coronaviruses, arteriviruses, and bunyaviruses mature by budding through either membranes of the golgi complex or the rough endoplasmic reticulum; vesicles containing the virus then migrate to fuse at the plasma membrane thereby releasing the virions by exocytosis (fig. . ) . uniquely, the envelope of the herpesviruses is acquired by budding through the inner lamella of the nuclear membrane; the enveloped virions then pass directly from the space between the two lamellae of the nuclear membrane to the exterior of the cell via the cisternae of the endoplasmic reticulum. satellite viruses are subviral particles that contain a dna or rna genome coding for a capsid protein, but that are absolutely dependent upon the presence of another virus for replication. the vast majority are found among plants but a few have an impact on medical virology and public health. replication of the dependoviruses (family parvoviridae, genus dependovirus-adeno-associated viruses, now used as vectors for delivering heterologous genes of interest, e.g., vectored vaccines) for example, is dependent upon co-infection with an adenovirus-the adeno-associated virus produces coat protein but not the enzymes necessary for genome replication. satellite viruses share little or no nucleotide sequence homology with the helper virus, yet can sometimes interfere with the replication of the helper virus. viroids are small, rod-like single-stranded rna molecules with a high degree of secondary structure, approximately to bases in size, all sharing a common structural feature of a conserved central genomic region essential for replication. the rna forms a hammerhead structure with the enzymatic properties of a ribozyme, an autocatalytic, self-cleaving molecule. this ribozyme function is used to cleave multimeric rna structures produced during the course of replication (chapter : hepadnaviruses and hepatitis delta). most are plant pathogens, remarkable in not coding for any protein. first described by theodore diener, it has been suggested that viroids may represent crucial intermediate steps in the evolution of rna-based life forms from inorganic precursors. hepatitis delta virus (hdv) is a unique example of a defective virus that requires co-infection with hepatitis b virus to provide its outer hbsag-containing envelope. the hdv genome is a branched or rod-like, single-stranded rna molecule similar to plant viroids in conformation, but unlike other viroids it codes for a single protein, the delta antigen. this nuclear phosphoprotein exists in two forms generated by rna editing: the shorter form ( amino acids) is required for hdv genome replication whereas the larger form ( amino acids) is necessary for assembly and release from infected liver cells. the hdv genome is thought to be replicated by the host cell rna polymerase ii enzyme via a rolling circle mechanism to produce a consecutive series of concatemers of first, antisense rna and then, rna of genome sense. these are immediately cleaved by the ribozyme domain in the rna genome to generate new viral genomes: this is the only known example of a mammalian virus utilizing a ribozyme for genome replication (see fig. . ). a sample of any given virus inevitably contains a population of closely related genome sequences ("quasi-species"), replicating simultaneously at different and varying rates according to selection pressures. the molecular mechanisms involved in generating diversity include nucleotide substitution, insertion, or deletion; sequence duplication or deletion; genetic recombination; and genetic reassortment for viruses with segmented genomes. the error rate in nucleotide copying is approximately in nucleotides for dna viruses, and in nucleotides for rna viruses. this quasi-species population is then constantly varying its composition according to the most recent selection applied, providing the raw material for antigenic drift and for adaptation of the virus population to new hosts. the selection properties that are desirable in vivo include the ability to replicate to high levels, the ability to be transmitted from host to host, evasion of the immune response, and resistance to host antiviral mechanisms and/or antiviral therapy. not all of these properties are as necessary for successful replication in cell culture as they are in vivo, and virus stocks prepared in vitro are likely to include a different set of variants from those produced in vivo, say in experimental animal tissues. serial passaging of wild-type virus through cell culture or a foreign host species has been a long used and successful way to generate virus strains with lowered virulence for the original host, to be used as "modified live-virus" vaccines. many different viral genes may be involved in such adaptations; for example, mutations in viral polymerases may affect replicative ability or resistance to antiviral drugs, changes in antigenic epitopes can lead to immune escape, changes in receptor ligands that affect receptor preference can alter many aspects of pathogenesis, and so on. examples are discussed in chapter : emerging virus diseases. advances in our knowledge of virus replication, pathogenesis, diagnosis, vaccine production, and many other areas would not have been possible without the tools to quantitate how much virus there is in a given sample or preparation. with the greater use of antiviral drugs, most notably in blood-borne diseases such as hiv, hepatitis b virus, and hepatitis c virus, the stage of disease and effectiveness of treatment are now routinely assessed by quantifying the level of virus (viral load) in the blood. there are two approaches to measuring the titer of virus, either biological or physical. physical assays measure actual virus particles, and include electron microscopy, hemagglutination, and serological assays for the amount of viral antigens; biological assays measure some viral function, for example, infectivity, reverse transcriptase activity. tests performed on the same sample with different techniques will in some cases give significantly different results, and thus it is important to understand the reason for these differences. the difference between the amount of virus detected using a physical assay such as particle counting by electron microscopy and a biological assay, for example a plaque assay (fig. . ) , is often referred to as the particle to plaque-forming unit (pfu) ratio. in virtually all instances, the number of particles exceeds the number determined in a biological assay, often with ratios greater than : . this is due to a number of reasons; first, not all virions may be capable of replication due to intrinsic defects, for example, because only a partial or defective genome is present, or the particle is empty, faulty capsid assembly, or because of lethal mutations in the genome; second, virions may have undergone environmental inactivation; third, the choice of cells for virus isolation and growth may not mimic the optimum intracellular environment of the natural target organ; fourth, the interaction between a fully viable virus particle and a permissive cell may not always successfully initiate infection and an excess of particles may be necessary to achieve a statistical chance of success. perhaps no other procedure has contributed as much to virology as the development of the plaque assay. the test was originally developed a century ago by felix d'herelle in his initial studies of bacteriophages and was subsequently adapted to mammalian viruses in by renato dulbecco and marguerite vogt. the assay is elegantly simple: serial -fold dilutions of a virus sample are made in a cell culture medium. these diluted samples are then added to preformed monolayer cells and incubated under agar or a carboxymethyl cellulose layer to prevent the spread of secreted virus. after to hours (or longer, according to the virus of interest), plaques of necrotic cells become visible under the light microscope. at an appropriate time, fixation and staining, for example, with crystal violet, reveals clearly visible holes in the monolayer, each corresponding to a focus of necrotic cells initiated by one viable virus particle. serial dilution of the virus preparation facilitates the counting of discrete plaques so that, knowing the dilution, volume tested, and the plaque count, the concentration (titer) in the original sample can be determined. immunohistochemical staining procedures using specific antisera can be used as an alternative for visualizing plaques formed by non-cytopathic viruses. with the development of real-time pcr assays, the concentration of viral nucleic acid in a test sample can now be measured in virtually any context. by comparison with copy number controls, the concentration of nucleic acid in the treated sample can be determined. this type of assay does not detect empty capsids (those that do not contain viral nucleic acid), and, importantly, it does not necessarily relate to the infectivity of the sample. chapter : laboratory diagnosis of virus diseases, describes a further range of assays that are used in a diagnostic context, most of which can be also used both quantitatively and for research applications. this chapter has hitherto described how, during virus replication, non-infectious particles that lack the full genetic information essential for infectivity, are commonly produced-analogous to the assembly of defective motor figure . plaque assay for virus quantitation. cell monolayers are grown, typically in petri dishes, and when confluent the virus inoculum is allowed to adsorb to the monolayer in a small volume. the monolayers are overlaid with agarose to restrict diffusion of virus particles, and incubated to allow virus replication beginning with single cells and then spreading as more cells become successively involved. the vital stain neutral red is then added; this is taken up by viable cells, staining the monolayer red and leaving clear holes. plaque sizes and rates of development depend on the replication cycle and yield of the individual virus (see text). to determine the virus titer of the starting inoculum, serial -fold dilutions are inoculated on to duplicate monolayers. for those monolayers where discrete plaques can be distinguished (typically to plaques per dish), plaque numbers are counted and the original virus titer calculated by multiplying this plaque number by the dilution factor. by harvesting virus from a single plaque, a stock of genetically homogeneous virus can be obtained ("plaque-purifying"). spontaneous mutants in a virus inoculum may be recognized by plaques of abnormal size or morphology, and can thus be harvested and separated for further study. vehicles from a car assembly plant. one special example involves defective interfering (di) particles; these ( ) lack the complete genome of the wild-type virus (deletion mutants), but ( ) can still replicate in a new cell if the cell is also co-infected by wild-type virus which provides the missing function (by a process known as complementation). such defective particles may then also ( ) interfere with the ongoing replication of the wild-type helper virus, possibly due to possessing a competitive replication advantage over wild-type virus. the production of di particles is encouraged by infection at high multiplicity, where the chances of co-infection with full-length and defective viruses occurring in the same cell are increased. in fact, this phenomenon was first observed by preben and herdis von magnus in with influenza virus; they found that very high multiplicities led to poor virus replication and the production of "interfering" virus stocks. a cyclical pattern may be observed, where di particles increase in number as long as the culture has sufficient wild-type helper virus replication; however, as di particles then inhibit the numbers of wild-type virus, the whole virus population diminishes, fresh rounds of wildtype virus replication can then occur and so a milieu for a return to di replication is built up again. despite several suggestions, it is not clear to what extent di particles play a role in the pathogenesis of an ongoing infection in a host, for example, in modulating recovery from an acute infection or promoting chronic infection. timing is everything: finetuned molecular machines orchestrate paramyxovirus entry principles of virology hiv- assembly, release and maturation viral membrane fusion. virology - poliovirus cell entry: common themes in viral cell entry pathways . fields virology how viruses hijack the erad tuning machinery the rna synthesis machinery of negative-stranded viruses key: cord- -jj l ydl authors: girardi, erika; pfeffer, sebastien; baumert, thomas f.; majzoub, karim title: roadblocks and fast tracks: how rna binding proteins affect the viral rna journey in the cell date: - - journal: semin cell dev biol doi: . /j.semcdb. . . sha: doc_id: cord_uid: jj l ydl as obligate intracellular parasites with limited coding capacity, rna viruses rely on host cells to complete their multiplication cycle. viral rnas (vrnas) are central to infection. they carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. regardless of its origin or tropism, vrna has by definition evolved in the presence of host rna binding proteins (rbps), which resulted in intricate and complicated interactions with these factors. while on one hand some host rbps recognize vrna as non-self and mobilize host antiviral defenses, vrna must also co-opt other host rbps to promote viral infection. focusing on pathogenic rna viruses, we will review important scenarios of rbp-vrna interactions during which host rbps recognize, modify or degrade vrnas. we will then focus on how vrna hijacks the largest ribonucleoprotein complex (rnp) in the cell, the ribosome, to selectively promote the synthesis of its proteins. we will finally reflect on how novel technologies are helping in deepening our understanding of vrna-host rbps interactions, which can be ultimately leveraged to combat everlasting viral threats. viruses are obligate intracellular parasites that strictly rely on host cells to translate and amplify their genomes [ ] [ ] [ ] . viral rnas (vrnas) play a central role during infection as they bear all the necessary information for a virus to express its proteins, replicate and spread. unlike dna viruses where the messenger rna must be first transcribed for the synthesis of viral proteins, rna viruses use their rna molecules both for protein synthesis and as replication templates. viruses encode an array of vrna-binding proteins (vrbps), essential for different steps in the viral lifecycle, comprising translation, synthesis, packaging of the viral genome and cell-to-cell spread. importantly, vrna is never really naked in the cellular milieu. apart from its interactions with vrbps, it has evolved to co-opt specific sets of host encoded rbps. this is illustrated by the variety of described strategies that vrnas use to hijack key cellular machineries and evade the host's defense arsenal. unlike vrbps that promote infection, host encoded rbps can either have pro-or antiviral functions. indeed, a large body of work produced during the last decades describe the involvement of host encoded rbps in almost every known stage of vrna lifecycle. certain host rbps, ordinarily functioning in cellular tasks, are repurposed by vrnas to guide them through the viral lifecycle, including genome translation, synthesis, modification, localization and packaging. on the other hand, many host rbps have evolved dedicated antiviral functions, ranging from the recognition of the invading vrna to the restriction of viral replication. the ability of vrna to either subvert or get antagonized by cellular rbps determine the outcome of a viral infection. indeed, vrna-rbps interactions could dictate the permissiveness of certain cell types to infection, host range, tissue tropism, viral evolution, efficiency of viral replication, pathology of infection and immune clearance [ ] . vrnas interact with numerous and very diverse rbps during infection. this is illustrated by the rich literature on the subject that has yielded many discoveries in the last decades. for example, the study of intimate vrna-rbp interactions shaped our understanding of how rna interference (rnai) functions as the main antiviral defense system in plants and invertebrates, and how bacteria defend themselves against invading phages through the crispr system [ , ] . herein, we review the mechanisms at play when rna viruses infect their animal hosts, taking a vrna-centric view. focusing on viruses that are important human pathogens, we will describe how cellular rbps act on early steps after viral entry, driving mechanisms of vrna recognition, degradation and modification to limit or to promote viral replication and spread. we will further describe a number of crucial mechanisms during which vrna hijacks the ribosome to preferentially translate the viral program. finally, we will reflect on how new emerging techniques are allowing to get a better grasp on the diversity of host rbps-vrna interactions. elucidating the mechanisms by which rbp-vrna interactions influence viral infection, is advancing our understanding of cell biology by revealing unforeseen biological knowledge and may also provide new targets for host-directed antiviral therapies [ ] . indeed, recent corona, ebola and zika virus outbreaks remind us that new innovative antiviral approaches are clearly needed, particularly for emerging rna viruses with important epidemic potential [ ] [ ] [ ] . despite the fact that the molecular composition of rna is universal throughout life kingdoms, host immune pathways are able to differentiate and recognize non-self nucleic acids based on specific features, structures and modifications. moreover, despite the molecular mimicry set by rna viruses to resemble cellular mrnas and escape host recognition, the viral nucleic acid still needs to embark on a long journey through a hostile cell environment and must overcome the obstacles put in place by the host antiviral system in order to be translated and replicated. cellular innate immunity comprises a rather sophisticated set of host factors that discriminate molecular alterations with a high specificity and restrict rna virus infection through direct or indirect activity on the vrna. in vertebrate animals, a variety of cellular sensors have evolved to detect foreign rna sensing to activate the innate immune response, which involves transcriptional and post-transcriptional activation of genes of interferon-stimulated genes (isgs). pattern recognition receptors (prrs) are host-encoded proteins that sense molecular features of vrna molecules which are generally absent in the majority of cellular rnas. multiple molecular receptors are involved in the recognition of these so-called pathogen-associated molecular patterns (pamps) [ , ] . a major molecular signature and weakness of all rna viruses is the long double stranded (ds) rna molecules that are generated during replication. the sensing of dsrna is believed to be sequence-independent and rather depends on the distinct molecular features of the viral dsrna structure mostly absent in uninfected host transcriptome [ ] . among the first cellular proteins that detect the invading virus are the toll-like receptors (tlrs) (fig. ) , which are transmembrane glycoproteins that are constitutively expressed or pathogen-induced, located on the plasma membrane or intracellular endosomes which are preferential entry routes for vrnas (reviewed in [ , ] ). tlrs share strong similarities in their structures and organization. their n-terminal region recognizes ligands thanks to its extracellular or luminal domains and is connected to the c-terminal cytoplasmic domains (ctds) by a single membrane-spanning domain. among the tlrs identified in humans so far, tlr and tlr recognize ssrnas and tlr recognizes dsrnas [ , , ] . in tlr , the n-terminal region recognizes the ligand, dsrna, in the lumen of endosomes whereas the c-terminal signalling domain resides in the cytoplasm, and upon ligand recognition, tlr initiates the signalling process that culminates in transcriptional induction of specific immune genes. tlr was believed to be a major sentinel against viral infections since it responds to a common by-product of viral replication ( table ) . for instance, encephalomyocarditis virus (emcv) infection leads to a tlr -dependent innate stress response, which is involved in mediating protection against virus-induced myocardial injury [ ] . similarly, tlr recognizes dengue virus (denv), limiting its replication [ ] , while zika virus (zikv)-mediated tlr activation was shown to deplete neural progenitor cells [ ] . indeed, the protective versus pathogenic role of tlr in viral pathogenesis is debated [ ] . another group of molecular sentinels that senses foreign rna is represented by the ifn-induced intracellular cytosolic receptors, named retinoid acid-inducible gene i (rig-i)-like receptors (rlrs), which comprise dexd/h-box helicases such as rig-i (or ddx ), melanoma differentiation-associated protein (mda or ifih ) and laboratory of genetics and physiology (lgp or dhx ) [ ] (fig. , table ). all rlrs possess a central dexd/h-box rna helicase domain and a ctd which are necessary for detection of foreign rnas. rig-i and mda have as well two n-terminal caspase activation and recruitment domains (cards), which mediate downstream signal transduction, while lgp lacks them. although rig-i and mda share similar structural composition, their ability to detect vrna is not redundant but specific [ ] ( table ) . rig-i monitors the ′ ends of rna molecules via its ctd and helicase domain in order to initiate cytokine production in response to a wide range of viruses. triphosphate (ppp) or diphosphate (pp) at the uncapped ′ -end of rna molecules with partial base-pairing to a complementary strand of rna molecules are recognized and required for vrna is sensed by different families of receptors (e.g. rlrs, tlrs, pkr). vrna is edited by adars and methylated by host mettl proteins (m a) or viral methyl transferases ( ′ -o-me). vrna can also be degraded by ′ - ′ or ′ - ′ exonucleases (e.g. xrn and rna exosome respectively) or by endonucleases (e.g. dicer). rig-i activation [ ] [ ] [ ] [ ] . the typical structure of the panhandle of negative-strand viral genomes confers full rig-i ligand activity. rig-i recognizes many single stranded negative rna virus families, such as paramyxoviridae, rhabdoviridae, orthomyxoviridae, bunyaviridae and filoviridae [ ] [ ] [ ] . rig-i also detects some positive-sense rna viruses such as dengue and zika viruses of the flaviviridae family, by recognition of nascent viral genomes containing ′ -ppp groups prior to capping [ ] . mda senses both rna length and secondary structure by recognizing uninterrupted rna duplexes longer than a few hundred base pairs [ ] and forming filaments around dsrna to initiate signalling [ ] . mda is required for immunity against several classes of viruses, including single-stranded positive rna viruses such as picornaviruses [ , , ] , flaviviruses [ ] and coronaviruses [ ] (table ) . the third rlr family member lgp lacks the amino-terminal tandem cards and thus lacks signal-transducing activity. several studies indicate a disparate regulatory role for lgp in either triggering or dampening the innate immune signalling pathways following rna virus infection [ ] . on one hand, lgp can function as a feedback inhibitor of rig-i by sequestrating double-stranded but not single-stranded rnas. lgp negatively regulates sendai virus (sv)-induced irf- or nf-κb signaling acting as a natural inhibitor of antiviral responses, and as a repressor of rig-i signaling [ ] . however, lgp has a positive effect on mda -mediated antiviral response. lgp -deficient mice exhibited a defect in type i ifn production in response to infection by the encephalomyocarditis virus, the replication of which activates mda -dependent innate immune response [ ] . moreover, a recent study indicated that lgp can inhibit antiviral rnai in mammals by competing with and preventing dicer-mediated processing of dsrnas into sirnas both in vitro and in cells [ ] . interestingly, other dexd/h-box helicases, such as ddx , ddx , ddx or ddx , are also emerging as important sentinels that influence viral sensing in multiple ways. these helicases have been shown to bind vrnas, acting either as antiviral effectors or contributing to viral replication [ ] . the presence and replication of viral nucleic acids in vertebrate cells can also induce the activation of other antiviral enzymes that are both sensors and effectors ( table ) . these also recognize viral signatures; however, their main function is not necessarily to only induce a transcriptional immune response, but rather to directly attack vrna by degrading it or inhibiting its translation. for this reason, these are not usually referred to as receptors [ , , ] . among them, double-stranded rna (dsrna) activated protein kinase r (pkr; also known as eif ak ) or adenosine deaminase and ′ - ′ -oligoadenylate synthetase class of enzymes (oass), can bind dsrna in a sequence independent manner, recognizing the structure as a prr (fig. first, pkr is a serine-threonine kinase constitutively expressed in all tissues at a basal level as an inactive monomer. viral dsrna binding to the two n-terminal rna binding motifs induces a conformational change that allows atp binding to the c-terminal kinase domain. pkr can be activated by dsrna from reoviruses [ ] but can also recognize dsrna from replication intermediates of + rna viruses [ ] ] (table ) . moreover, it has been shown that highly structured rna elements from retroviruses such as human immunodeficiency virus (hiv) transactivation-response region (tar) rna hairpins [ ] or dsrna formed by the antiparallel mrna transcripts of some dna viruses can also activate pkr [ ] . upon dsrna binding pkr dimerizes and autophosphorylates threonine residues in its activation domains, thereby stabilizing the dimers and increasing kinase activity [ ] . this in turn leads to a series of events that inhibit viral translation and replication, as will be detailed later on in this review. interestingly, pkr is an interferon stimulated gene (isg) itself and its transcription can be up-regulated upon type i interferon production by virus-infected cells, stimulating its accumulation in neighboring cells. pkr activation following infection of interferon-primed neighboring cells inhibits global protein synthesis and reduces viral spread, making this activation a key player in the innate response to viruses [ ] . therefore, pkr can be considered a major molecular sentinel of antiviral innate immunity, recognizing a diverse set of stress-inducing stimuli and mounting an appropriate (fig. ) . the second known class of dsrna sensors and effectors is represented by the ′ , ′ -oligoadenylate synthetases (oases) (fig. , table ), which also act as prrs for the detection of many rna viruses [ ] . the four human oas genes, oas , oas , oas and oasl (oas-like), are constitutively expressed at low levels in the cytoplasm and are activated in response to viral dsrna. as an example, oas protein accumulates in the cytoplasm as an inactive monomer [ ] and following activation by viral dsrna, it forms a tetramer that synthesizes ′ , ′ -oligoadenylates ( - as). these - as activate the ribonuclease l (rnasel) to cleave cellular and viral rnas and supress viral replication (see below). overall, the functional redundancy, provided by multiple host proteins that recognize different features of vrnas, is a successful strategy for the host to fight and contain the infection [ ] . similar to pkr, the oas-rnasel axis can be antagonized by some rna viruses. for example, certain corona and rotaviruses encode proteins belonging to the phosphodiesterase family, able to cleave - a molecules, thereby preventing activation of rnasel [ ] . in concert with innate immune sensors linked to ifn activation, mammalian cells can also depend on intrinsic factors that participate in the direct antiviral response to non-self nucleic acids. accumulating evidence suggests that the cellular rna surveillance pathway restricts viral infection by modulating vrna stability [ ] . the presence of specific molecular features on the vrna and the binding of trans-acting antiviral host rbps can drive vrna through the host mrna quality control pathways (fig. ). for instance, in mammalian cells, the non-sense mediated decay (nmd) pathway has recently been shown to function in the restriction of positive-sense (+) rna viruses such as alphaviruses in mammalian cells [ ] . in fact, alphavirus rna genome resembles cellular mrnas and is translated into non-structural viral proteins (nsps) but has an untranslated ′ utr, much longer than typical cellular mrnas, which is detected and degraded by the nmd pathway [ ] . the ′ to ′ rna decay machinery can also directly target vrna for degradation in the cytoplasm. in this pathway, the monomethyl guanosine (m g) cap structure is cleaved from mrnas by decapping enzymes exposing the ′ monophosphorylated product to progressive ′ → ′ exoribonucleolytic degradation by xrn . the hepatitis c virus (hcv) genomic rna, which lacks a ′ cap and is therefore susceptible to xrn- mediated decay, uses an unusual mechanism to overcome this pathway [ ] . binding of the liver-abundant mir- to the ′ utr of hcv in association with argonaute- (ago ) protein stabilizes the vrna and slows xrn decay of the viral genome in infected cells [ , ] ( fig. ) . other flaviviridae are able to take advantage of their susceptibility to xrn- mediated vrna decay. dengue, west nile, yellow fever or zika vrnas contain a highly structured - bp-long non-coding rna in their ′ end. an incomplete ′ - ′ degradation of the viral genome by the cellular xrn exonuclease produces the so called small flaviviral rna (sfrna). during rna degradation, xrn is stalled at the ′ utr extremity of flaviviral vrnas, more precisely at stem-loops/pseudoknots, causing the accumulation of different species of sfrna. although, sfrna does not seem to have a direct role in viral replication per se, it contributes to viral pathogenicity in vivo partly by interfering or evading innate immune responses, by sequestering cellular rbps and inhibiting their endogenous functions [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, sfrna seems to function in a species-specific manner. while it facilitates replication and pathogenesis by inhibiting ifn-signalling in mammalian hosts, a recent report shows that zikv sfrna possess an anti-apoptotic function that helps the virus to disseminate and reach saliva in mosquito hosts, thus promoting productive infection and transmission [ ] . it has also been proposed that sfrna could be a suppressor of rnai [ ] . in the ′ decay pathway, deadenylated mrna degradation is mediated by a cytoplasmic multisubunit ′ -to- ′ exoribonuclease complex, namely the rna exosome ( fig. ). this degradation pathway is emerging as a crucial effector for recognition and degradation of specific vrnas [ ] . the exosome interacts with a variety of rbps, some of which are exported to the cytoplasm in response to viral infection and serve as adaptors to specifically target particular rnas. several antiviral rbps have been found to interact with the exosome, suggesting that their mechanism of action may involve exosomal degradation. for instance, the conserved rna helicase ddx translocates from the nucleus to the cytoplasm upon rift valley fever virus (rvfv) infection, binds a specific stem-loop structure in the vrna and recruits the exosome to degrade the vrna [ ] . another example, is the zinc-finger antiviral protein (zap), which binds vrnas containing a zap response element (zre) and induces rna degradation via interaction of its n-terminal domain with host decay machinery mediated [ ] (fig. ). zap is constitutively expressed in many cell types and it is induced upon pathogen-sensing and in response to interferon. zap exhibits broad antiviral activity against a broad spectrum of viruses, such as alphaviruses, enteroviruses, filoviruses, influenza and porcine reproductive and respiratory virus [ ] but also retroviruses [ ] . for instance, zap binds regions of hiv- viral rna containing cpgs and induce degradation of viral rnas via interaction with the essential cellular cofactor khnyn [ ] . moreover, zap antiviral activity has been linked to its subcellular localization in stress granules, which are induced upon infection by sindbis virus (sinv) for example [ ] . interestingly, trim , an e ubiquitin ligase with no previous known role in rna binding is emerging as a regulator of many antiviral factors, including zap [ , ] . other ribonucleases can exhibit broad-spectrum antiviral effects through viral rna binding and degradation. the ribonuclease mcpip is involved in the suppression of japanese encephalitis virus (jev) and denv, but also other positive-sense rna viruses, such as sinv, hcv and emcv, and negative-sense rna virus, such as influenza virus [ , ] . finally, two conserved endoribonucleases have been shown to play a major antiviral role in different animal species, namely rnase l and dicer ( fig. , table ). rnase l is composed of three major domains: an n-terminal regulatory ankyrin repeat domain (ard), a protein kinase (pk)-like domain, and a c-terminal ribonuclease domain (rnase). in the absence of - a, the ard represses the rnase domain. binding of - as, generated by oases, to the ard alters the conformation of rnase l, thereby exposing protein-protein interaction domains and releasing the rnase domain from internal inhibitory sequence, triggering its dimerization and activation. rnase l cleaves single-stranded rnas at the uu/ua dinucleotides, which are relatively rare in the coding regions of cellular mrnas compared to viral rnas [ ] . in the case of hcv infection, rnase l degradation of vrna produces small rna cleavage products with ′ -monophosphate ( ′ -p) and ′ -hydroxyl ( ′ -oh) at their termini, which fold back into potent pamps recognized by rig-i, amplifying the innate immune responses [ ] . interestingly, some hcv genotypes escape rnasel cleavage by accumulating silent mutations at ua and uu dinucleotides, which are preferentially targeted by the nuclease [ ] . the rnase iii endoribonuclease dicer is active against different rna viruses in both vertebrate and invertebrate animals (table ) [ , [ ] [ ] [ ] [ ] [ ] . rna interference (rnai) is a primordial form of antiviral immunity mediated by small rnas and is the major immune defence in insects, plants and nematodes species [ , ] . in the antiviral rnai pathway, dicer is able to bind and cleave viral dsrnas into ~ nt-long small interfering rnas (sirnas). the sirnas are subsequently loaded into the argonaute-containing rna induced silencing complex (risc) and program risc to target vrna in a sequence-specific manner, inducing vrna degradation or translational arrest. although evidence of dicer activity against certain rna viruses have been reported in undifferentiated mouse stem cells [ ] , and in some other specific context [ , ] , the physiological antiviral role of rnai and its interplay with the interferon response in vertebrate animals is still an area of active debate [ ] [ ] [ ] . in recent years, it has been revealed that the rna code is much more complex than the primary rna sequence. in particular, covalent rna base and sugar modifications recently emerged as an additional layer in the regulation of gene expression. such modifications can change the rna structure and its interaction with other rnas or proteins, thereby regulating important steps in rna metabolism including splicing, rna stability and translation. these rna modifications are also utilized by host cells as a way to distinguish self-rnas from vrnas [ ] [ ] [ ] . it is therefore not surprising that viruses evolved strategies to modify their own genomes and transcripts or hijack dedicated cellular enzymes responsible for these modifications. here we briefly describe three rna modifications known to be present on vrnas (adenosine-to-inosine editing, n -methyladenosine (m a) and ′ -o-methylation) [ ] [ ] [ ] . we expect that other rna modifications may have the ability to also influence vrna stability, translation and immune potential. deamination of adenosines to inosines (a-to-i editing) is a covalent rna modification occurring on dsrna structures, by the adenosine deaminase acting on rna (adar) enzymes (fig. , table ). among the three mammalian adars, adar is expressed in its constitutive nuclear form adar p and the ifn-inducible cytoplasmic one, adar p . both enzymes consist of a c-terminal deaminase domain, three consecutive dsrna binding motifs and one or two copies of n-terminal z-dna binding domains [ ] . the consequence of the rna modification by adar is two-fold: first, if editing takes place in coding sequences, inosines will be misinterpreted by the translation machinery and will be read as guanosines. second, a-to-i conversion have the capacity to destabilize dsrna structures by changing the watson-crick base pairing and impairing recognition by cytoplasmic antiviral receptors, such as rlrs, pkr and oas . for these reasons, adar proteins play a pivotal role in masking self rnas against innate immune detection by selective labelling [ ] . given the opposite effects of adar and antiviral sensors in the control of innate immune responses to endogenous duplex rna, the ato-i editing contribution in antiviral defence remains unclear. a bias toward a-to-g mutations dependent on adar has been described for a wide range of viruses [ ] . this could potentially lead to the synthesis of dysfunctional viral proteins and destabilize key rna structures such as dsrna replication intermediates. although several studies have reported high a-to-i editing levels indicative of a potential mutagenic and antiviral effect, adar activity seems to be rather proviral due to its dampening effects on the innate immune system [ ] . one clear example where adar plays a proviral role is the case of hepatitis d virus (hdv). hdv contains only one orf responsible of expressing the hdv delta antigen (hdag), essential for hdv replication and spread. two forms of hdag are expressed: a short form s-hdag and one that is amino acid longer (l-hdag). hdv needs both to be expressed in order to replicate and spread and this depends on adar . in fact, adar recognizes a specific structure on the rod-like rna of hdv antigenomic rna and converts adenosine to inosine. replication of this mutated rna leads to the substitution of guanosine in its genome, specifically leading to the replacement of an amber codon (uag) for the small delta antigen by a tryptophan codon (ugg) which produces a protein that is amino acid longer. the synthesis of this longer protein is crucial for hdv spread, therefore adar is a proviral host rbp essential for hdv infection [ ] . in mammalian cells, n -methyladenosine (m a) is the most prevalent and dynamically modulated internal mrna modification. such a modification is reversible via the activity of two key enzyme classes: methyltransferases or "writers" (e.g. methyltransferase like (mettl ) and (mettl )) and demethylases or "erasers" (e.g. (fto) and alkbh ) (fig. , table ). the m a writers and erasers can alter the cellular rna fate by destabilizing local rna structures or by affecting protein binding to modulate host rna stability or translation efficiency. in particular, the m a readers such as yth domain-containing family proteins (ythdf -ythdf ) play crucial roles [ ] . recent reports show that m a could be a regulator of the immune system, playing a role in the discrimination between endogenous and exogenous rnas [ ] . indeed, several studies have identified m a modifications on a variety of vrnas and this enhances global viral gene expression by increasing rna stability and translation [ , ] . interestingly, mapping of m a on the rna genomes of flaviviridae, including dengue, zika, yellow fever, and west nile virus, identified conserved regions modified by m [ ] a, suggesting that this modification is a conserved regulatory mark across flaviviridae genomes [ ] . another case where m a appears to function in innate immune discrimination between self and non-self rnas is given by the human metapneumovirus (hmpv) [ ] . m a methylation of vrna promotes hmpv replication and gene expression. in the absence of m a residues on the vrna, viral infection is compromised due to a rig-i-dependent increase of type i interferon [ , ] . this notion is reinforced by the recent report of the effect of the loss of the m a writer mettl in the mouse hematopoietic system. indeed, mettl knock-out results in the upregulation of mda -rig-i, pkr and oas-rnase l pathways due to the aberrant accumulation of endogenous dsrna [ ] . therefore, m a modification appears to prevent formation of certain dsrna, so it would be interesting in future work to check whether this has implications during viral infection. the ′ o-methylation ( ′ o-me) modification is a highly abundant chemical modification found in the rna of virtually all eukaryotic species, whereby a methyl group is added to the ′ -oh of the ribose ring by cellular ′ -o-methyltransferases ( ′ o-mtase) (fig. ). ′ o-me is enriched in the first and second transcribed nucleotides next to the n methylated ′ cap structure of higher eukaryote mrnas. while n- methylation is important for stability and translation of the mrna, the ′ -o-methylation has been shown to antagonize the innate immune response by allowing endogenous mrnas to escape recognition by immune sensors [ , ] . notably, ′ -o-me of the first (cap ) and often the second (cap ) nucleotide abolishes interaction of host mrnas with rig-i and mda [ ] . the functional importance of ′ -structures and modifications in mrnas is supported by the fact that many cytoplasmic rna viruses (including picornaviruses, flaviviruses and coronaviruses) have evolved alternative ′ elements. these include small viral proteins linked to the ′ end of genomic rna, or encode functions associated with the formation of a ′ cap that are homologous to those found in eukaryotic cells. many viruses have evolved mechanisms to generate their own cap structures with methyl ribose in ′ -o position in addition to the one at the n position of the capped guanine to evade innate immune recognition. for instance, the coronavirus ′ -o-methyltransferase activity associated with the highly conserved viral nonstructural protein (nsp ) is necessary to inhibit mda recognition of vrna and activation of ifn pathway [ ] . ′ -o methylation of the ′ cap of vrna also subverts mammalian antiviral responses by evading restriction by interferon-induced proteins with tetratricopeptide repeats (ifits). ifits are cytoplasmic antiviral proteins which differ in specificity against rna structures. among them, ifit binds ′ ppp rnas [ ] and non- ′ -o methylated viral capped transcripts [ , ] to inhibit their translation by out-competing the cellular translation initiation apparatus [ ] (table ) . members of the flaviviruses and coronaviruses are able to evade ifit sensing by adding a ′ o-methyl cap structure to their own mrna via viral proteins [ , ] . interestingly, alphaviruses whose genomic rna has a ′ cap lacking ′ -o methylation can avoid ifit immune restriction and efficiently replicate thanks to a secondary structure within their ′ untranslated region (utr) which alters ifit binding and function [ ] . once it has managed to escape host cell sensing and degradation pathways, vrna must compete with endogenous mrnas for accessing the same protein synthesis machinery. rna viruses have evolved several mechanisms to bypass the highly regulated translation cycle, particularly at the initiation phase and most of these mechanisms rely on peculiar vrna-host rbps interactions. importantly, changes in abundance, modification and activity of translation factors could regulate both vrna and host mrna translation [ ] . therefore, rna viruses often employ strategies that favor the selective translation of their genomes. they can accomplish that by targeting, modifying or cleaving host translation factors and/or by usurping the identity of endogenous mrnas to have a privileged access to the ribosome [ ] . canonical cellular mrnas are capped at their ′ end cotranscriptionally. the cap structure, consisting of a -methylguanosine (m g) linked to the ′ nucleoside of the mrna chain through a ′ - ′ triphosphate bridge, is crucial for mrna stability and translatability. specifically, the cap protects mrna from ′ - ′ exonucleases and ensures recognition by the cap-binding complex eukaryotic translation initiation factors (eif f) [ ] (fig. ) . interestingly, vrna caps can be stolen ("cap-snatching") from cellular mrnas or synthesized using either a host-or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism [ ] . recognition of the cap by the eif f subunit eif e is thought to prepare mrnas for recruitment of the s complex containing multiple eifs, the initiator methionine trna (met-trna i met ) as a ternary complex with eif and gtp (eif -tc), and the small s ribosomal subunit (fig. ) . the polyadenylated ′ mrna end is recognized by a poly(a)binding protein (pabp), which is bridged to the ′ end via pabp interactions with the eif g subunit of eif f end. the assembled s complex scans the ′ utr of the mrna to locate the initiator start codon (aug) (fig. ) . after aug recognition ribosomal s subunits joining trigger eifs release to form the s ribosome where elongation can proceed for synthesis of the polypeptide chain. translation is one of the most energy-expensive processes in the cell and is therefore tightly regulated. cells rapidly reprogram gene expression when subjected to stress to conserve energy and minimize damage [ ] . the arrest of bulk protein synthesis is a classical reaction cells employ during viral infection and it is often characterized by a specific inhibition of translation initiation. in the never-ending arms-race, many viruses have however evolved mechanisms to overcome the cellular stress they induce. generally, translation initiation is inhibited either by the disruption of the formation of the eif f complex or by the inhibition of eif -tc recycling [ ] (fig. ) . one major cellular regulator of eif f complex formation is the mechanistic target of rapamycin (mtor) pathway and will not be discussed here [ ] [ ] [ ] . the second major mechanism that causes translational arrest is the disruption of eif -tc recycling, caused by phosphorylation of the α subunit of eif . mammalian cells encode four known eif α kinases among which pkr (table ) (see above). the other three eif α kinases: haeme-regulated inhibitor (hri), general control non-derepressible protein (gcn ) and pkr-like endoplasmic reticulum (er) kinase (perk), respond to various cellular stresses including heat shock, amino acid deprivation, and er stress [ ] (fig. ) . these cellular stress states can be indirect indicators of viral infections. for instance, flaviviruses like dengue and zika viruses, which replicate in close association to er-membranes, induce er stress that results in perk activation and eif α phosphorylation early during viral infection [ , ] (fig. ) . in some cases, gcn can also be activated by vrna to arrest translation. for example, gcn can recognize two regions of sindbis virus (sinv) genomic rna and inhibit vrna replication by blocking early viral translation. strikingly, mice lacking gcn are extremely susceptible to intranasal sinv infections, demonstrating high virus titers in the brain compared to similarly infected control animals [ ] . alphaviruses like sinv take advantage of eif inactivation and can be totally insensitive to translational arrest by eif phosphorylation. subgenomic mrnas of sinv and another alphavirus, semliki forest virus (sfv), initiate translation in the presence of high levels of phosphorylated eif α, utilizing a highly stable rna hairpin loop downstream of the aug initiator codon that stalls the ribosomes on the correct site to initiate translation of their mrnas, thus bypassing the requirement for a functional eif [ ] . as mentioned earlier, dsrna is detected by and activates pkr for the rapid inhibition of translation initiation [ ] . following activation by dsrna binding, dimeriziation and auto-phosphorylation, pkr phosphorylates eif α which locks it onto its guanine nucleotide exchange factor eif b, thereby preventing the regeneration of the eif -tc that is critical for canonical translation initiation [ ] (fig. ) . although initially identified because of its ability to regulate translation in response to dsrna, pkr can also be activated by other cellular stresses such as oxidative stress, cytokines or following the stimulation of tlrs [ , ] . furthermore, upon activation, pkr not only inhibits translation initiation, but also modulates a plethora of different signal transduction pathways, including the activation of p mitogen-activated protein kinase (mapk), c-jun n-terminal kinase (jnk), signal transducer and activator of transcription and (stat and ) and the tumor suppressor p [ ] . pkr can indirectly activate the nuclear factor κb (nfκb), which has among other functions an important role in type i ifn induction [ ] . selective translation is a common strategy used by rna viruses to maintain intact their protein synthesis during global translation shutoff. we will briefly describe mechanisms used by viruses to promote the selective translation of their genomes during global protein shutdown. sequence, structure and chemical modifications present in the viral genome can determine the identity of the interacting rbps serving as translation factors. selective translation can therefore be achieved when vrna shunts the lengthy and elaborate multi-step mechanisms that yield assembled and productive s ribosomes to gain a privileged access to the ribosome. other strategies, such as targeting and degrading translation factors, ribosomal rnas or messenger rnas might also confer a selective advantage for vrnas over endogenous ones. we will discuss mechanisms that target the translation initiation step, nevertheless it is important to note that viruses are also able to trick capped rna is recognized by eif f complex recruiting the s complex to the ′ utr of mrna. this is followed by scanning until a start codon in encountered, followed by the formation of s ribosomes before elongation. lower panel: depicted are different modes of ires-dependent translation (i, ii, iii and iv). note the minimal requirement for initiation factors depending on ires class. these can either act on endogenous mrnas, eif f, eif , eif or the ribosomal subunits. in red are listed the four known cellular eif alpha kinases: gcn , hri, perk and pkr. translation elongation or termination steps [ ] . while some positive-sense rna viruses like flavi or coronaviruses possess a cap-structure on the ′ end of their vrna, some others like caliciviruses or picornaviruses (e.g. poliovirus or rhinovirus) decorate their ʹend with a covalently bound viral protein, vpg. in some cases, vpg proteins linked to the ′ end of positive-strand rna genomes (e.g. feline calicivirus (fcv) or human norovirus) plays the role of a cap structure, recruiting ribosomes through interaction with eif [ ] . in fact, modifications at the ′ end of vrnas largely dictate the translation initiation mode that could either be cap -dependent or -independent. coronavirus mrnas are thought to undergo a classical cap-dependent translation initiation mechanism. a small compound that prevents the formation of the eif f complex, a hallmark of cap-dependent translation, by blocking the interaction between eif e and eif g is able to significantly decrease human coronavirus e replication and infectious virus titers [ ] . to favor the translation of their vrnas over endogenous mrnas, severe acute respiratory syndrome coronaviruses (sars-cov) non-structural protein (nsp ) is able to efficiently and selectively inhibit host mrna translation by binding s subunits [ ] (fig. ) and promote host mrna degradation. for example, sars-cov- nsp protein induces template-dependent endonucleolytic cleavage of mrnas, however viral mrnas seem to be resistant to nsp -induced rna cleavage [ ] . one mechanism permitting this selectivity have been shown for middle east respiratory syndrome coronavirus (mers-cov) where nsp selectively target mrnas transcribed in the nucleus and spares mrnas of cytoplasmic origin, notably vrnas [ ] . flaviviruses are also thought to undergo a canonical cap-dependent initiation of translation requiring both the eif f complex and pabp [ ] [ ] [ ] . interestingly, in contrast to most cellular mrnas, flavivirus vrna lacks a ′ poly-a tail; however, pabp is still able to associate internally to denv ′ utr upstream of a conserved ′ stem-loop, pabp/ ′ utr interaction mimicking the role of mrna poly-a tail and presumably stimulating translation initiation ]. both utr regions ( ′ and ′ ) in flaviviruses possesses secondary structures shown to stimulate viral translation [ , , ] . interestingly, under cellular conditions where translation factors are limiting or eif e is inhibited, denv has been shown to alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that does not require a functional m [ ] g cap [ ] . although the mechanism by which flaviviruses undergo cap-independent translation initiation is not completely clear, it has been recently suggested that denv and zikv ′ untranslated regions could harbor internal ribosomal entry site (ires) functions [ ] . in fact, ires-dependent initiation of translation is the other major mechanism used by many viruses to initiate their translation, dispensing them from cap-dependence. ires sequences are specialized cis-acting rna elements present in viral ′ utrs capable of bypassing the extensive requirement of translation initiation factors to recruit ribosomal complexes and drive protein synthesis. iress therefore confer a considerable competitive advantage to viral mrnas, freeing them from host regulatory constraints and sustaining viral protein synthesis when canonical cap-dependent translation is impaired [ ] (fig. ) . there are four known types of viral iress (i, ii, iii and iv). although iress perform a similar function, there are no known universal ires sequences. in fact, ires elements present in the genome of different families of rna viruses lack overall conserved features [ , ] .the classification of viral iress in four types stems from their structural organization, their respective dependence on sets of translation initiation factors, and whether they use scanning or instead directly recruit ribosomes to the start codon [ ] (fig. ) . type i and ii ireses were first discovered in picornaviruses, such as poliovirus (type i) and encephalomyocarditis virus (type ii). these two ires classes interact with eif g c-terminal region which binds to eif and eif a [ ] . both require eif b, eif and met-trnai and are stimulated by the activity of eif , eif a and eif b [ ] (fig. ) . type iii iress, exemplified those of pestiviruses and hepatitis c virus ires, require even less translation initiation factors to recruit s ribosomes. in fact, the hcv ires bypasses the requirement for eif f and the scanning step of translation initiation. hcv ires is able to directly interact with eif and the ribosomal s subunit, placing the start codon present in the vrna in the ribosomal p-site [ ] (fig. ) . finally, type iv iress are amongst the most remarkable as they completely obviate the need for canonical initiation factors. these iress are common in the dicistroviridae family of viruses such as cricket paralysis virus (crpv) or drosophila c virus (dcv), which infect insects. these viruses contain a single positive-sense rna genome that encodes two non-overlapping open reading frames separated by a short intergenic region (igr). the igr region acts as an ires to initiate translation by recruiting s ribosomes in the absence of initiator trna i met or any canonical initiation factors, from a gcu alanine codon located in the a-site of the ribosome [ , ] (fig. ) . indeed, the igr rna structure mimics a trna, thus tricking the ribosome into translation initiation without the need of the eif -gtp-met-trna complex. using shortcuts for a privileged access to the translation machinery by tricking the ribosome is not the only strategy rna viruses employ to selectively favor the synthesis of viral proteins. another effective tactic is targeting and inhibiting the translation of cellular mrnas, which frees up a large pool of ribosomes, making them readily available for vrna translation. this is generally achieved by targeting host rbps, specifically translation initiation factors or ribosomal proteins. viruses that use ires-dependent translation for example, actively provoke the shut-off of host cap-dependent translation initiation, by targeting specific initiation factors. for instance, enteroviruses including rhino and polioviruses, encode proteases that cleave eif g, impeding the formation of the eif f complex and therefore cap-dependent host mrna translation [ ] (fig. ) . other picornaviruses, such as emcv suppresses cap-dependent translation by activating the translational repressor ebp [ ] . hypophosphorylated ebp binds eif e preventing, eif e-eif g interaction, thus eif f assembly. in fact, ebp hypophosphorylation is a strategy common to many viruses to inhibit host mrna translation (e.g., crpv, vsv) [ ] [ ] [ ] (fig. ) . targeting eif f-associated pabp proteins is also used by some viruses to influence host translation. for example, rotavirus nsp interacts with eif g and displaces pabp, therefore inhibiting host translation [ ] . likewise, enterovirus and calicivirus proteases cleave pabp, and the rubella virus capsid protein binds pabp to suppress cellular mrna translation [ , , ] . other translation initiation factors like eif can also be targeted by viral proteins. eif -binding proteins from measles and rabies viruses inhibit host protein synthesis [ , ] , whereas foot-and-mouth disease virus protease degrades eif a and eif b subunits [ , ] . also, the coronavirus spike protein has been shown to interact with eif f influencing translational control [ ] (fig. ) . although initiation factors are the privileged target of viral proteins to shut-off host mrna translation, some examples of viruses targeting translation elongation factors are also reported. for instance, it has been shown that ef a and eef are respectively inactivated by sars-cov- n protein and avian reovirus p [ , ] . in many of these examples, how vrna translation is able to proceed upon inactivation of these essential initiation or elongation factors remains unclear. viral rbps can directly target the ribosome to promote translation of their genomes and viral rna may also directly interact with ribosomal proteins to preferentially translate their genomes. denv ns protein interacts with rpl to promote viral protein synthesis [ ] . conversely, vrna can rely on specific nonessential ribosomal proteins, dispensable for translating most host mrnas. moloney leukemia virus and sinv stop codon readthrough and frameshifting efficiency, have been shown to depend on rpl [ ] . vsv cap-dependent mrna translation have been shown to be affected by rpl presence [ ] (fig. ) . also, some ires functions, like those found in polio, hcv or dicistroviridae, have been shown to be regulated by small ribosomal proteins such as rack and rps [ ] [ ] [ ] [ ] [ ] (fig. ) . interestingly, rack seem to be a privileged target of poxviruses to promote selective translation of viral mrnas [ ] . finally, rbps that are not necessary translation factors or ribosomal can also positively influence viral translation. for example, pelo has been shown to be a host factor needed for efficient translation of viral capsids of drosophila c virus (dcv) [ ] . vigilin and rrbp , two endoplasmic reticulum localized rbps have been shown to bind denv and zikv vrnas, promoting their translation, stability and replication [ ] . during the last decades, our knowledge of the intricate interactions of cellular rbps with vrnas have been a direct result of technical advances in modern molecular biology. when it comes to the study of rbps that are viral sensors or antiviral effectors, many major discoveries in how tlrs, rlrs, dicer or other rbps recognize and/or antagonize vrna, were made thanks to in vivo genetic screening technologies and innovations [ ] . both, hypothesis-driven reverse genetics approaches, and forward genetic techniques have been able to yield susceptibility or resistance phenotypes, revealing genes with unanticipated and important roles in recognizing and/or targeting vrnas. the involvement of tlr , , mda and rig-i in the innate antiviral response wouldn't have been possible without in vivo genetic manipulation and screening of mouse models [ ] [ ] [ ] . another classical example where genetics played a fundamental role in discovering important antiviral host rbps, is the study of the rnai pathway components. argonaute and dicer proteins' involvement in antiviral defenses across several animal and plant species was only possible thanks to genetic manipulation and screening techniques in model organisms [ , [ ] [ ] [ ] . importantly, biochemical and structural biology approaches including x-ray crystallography and cryo-electron microscopy (cryo-em) guided these discoveries and were able to determine structurally how sensor and effector rbps interact with vrna [ ] [ ] [ ] [ ] [ ] . likewise, recent technical innovations and improvements in rna isolation, manipulation and sequencing have enabled detection of new rna species and rna chemical modifications (e.g. m a, ′ -o-me) in vrnas, revealing an underappreciated role of these species and modifications in viral lifecycles [ , , ] . thus, the use of oxidative treatment on the rna prior to its sequencing helped to unravel the existence of ′ -o-methylated viral small rnas species that accumulate during sinv infection [ ] . more recently, the use of single cell rna sequencing allowed to determine at an unprecedented resolution the accumulation of viral transcripts in a dynamic manner [ ] . while this has so far been used to study dna viruses' transcription, it might also prove useful to study replication of rna viruses. we are still lacking a global picture of rna modifications that are present in viral rnas, and more importantly, we need to determine their exact contribution to the infectious cycle. to this end, it is crucial to develop and validate good antibodies targeting modified ribonucleotides, or to turn to emerging sequencing technologies, such as the oxford nanopore one, that allows direct rna sequencing. this strategy has recently allowed the direct detection of m a by sequencing of cellular rnas [ , ] . similarly, the last decades of research brought very detailed insights into how vrnas co-opt host ribosomes. this is partly due to the history of research in the mrna translation field. the ribosome being the most conserved and largest known rnp complex in living organisms, deciphering mrna translation has been an endeavor pursued since the birth of modern molecular biology [ ] . important knowledge in our understanding of mrna translation were made in the past thanks to technical innovations and improvements. for example, in vitro translation systems, reconstituted from rabbit reticulocytes, were instrumental to understand many aspects of endogenous mrna translational control [ ] . interestingly, thanks to such in vitro systems, we know since the ′ s the inhibitory role of viral dsrna or eif phosphorylation on translation initiation [ , ] . in vitro translation systems coupled to reporter assays were also crucial later on to decipher the different mechanisms by which ires-dependent translation proceeds [ , , ] . more recently, cryo-em approaches were able to paint a more detailed picture of how different ireses can interact and co-opt host ribosomes [ , , ] . the rapid development and improvement of cryo-em techniques is expected to generate further knowledge, surpassing ires-ribosome interactions. indeed, cryo-em techniques have been lately used to unravel the structural basis of flaviviral sfrna production for example [ ] . most recently, cryo-em structures of sars-cov nsp with human s ribosomal subunit revealed that nsp c-terminus binds to and obstructs the mrna entry tunnel, explaining how this protein is able to shutdown endogenous mrna translation [ ] . another very recent cryo-em study was able to identify residues of nsp crucial for mediating translation inhibition [ ] . in the future, these approaches promise to aid structure-based drug design not only against sars-cov but also other emerging rna viruses [ ] . it is finally important to note that other novel and innovative techniques developed to study translation such as single-molecule approaches [ ] or ribosome profiling by deep sequencing [ ] are revealing the complexity of vrna interaction with host ribosomes. for instance, single-molecule studies of ires-mediated translation have revealed insights into the dynamics of hcv and crpv ires interaction with ribosomes and clarified decades of biochemical research, providing an outline of the conformational and compositional trajectory of the ribosome during initiation [ ] . likewise, ribosome profiling revealed strategies that some rna viruses use to induce programmed ribosomal frameshifting (prf) that would have not been discovered otherwise. frameshifting events are 'programmed' because they occur at specific sequences, stimulated by cis-acting elements generally present in the vrna at rates that are orders of magnitude more frequent than nonprogrammed events [ ] . cardioviruses such as emcv and its relative theiler's murine encephalomyelitis virus (tmev) induce prf but are atypical due to the absence of an appropriately positioned stimulatory rna structure in their genomes and a failure to reconstitute prf in vitro, outside of the context of virus infection. remarkably, thanks to ribosome profiling, a recent study shows that emcv frameshifting is trans-activated by viral protein a. as a result, the frameshifting efficiency increases from to % (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins [ ] . in the future, ribosome profiling techniques are expected to reveal more secrets of vrna translation, especially for viruses that annexes organelles like the er to complete their translation [ ] . each time a technological leap is achieved the scope of what we know about vrnas interaction with cellular rbps has expanded. two global strategies have been used to study vrna-rbp interactions; protein-centric or rna-centric methods. protein-centric methods start with a known rbp of interest and characterize its interaction with rna. these approaches involve uv-crosslinking followed by antibody purification of the rbp of interest and identification of bound rnas, often by deep sequencing, broadly termed hits-clip (cross-linking immunoprecipitation followed by high-throughput sequencing) [ ] . these methods have been used in the past in the context of viral infections to characterize the rna species that bind either known host rbps [ , [ ] [ ] [ ] or of viral proteins [ , ] . we will not focus further on these methods here but readers can refer to excellent reviews on the subject in the context of viral infections [ ] [ ] [ ] . rna-centric approaches are more recent and focus on purifying an rna of interest to identify its associated proteins. a large number of the so-called "unconventional rbps" have been identified as associated to rnas thanks to these rna-centric approaches. for example, rna interactome capture (rna-ic), have largely expanded the repertoire of known rbps [ ] . rna-ic entails ultraviolet crosslinking of rbps to rna in live cells, followed by collective capture of rnps with polyadenylated (poly(a)) rna on oligo(dt) beads and identification of proteins by quantitative mass spectrometry (q-ms) [ , ] . these methods discovered new rbps that generally lack canonical rna-binding domains and are conserved across species, suggesting the existence of previously unidentified modes of rna binding and new biological functions for protein-rna interactions [ ] . this method was applied to study cellular rna-interactome during sinv infection and was able to identify over rbps that interacted differentially with host rna upon viral infection, amongst which, rnas encoding proviral and restriction factors [ ] . although rna-ic is able to identify rbps that directly interact with rnas, one limitation of this method is its reliance on oligo(dt) to pull-down poly(a) containing rnas. another novel method, comprehensive identification of rna binding proteins-mass spectrometry" (chirp-ms), addresses this limitation by using -mer oligonucleotide probes complimentary to the rna of interest. chirp-ms uses formaldehyde (fa) cross-linking prior to pull-down with biotinylated probes, able to hybridize with an rna of interest. unlike uv-crosslinking, the use of fa for crosslinking doesn't only identify direct rna binders but also their associated protein complexes. cross-linked rnp complexes are then captured with streptavidin beads prior to proteomic analysis [ ] . originally developed to identify rbps associated with long non-coding rnas (lncrnas) [ ] , chirp-ms was recently applied to identify rbps associated with denv and zikv vrnas [ ] . chirp-ms identified viral non-structural proteins ns and ns as highly enriched with denv and zikv vrna. furthermore, this method identified high confidence hits from the human proteome that were specifically and reproducibly associated with the denv or zikv vrna [ ] . many of those proteins were localized to the er, in line with the known biology of flaviviral rna, being translated and replicated at specific er sites [ ] . importantly, when viral infection is difficult to synchronize, chirp-ms is unable to differentiate at which step in the viral lifecycle vrna-rbps associations occur (e.g. entry, translation, replication, assembly). a recent approach addresses partly this limitation and is able to capture interactions with just the pre-replicated viral rna genome [ ] . viral cross-linking and solid-phase purification (vir-clasp) relies on infection of unlabeled host cells with -thiouridine ( su)-labeled viral genomes [ ] . irradiation of infected cells with nm light generates covalent cross-links between su-labeled viral genomes and interacting host or viral proteins. solid-phase capture of these complexes with solid-phase reversible immobilization (spri) beads leads to purification of total rna but only proteins covalently crosslinked to the su-labeled viral rna. this purification precedes (lc-ms/ms) identification of the crosslinked proteins [ ] . using vir-clasp, the study identifies early interactomes of different viral genomes (e.g. chikv, iav, zikv, vsv). the authors of the study find that incoming chikv vrna binds both proviral and antiviral factors. while on one hand, chikv incoming vrna hijacks the lipid-modifying enzyme fatty acid synthase (fasn) for pro-viral activity, it is on the other hand n -methyladenosine modified, binding to ythdf which suppresses its replication [ ] . the aforementioned rna-centric methods (rna-ic, chirp-ms, vir-clasp) all use crosslinking (uv or fa) to identify in vivo interactions by purifying the rna under denaturing conditions that remove noncovalent interactions, and subsequently extracting only the cross-linked proteins for identification [ ] . other approaches, that do not require crosslinking of rnps, use proximity proteomics to identify rbp-rna interactions in live cells. these approaches rely on 'promiscuous' biotin ligases that are able to label preferentially proteins that are within nm of distance with biotin [ , ] . originally developed to identify protein-protein interactions, this approach was recently adapted to identify rna-rbps interactions. the rna-protein interaction detection (rapid) method allows to use this spatial detection constraint to detect rbps bound to rna by tagging an rna of interest with a boxb aptamer to recruit a fusion protein of λ-n and a promiscuous biotin ligase [ , ] . the biotin sprayer binds the boxb motif through its λ-n domain and labels proteins proximal to its bound rna [ ] . rapid-ms was successfully used to identify rbps that bind zikv utr sequences. interestingly, the identified rbps show enriched expression in neural tissue, which is in line with the neurotropic nature of zikv [ ] . rapid pointed to a known rna-binding protein, qki, that is highly expressed in neural progenitor cells (npcs) and whose depletion reduces zikv rna levels by % [ ] . importantly, because these rna-centric methods rely on rna sequence specificity they can be transposed and applied to identify rbps associated with virtually any rna virus. after these proof-of-principle studies, all of these novel rna-centric approaches are expected to yield important fundamental knowledge about vrna interactions with host rbps, if applied to emerging or re-emerging rna viruses with epidemic potential such as ebola or sars-cov [ , ] . rna-protein interactions are central to most cellular processes. it is thus not surprising that rna viruses and cellular genes evolved intricate vrna-rbp interactions that determine many aspects of the viral life cycle [ ] . from the host cell perspective, the affinity of its rbps to the invading vrna, could be a double-edged sword. many cellular genes have evolved to specifically recognize and/or target vrnas and alert the immune system to mobilize cellular defenses. this is illustrated by the relative conservation of many vrna sensors and anti-vrna effectors across species, such as components of the rnai pathway or vrna sensors like rlrs and tlrs [ ] . likewise, many viral genes and rna structures have specifically evolved to co-opt fundamental host cell machineries, without which the virus in unable to replicate. this is exemplified by the convergent evolution of many rna elements in the viral genomes, with roles in hijacking ribosomes and the cellular machinery in general [ ] . in fact, structured rna elements, such as iress are shared amongst viruses from very different families, ranging from plant to animal viruses [ , , ] . also, viral enzymes that can interfere with endogenous mrna lifecycles favoring vrnas translation, are also found across a wide range of viral families [ , ] . during the last decades, since the rise of modern molecular biology we are witnessing a remarkable and accelerated development of tools and technologies that are enabling to delve deeper into fundamental cellular mechanisms. as mentioned in this review, each time a technical innovation emerged, our understanding of vrna-rbp interactions expanded. for example, proteins identified by the novel aforementioned rna-centric methods (e.g. chirp-ms, vir-clasp, rna-ic) are highly enriched with classical rbps as expected. however, hundreds of "unorthodox" rbps have also been found to bind vrnas [ , ] . some of those unorthodox' rbps have already known cellular functions and weren't expected to have an influence on viral lifecycles by binding vrnas. for example, cellular transport motors like dyneins have been shown to have an rna binding activity that might be responsible of rnas trafficking, uncoating, reverse transcription and in some cases viral replication factory assembly [ ] . another eloquent example comes from the interaction of er-resident multimolecular complexes, such as sec translocon complex or the oligosaccharyltransferase (ost) complex, with the lifecycle of flaviviruses [ , ] . although these complexes have known and established roles in protein translocation and glycosylation in the er, new unexpected rna-related functions of these complexes are just emerging [ ] . for example, flavivirus vrnas that are translated and replicate in close proximity to er membranes, seem to require "non-canonical" functions of the ost complex [ ] . this might involve tethering viral rna genomes to the er for efficient translation and replication [ , , , ] . future work will clarify which unconventional rbps are truly important for the viral infectious cycle. in the meantime, these systemwide approaches are uncovering new host rbps with unexpected new roles during viral lifecycles. understanding mechanistically the relevance of these newly discovered interactions is crucial in the future. alongside careful and rigorous biochemistry, this can be tackled with new complimentary cutting-edge methods (e.g. clip-seq, ribosome profiling) [ , , , ] to determine the footprints of rbps on viral and host rnas. other new approaches to visualize, track, target or detect rnas (e.g. crispr-cas ), if applied to vrnas, will clarify the role of the newly discovered host rbp-vrna interactions [ ] [ ] [ ] . finally, there are many reasons to look at the future of the field with optimism. indeed, continuing to dissect the intimate relationship between vrnas and host rbps with new tools, is expected to both expand our fundamental understanding of virology and cell biology, and to guide the development of much needed novel antiviral approaches. the authors report no declarations of interest. host factors in positive-strand rna virus genome replication human host factors required for influenza virus replication genetic dissection of flaviviridae host factors through genome-scale crispr screens diverse roles of host rna binding proteins in rna virus replication crispr-cas immunity in prokaryotes antiviral immunity directed by small rnas combating emerging viral threats the global distribution and burden of dengue virus: new clinical syndromes and its emergence in the 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virus matrix protein interplay with eif , new insights into rabies virus pathogenesis foot-and-mouth disease virus infection induces proteolytic cleavage of ptb, eif a,b, and pabp rna-binding proteins coronavirus spike protein inhibits host cell translation by interaction with eif f the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor alpha avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor (eef ) in vero cells dengue virus ns protein interacts with the ribosomal protein rpl : this interaction is required for viral translation and replication in huh- cells large ribosomal protein increases efficiency of viral recoding sequences a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas rack controls 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screen defines a signal peptide processing pathway required by flaviviruses multifunctional roles for the protein translocation machinery in rna anchoring to the endoplasmic reticulum programmable inhibition and detection of rna viruses using cas development of crispr as an antiviral strategy to combat sars-cov- and influenza rna editing with crispr-cas enterovirus apro targets mda and mavs in infected cells mda detects the double-stranded rna replicative form in picornavirus-infected cells activation of the antiviral kinase pkr and viral countermeasures activation of rnase l is dependent on oas expression during infection with diverse human viruses zika virus infection induces rnai-mediated antiviral immunity in human neural progenitors and brain organoids the authors would like to thank dr. alex g. johnson for his thorough and critical reading of the manuscript. the authors apologize to colleagues whose work could not be cited due to space limitations. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.semcdb. . . . key: cord- -z vsjmxn authors: colón-lópez, daisy d.; stefan, christopher p.; koehler, jeffrey w. title: emerging viral infections date: - - journal: genomic and precision medicine doi: . /b - - - - . - sha: doc_id: cord_uid: z vsjmxn the emergence of viral infections is driven by multiple factors including changes in human behavior, population growth, reservoir host distribution, viral diversity and environmental changes. effective surveillance methods, diagnostic assays and containment measures are pivotal to preventing widespread outbreak of a new viral infection. however, the limited understanding of some emerging viruses poses numerous challenges for effective intervention. in this chapter we discuss various genomics-based methods and strategies to overcome these inherent challenges of emerging and re-emerging viral infections with a focus on current viral threats. we also provide an outlook on the use of genomic tools in personalized medicine and potential solutions to current and foreseeable challenges. the mastomys natalensis rat, are increased in west africa during the dry seasons when the rats are more likely to be inside individuals' houses [ ] . fluctuations in climate resulting in increasing crop yields, and the associated rat population, followed by a severe drought can lead to increased cases of lassa fever. furthermore, climactic changes are altering vector-borne infectious disease risks due to expanded vector ranges. for example, there is an increased risk of lyme disease in canada due to tick range expansion [ ] , and climate change is expanding the mosquito range, and the associated infectious disease risk, of aedes albopictus in the united states [ ] . rna viruses represent a significant source of viral outbreaks due partly to the higher mutation rate and error prone nature of rna replication itself [ ] . newly introduced mutations into a viral genome can confer new pathogenic characteristics such as enabling transmission to new species or increased virulence. for instance, mutations within the severe acute respiratory syndrome coronavirus (sars-cov) genome allowed transmission from bats to humans [ , ] . the better understanding about the viral agents responsible of causing outbreaks in humans and the underlying factors driving emergence, the better the chances of preparedness and effective intervention for prevention of a new global epidemic ( figs. and ) . this chapter provides an overview of the genomics and precision medicine methodologies relevant to the topic of emerging and re-emerging viral threats. the chapter is divided up into two main sections: viral genomics and host genomics, and include examples of their applications in pathogen detection, population susceptibility, diagnostics, and outbreak response. the seminal investigations on the transmission of yellow fever virus by walter reed and colleagues in [ , ] ushered in an era of discovery and classification of a wide variety of viral pathogens including smallpox virus, poliovirus, and measles virus. technological advancements including viral cell culture, plaque assays, enzyme-linked immunosorbent assays, and monoclonal antibodies pushed human virology forward. a second wave of viral discovery and characterization is currently ongoing, harvesting the power of next-generation sequencing to identify unknown viruses in acute febrile illness patients [ ] [ ] [ ] . increasingly, efforts are underway to identify the next emerging pathogen by sequencing and identifying the viruses circulating at the human/animal/pathogen interface [ , ] . while detection of viruses in wildlife does not predict transmission, identifying mutations or gene acquisitions through surveillance and continued discovery will provide valuable insight in the future. a major hindrance for precision medicine toward emerging zoonotic pathogens is the limited global characterization of potential threats. taxonomic placement of viruses is problematic due to the lack of universal genes, such as the bacterial s rna gene, limiting classification to viruses with defined biological characteristics [ ] . in fact a mere total species are currently classified by the international committee on taxonomy of viruses (ictv) which pales in comparison to the estimated , viral species infecting mammals alone [ ] . metagenomic sequencing has highlighted this significant gap in classified vs unknown viromes, and while biological characterization will not also always be defined, knowledge can be gained from genomic sequences including evolutionary relationships and genome structure [ ] . continuing to gather viral genomic information from environmental, plant, and animal samples will continue to fill gaps in our knowledge base and further prepare us for detecting and predicting emerging pathogens. metagenomic sequencing has been used for the discovery of previously unclassified human pathogens. in lujo virus was discovered in south africa sequence diversity of lassa virus complicates molecular diagnostic tool development. a phylogenetic tree (a) was generated using and alignment of full length and near full length lassa virus s segment nucleic acid sequences available in genbank. diversity of lassa virus is linked to geographic location as shown here for viruses sequenced from nigeria, sierra leone, guinea, mali, and togo. selected sequences from each country [starred in (a)] were aligned in (b). the forward and reverse primers (red arrows) and the probe (green arrow) from a previously published lassa virus josiah real-time rt-pcr assay [ ] are indicated. the primers and probe are exact matches to the lassa virus josiah sequence; however, contain mismatches within other lassa virus isolates, including ones found in sierra leone. [ ] . five patients with undiagnosed hemorrhagic fever were discovered after air transfer of a critically ill index case. the disease resulted in a case fatality rate of %. unbiased pyrosequencing of rna extracts from human samples identified approximately % of an arenavirus genome which was thereafter gap filled using primers designed off the sequenced genome. phylogenetic analysis confirmed the identification of a novel old world arenavirus [ ] . similarly, deep sequencing identified bas-congo virus, of a novel rhabdovirus, in three human cases in the democratic republic of congo [ ] . this virus was associated with high fever and rapid death and was found to have only % amino-acid identity with other rhabdoviruses. the discovery and characterization of these viruses has allowed novel diagnostics to be developed, including real-time pcr, leading to a better preparedness for future outbreaks. sequencing human samples from outbreaks has resulted in the discovery of several viruses; however, % of emerging human pathogens result from wildlife [ ] . analysis of samples from non-human hosts including arthropods, bats, rodents, and domestic animals offers potential insight into the transmission, evolution, and treatment of these pathogens [ ] . deep sequencing on more than invertebrate species resulted in the discovery of phylogenetically distinct viromes filling in phylogenetic and evolutionary gaps [ ] . characterization of bacterial and viral relationships in mosquito arthropods demonstrated a symbiotic relationship between the bacterium and host, limiting dengue virus infection and potentially revealing new antiviral strategies [ , ] . the discovery of middle-east respiratory syndrome-coronavirus (mers-cov) in camels demonstrated these animals as a potential reservoir and host for transmission [ , ] . while detection of viruses in wildlife does not predict transmission, identifying mutations or gene acquisitions through surveillance and continued discovery will provide valuable insight in the future. in addition to pathogen discovery, metagenomic sequencing is increasingly used to monitor viral genomic changes as an outbreak is occurring [ ] [ ] [ ] [ ] . the ebola virus outbreak in west africa resulted in , cases and , documented deaths, and genomic sequencing was applied in near real-time to provide information to aid in containing the outbreak [ , ] . sequencing results early in the outbreak provided valuable insight into origination and transmission routes demonstrating the outbreak started from a single introduction in guinea in december and was sustained by human to human transmissions [ ] . sequencing provided molecular evidence that ebola virus was transmissible by sexual intercourse leading to changes in cdc recommendations for survivors [ , ] and establishing programs to support national testing of semen and other body fluids in male survivors [ ] . rapid outbreak sequencing allowed for the identification of transmission chains in sporadic clusters following the outbreak [ ] , further adding insight to end the epidemic. sequencing data from sierra leone early in the outbreak also found increased ebola virus diversity that could have an impact on sequence-based therapeutics, vaccines, and diagnostic assays being fielded at the time [ ] . having such rapid sequencing information during an outbreak would inform responders about the potential efficacy of diagnostics and sequence-based countermeasures, either reassuring decision makers or informing them of the need to modify strategies based on the findings. while sequencing has found a niche in the discovery and epidemiologic tracing of emerging infections, its roll for diagnostics is still in its infancy. emerging pathogens often occur in austere environments or countries with limited infrastructure not conducive to sequencing technologies. sequencing still has significant hurdles in decreasing the technical and mechanical requirements for routine clinical use. however, next-generation sequencing offers limitless potential due the necessity for little to no prior knowledge in sample composition. newer technologies such as nanopore sequencing are looking to minimize these hurdles by creating smaller foot-prints and near real-time sequence analysis with minimal sample preparation [ ] ; however, difficult paths to their acceptance beyond laboratory derived tests to full regulatory approval remain. knowledge of viral genomic sequences in designing rapid point of care molecular diagnostics such as pcr is invaluable. the last decade has seen a significant increase in the use of pcr as a primary diagnostic due to its speed, sensitivity, and cost. however, due to the lack of available genomic sequences and high genetic variation within certain viral species, finding a conserved target can be difficult. the advent of multiplex pcr has allowed more targets to be captured in a single assay, lowering costs and increasing throughput. for example, this technique has been used to subtype influenza a and b viruses [ ] . other diagnostic devices such as the biofire filmarray can run multiple pcrs at once on a single device allowing the user to select between different panels [ ] [ ] [ ] [ ] [ ] . whether singleplex or multiplex, pcr remains the most rapid and sensitive method for the detection of viral genomes directly from clinical matrices. coordinating with detection, viral genome sequences are increasingly being used to predict and guide antiviral therapy. during the ebola virus outbreak, sequence analysis of the viral genome over time demonstrated changes which could make the pathogen resistant to therapeutics such as sirnas, phosphorodiamidate morpholino oligomers (pmos), and antibodies [ ] . the discovery of the crispr/cas system and its ability to destroy dsdna has brought to light its potential in mutating essential sites or removing proviral dna from infected cells during and hiv infection crispr [ ] . similarly studies involving herpesviruses, large dsdna viruses, have demonstrated the ability to destroy latent herpesvirus from infected cells [ , ] . these methods require prior knowledge of the viral genome promising a future where antiviral therapies are targeted specifically toward an individual's own specific infection. emerging and re-emerging viral diseases pose unique challenges to the use of omics and precision medicine tools. how does one predict when and where a pathogen will jump a species barrier or emerge into a new population that could rapidly spread into new geographic regions? to further complicate the scenario, a new human viral infection may not present with obvious signs of an infection. such a virus may get introduced and transmitted across populations while remaining asymptomatic or unrecognized for long periods of times, like the hepatitis c virus (hcv) and hiv [ ] , for instance. viruses rely on their small-sized genomes to encode enough information to hijack the host's cellular machinery for target-cell recognition and entry, genome replication, protein synthesis, viral participle assembly, and propagation. evolutionarily, hosts evolved conserved mechanisms to generate an immune response to detect and limit an infection and to prevent re-infection in the future. the early innate immune response functions to slow the infection once the immune system recognizes there is an infection (extensively reviewed in [ ] ) and guides the subsequent adaptive immune response to the pathogen. successful control of these immunological processes depend on tight control of the host's immunological and inflammatory pathways by regulating gene transcription. these processes result in measurable changes in the relative expression levels of coding and non-coding rna and protein, even when an infection is asymptomatic [ ] [ ] [ ] [ ] [ ] or difficult to diagnose [ ] . the development of low-cost and less-time consuming genomics had facilitated the study of pathway-specific transcript alterations during viral infections. consequently, various transcriptomic-based assays have been developed to study, characterize, and identify signs of infection using transcript signatures. for example, zaas and colleagues identified a unique immunological gene expression profile capable of differentiating viral from bacterial respiratory infections in humans [ , ] , laying to foundations for appropriate antibiotic use without direct pathogen identification. traditionally, diagnosing viral infections is made based on clinical symptoms, pcr and serological testing, and virus isolation. while the world of omics is expanding with new and improved platforms, host transcriptome-based assays are becoming more feasible and widely available. those of more relevance to this chapter are targeted and agnostic next-generation sequencing (ngs) platforms for genome-wide association studies (gwas) and transcriptomics as well as microarray-based assays for amplification-independent profiling of transcripts. gwas have direct applications to precision medicine whereas host-based transcriptomic assays have shown to have multiple applications for clinical diagnosis, and pathogen identification and characterization. these technologies were not specifically developed for emerging or re-emerging viruses but can be leveraged for these purposed based on their applications. gwas may provide insights into the genetic factors driving population susceptibility to viral infections [ , ] . this information can be utilized to identify populations within geographic regions at higher or lower risk of being infected with a new pathogen. higher-risk populations may include those with increased direct exposure to animal reservoirs of zoonotic pathogens or their arthropod vectors. other population subgroups without defined topographical relationships can be determined as well. a few examples of these are demographic, age and, gender groups that can be classified based on genetic polymorphisms and other susceptibility markers. in this setting, gwas can provide guidance in outbreak preparedness and intervention. the information provided by gwas-based studies is not focused on the directed response to an infection but on the information already stored within the host's genome. on the other hand, transcriptomic approaches identifies the interactions between the pathogen and the host's genome by evaluating transcript levels, typically mrnas. the most commonly used transcriptomic methods are rna-seq [ , ] and microarrays [ , ] . rna-seq is generally suitable for unbiased transcriptomic profiling and provides better understanding of global transcriptomic changes. this agnostic method is appropriate for identifying changes in the human transcriptome as a result of an emerging viral infection to show specific mechanisms of immune response evasion and other effects in the host's biology at the transcriptomic level. conversely, targeted-approaches depend on previous knowledge of host-transcriptomics. studies with human cohorts and animal models have developed gene signatures that discriminate between viral and bacterial respiratory infections [ ] [ ] [ ] [ ] [ ] . other groups have developed sepsis classifiers to guide treatment options in clinical settings [ , ] . a goal of developing these disease classifiers is to use them to predict disease outcome prior to the onset of symptoms and to assist in decision making when a symptomatic disease with an unknown etiology is suspected. effective and accurate discernment between a viral and a bacterial infection can make the difference in administering the appropriate therapeutics and potentially saving lives. when a specific viral species can't be identified, a gene classifier may be useful to determine potential disease outcomes such as lethality. as technology continues to advance, the linkages of genomics and precision medicine with emerging infectious diseases will continue to strengthen. improvements with sequencing accuracy and speed are pushing pathogen-specific genomics to near real-time [ , ] , allowing rapid access to critical information within a timeframe that can impact an outbreak response [ , ] . confidence in the results from sequencing is leading to clinically actionable diagnostic patient testing [ ] , and it is reasonable to foresee rapid, pathogen agnostic diagnostic assays routinely utilized in the clinic. population growth, expansion into rural geographic regions, and rapid travel has greatly expanded the pathogen/host/environment interactome, putting a greater number of people at risk for formerly exotic infectious diseases such as ebola virus or even the next unknown, soon-to-emerge pathogen. applying genomics and the host transcriptome to generalize the response to a viral or bacterial pathogen would allow for more appropriate antibiotic use without requiring direct pathogen detection. besides the benefits to antibiotic stewardship in an era where 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advances in the applied virology of zoonoses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: no mbg d the aim of this manuscript is to describe how modern advances in our knowledge of viruses and viral evolution can be applied to the fields of disease ecology and conservation. we review recent progress in virology and provide examples of how it is informing both empirical research in field ecology and applied conservation. we include a discussion of needed breakthroughs and ways to bridge communication gaps between the field and the lab. in an effort to foster this interdisciplinary effort, we have also included a table that lists the definitions of key terms. the importance of understanding the dynamics of zoonotic pathogens in their reservoir hosts is emphasized as a tool to both assess risk factors for spillover and to test hypotheses related to treatment and/or intervention strategies. in conclusion, we highlight the need for smart surveillance, viral discovery efforts and predictive modeling. a shift towards a predictive approach is necessary in today’s globalized society because, as the h n pandemic demonstrated, identification post-emergence is often too late to prevent global spread. integrating molecular virology and ecological techniques will allow for earlier recognition of potentially dangerous pathogens, ideally before they jump from wildlife reservoirs into human or livestock populations and cause serious public health or conservation issues. generated concern for the environment and thrust ecologists into a new political field where preserving the integrity of our global ecosystems was the priority [ ] . even so, the society for conservation biology was not established until [ ] . as a part of this transition, ecology shifted from a descriptive science to one of prediction, reflecting the hope that ecologists might mitigate changes which can have negative impacts upon the ecosystem. ecologists have branched out into the study of parasites and disease as it has become increasingly apparent that parasites are inextricably linked to the ecology of their hosts and environments, to the point where they have been a driving force in the evolution of sexual reproduction and in the shaping of biodiversity [ , ] . over the past years, disease ecologists have developed the study of parasites and pathogens in the wild. this knowledge has been synthesized into mathematical models which describe the dynamic properties of ecosystems and predict how parasites and pathogens flow through them. [ , ] . these models are becoming more commonly integrated into epidemiological studies that seek to predict outbreaks or periods of time when cross-species spillover risk is highest. parallel to this progress, the field of virology, particularly the subfields of molecular virology and viral evolution, have also been burgeoning, largely due to advances in technology that have made molecular assays and genetic sequencing more accessible to a greater number of scientists. the development of high-throughput sequencing has greatly increased our ability to efficiently detect known viruses as well as to discover new types of viruses, thereby improving our understanding of viral diversity, pathology and evolution. this increased capacity has spawned the development of new fields of study. for example, phylodynamics allows researchers to determine the origin of circulating viruses in space and time. mutations among viral strains can be used to investigate interactions among host species as well as long-range host movement via corridors and flyways. phylodynamic analyses can also inform livestock management practices, as was the case with foot and mouth disease in the united kingdom [ ] . conducting viral surveillance in animal reservoirs and invertebrate vectors can help explain circulation within host species; observed patterns of zoonotic transmission; and even allow for the prediction of periods of increased risk of zoonotic transmission (e.g., rift valley fever and rainfall [ ] ; west nile virus (wnv) and american robin (turdus turdus) migration [ ] ; as well as hantavirus in mice [ , ] ). understanding viral ecology in wildlife reservoirs and identifying high-risk human-wildlife interfaces is especially critical in the context of ever increasing globalization, whereby transportation networks facilitate rapid spread of pathogens well beyond bounds where traditional epidemiological methods can be effective [ ] [ ] [ ] . the influenza a (h n ) pandemic spread from the presumptive point of emergence in la gloria mexico to new zealand in just under a month [ ] while sars radiated from guangdong, china to different countries within several months [ ] . the negative impacts of emerging infectious diseases are not limited to humans. indeed, wildlife conservationists have documented several mass mortality events in other animal species. western lowland gorillas (gorilla gorilla gorilla) have been decimated by ebola virus [ ] and an especially virulent calicivirus, rabbit hemorrhagic disease virus, spread through both domestic and wild rabbit populations, resulting in tens of millions of deaths [ ] . in some instances the viruses have attenuated, while in others the animal populations have been brought to the brink of extinction. importantly, the risk from disease to humans and animals should not be separated. the global transportation network facilitated the introduction of infected vectors (e.g., mosquitoes) into new york and wnv caused both avian and human mortality, and this virus has subsequently spread across the united states [ ] . table . definitions of key terms used in the text. the formation of a hybrid population through the mixing of two ancestral, or long-separated, populations. a method for estimating historical population dynamics from a sample of sequences without assuming a predefined demographic model. coalescent theory a mathematical framework which describes the distribution of gene trees in populations. it provides mathematical methods for connecting demographic or ecological models with a phylogenetic tree. demography is the statistical study of populations. in the field of ecology, demography encompasses the study of the size, structure and distribution of populations, and spatial and/or temporal changes in them in response to birth, migration, aging and death. however, here we use a more rigid definition of demography-as the pattern and rate of population growth. the number of breeding individuals in an idealized population that would show the same amount of dispersion of allele frequencies under random genetic drift, or the same amount of inbreeding, as the natural population under consideration. the analogue of ne for viruses, the ‗effective number of infections' is related to the number of infected host individuals and to the number of new transmission events [ ] . a discipline which uses next-generation sequencing technologies to characterize the entirety of genomic material found in environmental samples. the molecular clock is derived from the hypothesis that sequence evolution, while random, occurs at stable rate such that the time since the divergence of two or more sequences can be estimated. recent ‗relaxed molecular clock' analysis can account for variation in the rate of sequence evolution through time or between lineages. the relations among a set of sequences showing which shares a most recent common ancestor with other sequences. the study of the principles and processes governing the geographical distribution of genealogical lineages. in the field of population genetics, population structure is defined as the absence of random mating within a population. this is the definition used here. in ecology, population structure is defined by several key parameters including number of individuals in a population, age distribution of individuals, probabilities of survival (or mortality), and rates of fecundity. a process that occurs in segmented viruses by which one or more segments ‗swap' to create a new viral genome. this drives the process of antigenic shift in influenza a viruses. the process by which new genotypes are created by the combination of distinct lineages. in sexual organisms it occurs during meiotic division, by the exchange of dna between different chromosomes or ‗crossing-over'. viral recombination occurs during viral replication and is an important factor in viral evolution (for more details see worobey and holmes, [ ] ). mutation of a virus such that it changes ‗back' to its wild-type state. the timing of an organism's schedule of reproduction and death. species with long life histories, also known as ‗k' strategists, tend to have low reproductive rates, stable populations, long generation times and long lifespans. where the parasite population is not randomly distributed among hosts, such that the variance is greater than the mean. the macroparasites in a host population are often best described by the negative binomial distribution such that a minority of hosts possess the majority of the parasites. r (basic reproduction number) in the case of viruses and other microparasites, r is the average number of secondary infections which an infection produces. as such it is a measure of parasite fitness. a host species that can independently maintain a disease and act as a source of infection to other host species. infection in reservoirs is usually more persistent and less harmful than that of other hosts. zoonotic disease a disease transmissible from animals to humans or vice versa. globalization, host ecology, host-virus dynamics, climate change, and anthropogenic landscape changes all contribute to the complexity of zoonotic viral emergence and disease, and create significant conservation and public health challenges. comprehensive and collaborative scientific approaches that transcend disciplinary boundaries are necessary to address these challenges. it is the goal of this paper to review new methods for understanding viral dynamics and illustrate how and when these techniques can be used by not only public health officials, but also disease ecologists and conservation biologists. the phylodynamic paradigm, established in [ ] , exemplifies the power of a multidisciplinary approach. it unites the ecological and evolutionary study of viruses and builds upon advances in sequencing technologies and coalescent theory, by which gene genealogies are reconstructed backward in time [ ] . the analysis of phylogenetic trees enables researchers to address many of the primary questions posed by disease ecologists (figure ). in some cases this approach can provide an estimate of a virus's basic reproductive number (r ), which is a measure of parasite fitness [ ] . phylodynamics has far-reaching applications for the control of viruses in both human and animal populations, in addition to being vital to our understanding of the interconnectedness between them. phylodynamic studies can be used to identify reservoir species as well as defining the spatial and temporal origin of emerging infectious diseases [ ] . they can also help to elucidate how these viruses spread following their emergence [ ] . firstly, chains of viral transmission can be extrapolated from the branching topology of phylogenetic trees. one example of the utility of this approach is with rabies virus. rabies causes thousands of human deaths a year in africa and has been implicated in the decline and local extinction of several populations of african wild dog (lycaon pictus) [ , ] . lembo et al. analyzed sequences of rabies virus from the serengeti, revealing that domestic dogs were the reservoir of the virus and that they had transmitted it to other resident carnivore populations on repeated occasions [ ] . this work has applicability in that it can be used to design efficient and effective vaccination strategies, both to alleviate current distress and prevent future outbreaks [ ] . secondly, by mapping the geographical origin of each sequence onto the nodes of phylogenetic trees, the geographical origin of a virus might be identified. wallace et al. ( ) [ ] used this phylogeographic approach to identify guangdong province, china as the most parsimonious origin of highly pathogenic h n strain of avian influenza and to delineate the most likely pathways of viral spread [ ] . however, a recent bayesian analysis of this data did not support the conclusion that h n had dispersed from guangdong to indonesia [ ] . instead, the bayesian analysis suggested it had spread to indonesia from guangxi or hunan in china. this example demonstrates that different statistical techniques may yield different conclusions. as yet, neither the bayesian nor the frequentist method is universally considered to be superior and there is much room for improvement as statistical phylogeography develops as a field. phylogeographic tools have also been applied by walsh et al. ( ) [ ] to locate the putative origin of the zaire strain of ebola virus. in an attempt to resolve the controversy over the time of emergence and spreading trajectory of ebola in the congo basin, they then used spatial data and two different tests for the impact of selection on the virus genome [ ] . where a virus is expanding in range, as the zaire strain of ebola virus appears to be, using a ‗landscape genetics' approach may help identify geographical barriers to viral spread and help identify vulnerable human or wildlife populations lying in the path of infection [ , ] . these phylogenies may reflect transmission chains, however sampling must be sufficient for them to do so, while recombination may obscure ‗true' relationships between viral sequences. (b) simple molecular clock theory, predicated on the neutral theory of molecular evolution [ ] assumes that mutation occurs at a constant rate over time, thus the time that has elapsed since a pair of virus strains diverged from a common ancestor may be quantified. methods that account for differences in the evolutionary rates of different strains, and for variation in these rates through time, have been recently developed [ ] . here variants represented by thick lines evolve much faster than those represented by thin lines. (c) using a phylogeographic approach, the location at which a sequence was sampled may be mapped onto the viral phylogeny and the likely spreading trajectory of the virus inferred. while parsimony approaches have been popular, powerful bayesian methods that account for uncertainty of dispersal process and historical phylogeny have been developed to reconstruct viral dispersal events [ ] . crosses on the phylogeny represent such viral dispersal events, in this example. (d) coalescent theory provides the basis for many phylodynamic approaches. here, circles on the same row represent temporally simultaneous infections. working back from sampled infections (red circles), lineages can be traced back to the most recent common ancestor (black circle) via hypothetical, unsampled ancestors (grey circles). the time it takes for sampled lineages to coalesce is dependent on a variety of variables (i.e., viral effective population size, population structure, selection, stochastic infection die-out and recombination). a variety of methods are available to test for selection and recombination. phylodynamic analyses are not without limitations. dense and representative sampling at a scale equivalent to epidemiological surveys is required to fulfill the potential of phylodynamics for understanding epidemics of rapidly-evolving viruses [ ] . the construction of phylogenetic trees from these viral sequences may be complicated by recombination and, in the case of segmented viruses, reassortment of viral genomes [ ] . as a result of these processes, the genes on a single viral sequence may have very different origins (see the discussion of the different origins of the hemagglutinin and neuraminidase segments of h n avian influenza in lemey et al. [ ] ). therefore, concatenated analysis of multiple genes may be confounded. the ability to construct phylogenies is further limited by the total viral genetic information available. genbank is a vast public database that contains records of genetic sequences; however, its usefulness is dependent upon the willingness and/or ability of individuals and organizations to submit viral sequences. governments and industrial institutions may be reluctant to report sequences of economically important viruses (i.e., avian influenza) due to the potential negative economic impacts that may ensue. although phylodynamics is currently encumbered by the aforementioned factors, there is hope for progress. advancements in coalescent theory will help us to deal with the phylogeny construction problems caused by recombination and reassortment. they will also facilitate better utilization of genomic and spatial data, provided these advancements are also accompanied by a simultaneous increase in computing power, which is also currently limiting. the utility of phylodynamics is not limited to questions of interest to virologists and disease ecologists. this approach may also inform investigations of host population biology and, in so doing, aid in the development of conservation policy. host molecular markers (e.g., microsatellites, mitochondrial dna) are used by conservation biologists and ecologists to infer population structure, historical demography and other critical features of wildlife populations [ ] and have proved particularly powerful when analyzed in combination (i.e., [ , ] ). recently, it has been demonstrated that the pathogens of host populations might also be useful to this end. research using helminths and bacteria has revealed patterns of ancient human migration and dispersal [ , ] identified ancient refuges of rodent and bird taxa [ , ] and shown that there was past contact between contemporary non-sympatric bat species [ ] . however, there have been few attempts to utilize viruses to this end (save [ , ] ). it is surprising that viruses have not been used more for the inference of host population biology since some of their characteristics make them ideal for doing so. most viruses have large population sizes and short generation times, and many replicate using a highly error-prone rna-dependent rna polymerase, causing them to accumulate many more mutations (nucleotide changes) per unit time than the host genomes [ , ] . consequently, viruses may provide information about host demographics on a shorter timescale than molecular markers of the host. one of the signature tools of phylodynamics, the bayesian skyline plot, might also be utilized to infer changes in historical population size of the host. these plots incorporate the use of a molecular clock and coalescent theory to infer historical changes in virus population sizes without assuming a predefined demographic model [ ] . it is important, however, that the timescale over which the evolutionary dynamics of the virus population can be reliably reconstructed is appropriate for the parameters of interest in the host population. unfortunately, the very characteristics that make viruses useful for estimating host population structure and demography may also impede the analyses. multiple substitutions can occur quickly in the viral genome and this will obscure the host population's actual evolutionary history. meanwhile, variations in the transmission mechanisms of viruses (vertical vs. horizontal) can alter the ability to accurately infer a virus' relationship to a host population. cross-species transmission is also problematic in that it can cause pathogen phylogenies to inaccurately reflect the history of their hosts [ ] . therefore, before the genetic information contained within a virus population can be used to infer the population structure and demography of the host, it is critical to test for congruence in the evolutionary history of the host and virus populations. this is accomplished by statistically comparing the respective phylogenies within the relevant timescale. viruses with high host specificity have a greater likelihood of exhibiting such congruence. feline immunodeficiency virus (fiv) is known for high host specificity and is, thus far, the only virus to have been used to elucidate changes in host population structure and size. from a phylogenetic analysis of fivpco, the fiv type specific to the cougar (puma concolor), biek, drummond, and poss ( ) [ ] inferred that the north american population of cougars became subdivided during the last century but subsequently expanded in both size and range. subsequently, antunes et al. ( ) [ ] used the distribution of fiv ple subtypes in the serengeti to infer that recent admixture has occurred between the region's lion (panthera leo) populations. these recent changes in felid population size and structure could not have been inferred from host genetic data. there is great potential for the further use of this technique by conservation biologists and ecologists, and for it to complement existing methods which utilize host genetic data. host genetic markers are used to define management units for conservation purposes [ ] . many ‗flagship' endangered species have long life histories [ ] , a feature that correlates with both extinction risk in certain regions [ ] and difficulty in reconstructing recent demographic history from molecular markers. because of the latter, the use of viral genetics to define management units may be an important avenue of exploration. in addition to aiding the definition of management units, viral data could be used to analyze the consequences of management activities and other environmental changes on target species. where viral genetic diversity exists in a spatially heterogeneous distribution, viral movement patterns could be used to study the migratory behavior of animals (as macroparasites have been [ , ] ). as such, researchers could monitor the use of wildlife corridors and the efficacy of control measures aimed at limiting the range of a host species [ , ] . where viruses can be readily amplified from non-invasively collected samples (see [ ] ), the above objectives could be achieved in a cost effective manner with minimal disturbance of the study species. viruses with specific transmission routes may also serve as proxies for behaviors related to transmission (i.e., sexually transmitted diseases). similarly, where cross-species transmission occurs, viruses might be indicative of types of sustained, direct contact between different, sympatric taxa which facilitate such transmission, for example predator-prey interactions [ ] . at the broader ecosystem level, inferences about long-term evolutionary processes might also be made by examining the phylogeographic structure of numerous host and virus populations of a region (see [ ] ). a major hurdle for both virologists and ecologists is defining the biodiversity of life. at present, scientists do not know the actual number of mammal species, much less the diversity of viruses they harbor [ ] . indeed, the diversity of viruses known to infect the house mouse (mus musculus), a staple in biomedical research, is not yet completely known. recent advances in genetic sequencing, including high-throughput sequencing and other -next-generation sequencing‖ techniques, as well as masstag pcr and microarray multiplex assays [ , ] have made the study of microbial diversity feasible [ ] . these technologies have facilitated a movement from classical virology, where the focus was on disease etiology, toward a broader discipline that considers the rest of the viral diversity or -the virosphere‖. metagenomic studies have used next-generation sequencing to study biodiversity in substrates such as ocean water and soil [ , ] . metagenomics has also been used to screen human and animal clinical samples in order to determine etiologic agents of disease or to describe the microbial flora normally present in a vertebrate host-in many cases the result has been the discovery both of novel pathogens and novel associations between clinical disease and agent [ ] [ ] [ ] [ ] . as technology becomes more affordable, and thus accessible, there will be increasing opportunities to ask large-scale questions such as: how does the virosphere vary across space and time? how does it vary across species? can we use this to define risk of cross-species transmission or to inform conservation efforts? and how might co-infections with these undiscovered viruses influence the dynamics of the more well known viruses? the paucity of information about viral diversity within a host poses problems for research progress. it is difficult to understand viral pathogenesis and transmission without completely understanding the dynamics of co-infections. indeed, it is currently difficult to ascribe a host's symptoms to an individual virus with any certainty because a virus' actions, and even its ability to infect the host, could be a function of another (possibly undetected) co-habitant of the host. evidence of interactions between co-infecting species has been clearly demonstrated [ , ] and it will be critically important to elucidate the interactions that occur between multiple pathogens as well as the combined effects they may have on a host's immune system. broadening our understanding of the diversity of pathogens that exist in human and animal hosts through wildlife and domestic animal surveillance will significantly improve our ability to recognize novel zoonotic agents in the context of a disease outbreak. phylogenetic information obtained from comparative sequence analyses can improve our understanding of the impact of sequence mutation on virulence, as well as inform decisions about vaccine development. a final noteworthy benefit of viral discovery efforts is that these techniques should be important for identifying candidates for future vaccines as a virus's most worthy competitor is often another virus. from a health perspective, vaccination is arguably the most important technology that has arisen from the study of viruses. vaccination offers a direct means of intervening in a host-pathogen system and it has become routine in many parts of the world. efforts to this end have resulted in the eradication and/or control of smallpox, polio, mumps, measles, rubella and most recently, rinderpest. vaccines take several forms including live-attenuated viruses; inactivated whole viruses; inactivated toxins and viral protein subunits, and these are often delivered in combination. while live-attenuated vaccines have been predominant, a new generation of techniques including gene delivery and nano-technologies are being used to develop highly-efficacious and safer vaccines, that have less risk of reversion [ ] . new types of administration methods are also being developed with oral, aerosolized and nasal vaccines currently on the market. these less invasive administration techniques decrease labor costs associated with administration and offer increased capacity for mass-dispersal of vaccines to both humans and free-ranging wildlife [ ] . vaccination campaigns aimed at both protecting threatened species and decreasing public health risks via animal vaccination have taken place. swiss health officials were pioneers in this field, using oral vaccines to control rabies in wild red foxes (vulpes vulpes) [ , ] . these vaccines were inserted into chicken heads which were distributed in the wild beginning in [ ] . as can be observed from the supplemental movie (video s ), their initial barrier approach evolved into a large-scale treatment of infected areas and resulted in rabies being successfully pushed back to and then eliminated from the swiss alps [ , ] . following the success of these trials, campaigns were conducted in western europe [ ] and canada [ ] with similar results, though the situation in the united states has proven more challenging. other successful vaccination examples include canine distemper virus in black-footed ferrets (mustela nigripes; [ ] ) and ethiopian wolves (canis simensis; [ ] ), as well as rabies in florida panthers (felis concolor coryi; [ ] ) and african wild dogs [ , ] . a caveat to this success is that there is growing evidence that vaccination against a specific strain of pathogen can result in inadvertent selection for related co-infecting strains. thus vaccination can influence the dynamics of a pathogen [ ] . the possibility of inadvertent viral strain selection highlights the importance of understanding the long-term evolutionary and ecological consequences of vaccination. indeed, where threatened or endangered animals are concerned, mishaps may prove disastrous. attenuated canine distemper vaccines did not provide immunity to critically endangered black-footed ferrets, while the use of a live canine distemper virus vaccine resulted in clinical distemper arising in one of the few remaining populations [ ] . ideally, long-term clinical trials with suitable animal models might avert these problems. these trials should also be used to provide an a priori understanding of how vaccination might shape future evolutionary processes. in contrast to the ferret experience, efforts with the endangered ethiopian wolf serve as an example of a successful vaccination program. wolf populations were suffering severe mortality due to rabies and distemper acquired from the wild dogs that shared their home range [ ] . on the basis of a spatially explicit individual-based model, which indicated rabies could be controlled in dogs given just over % coverage [ ] , knobel et al. executed an intensive vaccination plan [ ] . both the extent and duration of outbreaks in the treated areas were limited and, although monitoring and continued vaccination are required, the situation appeared to be under control in [ ] . wildlife vaccination campaigns are also being investigated as tools to limit public health risks. tsao et al. vaccinated mice in an effort to break the cycle of lyme disease and reduce the risk of emergence in human populations, in which it causes tens of thousands of deaths per year in the us [ , ] . in the same vein, griffing et al. [ ] have tested the efficacy of vaccinating american robins to interrupt the wnv transmission cycle. this species can absorb up to % of the potentially infective mosquito bites in early spring and is thus a key host in the wnv system [ ] . targeted vaccination of this single species could potentially result in herd immunity and reduce the risk of human infection as well as decreasing wildlife mortality. these works exemplify how ecological knowledge can be used to identify and exploit some of the heterogeneities which so often dominate the dynamics of pathogens. while the lasting efficacy of wildlife vaccination efforts has yet to be demonstrated with either endangered species or in breaking the transmission cycle of human pathogens, an increasing number of researchers are drawing attention to systems where it seems feasible [ , ] ; demonstrating that intricate knowledge of host and virus ecology can greatly reduce the amount of vaccine coverage that is necessary to control these viruses. the problems entailed by the sheer number of viruses, viral resistance, the explosive potential for spread, and the economic burden, make it clear that currently available vaccination methods do not provide a sustainable solution for either human or animal disease. the unambiguous indication is that researchers need to work towards the goal of developing a predictive framework where risk can be defined for different scenarios and not only to rank pathogens, and species, but also, places and times of year that can be identified as more or less precarious for global health. pending questions include: which geographic areas will experience more disease and conservation problems? which areas pose the highest risk for pandemic spread of pathogens? what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission events? and what are the relative roles of genetic relatedness and contact rate for transmission? some modeling work and reviews of historic data have been informative [ , ] , but novel uses of phylogenies of both viruses and hosts (as discussed above) provide promise for progress to this end, especially when coupled with high quality surveillance data. once we have this information, scientists will be able to design -smart surveillance‖ strategies whereby valuable vaccine resources can be efficiently targeted and efficiently distributed. ecological studies can effectively inform conservation as well as public health policy. gaining knowledge of reservoir host ecology can be critical for the development of eradication strategies. most viral disease systems are dominated by heterogeneities and identifying and understanding these can be crucially important when trying to interrupt the chain of events that leads to persistence. ecological studies of wnv have shown how forest fragmentation and decreased biodiversity can alter transmission among avian hosts as well as to humans [ ] . likewise, researchers have used satellite imagery to identify habitat characteristics that accurately predict the prevalence of sin nombre virus [ , ] , a hantavirus that uses the deer mouse (peromyscus maniculatus) as a reservoir host and it occasionally infects and kills humans [ ] . these studies epitomize the type of effort scientists will need to successfully fight viral pathogens in the future. however, piecing together emerging disease and conservation problems ex posto facto is only of limited value. increased pathogen surveillance and ecosystem process monitoring may provide the insight necessary to mitigate problems before they become serious human health or conservation concerns. this is especially the case for zoonotic viral pathogens where the reservoir hosts are known and a targeted approach is feasible. rodents rank as the number one reservoir of emerging and re-emerging zoonotic viruses [ ] . conveniently, these small mammals also present a manageable system for studying disease dynamics [ ] [ ] [ ] . individuals can be marked and sampled individually through time. their locations as well as their contacts with other individuals can be measured. as such, wild populations of rodents can be valuable as model disease systems to address relevant questions like: are there key hosts for transmission? how does prevalence vary seasonally or over time? what is the contact rate between the reservoir and humans? how do these pathogens flow through populations? the answers are of critical importance because they provide an indication of when and where there is increased risk of a zoonotic event whereby a human becomes infected, or when a species becomes at genuine risk of extinction. by monitoring and manipulating wild populations, one might also be able to identify factors that may increase a pathogens chance of emerging. for instance, what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission? and what are the relative roles of genetic relatedness and contact rate for transmission? long-term monitoring and surveillance in reservoirs will also enlighten us to the kind of aggregations and other heterogeneities that exist through time and that and can be exploited with efficient vaccination campaigns. we are experiencing a global increase in the rate of emerging viral zoonoses, which are primarily driven by anthropogenic activities such as land-use change, agricultural intensification, and driven by global travel and trade [ ] . in order to adequately understand, predict and ultimately interrupt the processes by which zoonoses cross the species barrier from their natural reservoirs to humans, and then become established as human pathogens, comprehensive scientific studies that use the tools of ecology, virology, microbiology, and epidemiology are needed [ ] . the study of disease ecology has become an established discipline with advances in both the formulation of new theory as well as the integration of molecular virological techniques that provide important information about epidemiology, ecology and viral evolution, all of which has been applied to both health and conservation [ ] [ ] [ ] . because ecological systems are rife with heterogeneities and often have non-intuitive processes underlying their dynamics, it is critically important for scientists to use a comprehensive approach to understanding the population processes of an ecosystem before successful intervention strategies can be developed or implemented. admittedly this is a daunting task and it is often the case that scientists need to operate with less than complete information. where this is the case, a modeling approach is necessary to identify key processes that allow successful interventions. technological advances in molecular virology and genetics, as well as the expanded use of mathematical models in epidemiology and disease ecology have dramatically changed our ability to manage both conservation and health. finally, it is only with this type of interdisciplinary approach that considers free-ranging wildlife, domestic animals and humans as inextricable components of a single disease system, that progress can be made that will satisfy both conservation and public health needs. this is the essence of conservation medicine [ ] and indeed we believe a -one health‖ approach to infectious disease is necessarily the way forward. supplemental movie (video s ). are we in the midst of the sixth mass extinction? 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the potential role of pterygodermatites peromysci in the population dynamics of free-living mice, peromyscus leucopus predicting the emergence of human hantavirus disease using a combination of viral dynamics and rodent demographic patterns anthropogenic environmental change and the emergence of infectious diseases in wildlife collaborative research approaches to the role of wildlife in zoonotic disease emergence ecology of infectious diseases in natural populations the ecology of wildlife diseases conservation medicine and a new agenda for emerging diseases this work was supported in part by nih/nsf -ecology of infectious diseases‖ awards from the john e. fogarty international center (grant r -tw - s ) and an nih award (k ai ) from the national institute of allergy and infectious diseases. we thank c. j. wale, b. wale and h. ewing for their help in proofing the manuscript.